Note: Descriptions are shown in the official language in which they were submitted.
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LIGNOCELLULOSE POLYMER PROBES AND METHODS
BACKGROUND OF THE INVENTION
[0001] This application claims the benefit under 35 U.S.C. 119(e) of prior
U.S. Provisional
Patent Application No. 62/246,231, filed October 26, 2015, which is
incorporated in its entirety by
reference herein.
[0002] The present invention relates to detection, characterization, and
quantification of specific
polymers, for example, lignocellulosic polymers such as polysaccharides, in
biomass, and further
relates to detection of any polymer, such as oligosaccharides, for instance,
for a variety of uses as
described herein.
[0003] In the field of pulp and papermaking, industries rely on a number of
physical, chemical,
and biological treatments to enhance the value of their product. Treatments
include, for example,
mechanical shearing (refining) of the fibers to help develop desired paper
properties. Currently,
there is no practical way to predict the outcome of such treatments without a
thorough analysis of
pulp and paper properties. Such an analysis involves pilot or industrial scale
trials that are costly
and time-consuming.
[0004] Current methods for polymer detection and fiber surface
characterization include, for
example, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy
(SEM); time-of-
flight secondary ion mass spectrometry (ToF-SIMS) and Fourier transform infra-
red (FTIR). These
techniques involve specialized equipment, specific expertise, and lengthy
manipulation and
interpretation. These techniques also do not distinguish between cellulose and
hemicellulose. Other
approaches have been developed for indirect detection of polymers based on the
use of dyes, for
example, acrydin orange and phenanthren. They have been successfully used for
studying plant cell
morphology, but they employ sophisticated microscopes and are not
quantitative. Still other
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techniques detect hemicellulose using antibodies raised against representative
molecules. However,
such antibodies are expensive and only detect a segment of polymers that
resemble the molecules
used for antibody production. If the exact epitope recognized by a particular
antibody is not found
or accessible, then the polymers will not be detected. Antibody techniques are
lengthy and also
involve use of secondary antibodies.
[0005] Carbohydrate binding modules (CBMs) are a group of molecules
specialized for
biomass polymer detection. CBMs are proteins optimized for specific detection
of various targets,
for example, carbohydrates such as crystalline cellulose and xylan. In order
to detect binding of
such CBM to biomass, one can attach a reporter dye to it. Such techniques
involve microscopy.
However, none of these methods have been successfully used for rapid
characterization of fiber
surface polymers that would enable the prediction of the impact of various
treatments on pulp or
paper.
[0006] Accordingly, there is a need for better materials and methods for
detecting
lignocellulosic polymers in biomass, and for the detection, in general, of
polymers, such as
oligosaccharides, in various applications.
SUMMARY OF THE PRESENT INVENTION
[0007] A feature of the present invention is to provide materials and
methods for detecting and
measuring lignocellulosic polymers in biomass, such as in a time and cost
effective manner.
[0008] Another feature of the present invention is to provide materials and
methods for
determining the effectiveness of industrial treatments on pulp or paper
before, during, and/or after a
particular treatment is applied.
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[0009] A further feature of the present invention is to provide materials
and methods for
determining a physical property of pulp or a paper product indirectly based on
the presence and/or
content of lignocellulosic polymers in the pulp or the paper product.
[0010] An additional feature of the present invention is to provide
materials and methods for
simultaneously measuring the presence and/or amounts of multiple, different
lignocellulosic
polymers in biomass independent of separate or sequential testing procedures.
[0011] A further feature of the present invention is to provide materials
and methods for
detection of any polymer, such as one or more oligosaccharides.
[0012] Additional features and advantages of the present invention will be
set forth in part in
the description that follows, and in part will be apparent from the
description, or may be learned by
practice of the present invention. The objectives and other advantages of the
present invention will
be realized and attained by means of the elements and combinations
particularly pointed out in the
description and appended claims.
[0013] To achieve these and other advantages, and in accordance with the
purposes of the
present invention, as embodied and broadly described herein, the present
invention, in part, relates
to a lignocellulosic polymer detection probe including a binding module that
specifically binds at
least one lignocellulosic polymer and a reporter module that is
spectroscopically detectable. Either
or both of the modules can include a polypeptide. The binding module can be a
carbohydrate-
binding module (CBM). The reporter module can be a fluorescent protein. The
probe can contain a
plurality of probes with each probe specific to a particular lignocellulosic
polymer and each probe
containing a unique fluorescent spectral profile.
[0014] The present invention also relates to a complex including a probe
specifically bound to a
pulp or paper product including at least one surface available lignocellulosic
polymer.
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[0015] The present invention further relates to a pulp or paper product
including at least one
surface available lignocellulosic polymer and at least one probe specifically
bound thereto.
[0016] The present invention also relates to method of detecting a
lignocellulosic polymer. The
method can include contacting the probe with a biomass material, and measuring
a property
associated with the reporter module to determine the presence or absence of at
least one
lignocellulosic polymer in the biomass material.
[0017] The present invention further relates to a method of determining the
effectiveness of an
industrial treatment on pulp or a paper product. A lignocellulosic polymer
detection probe can be
contacted with a pulp or a paper product. The specific binding of the probe to
the pulp or the paper
product can be detected. The amount of at least one lignocellulosic polymer on
a surface of the pulp
or the paper product can be calculated. The effectiveness or need of an
industrial treatment on the
pulp or paper product can be determined based on the amount of the at least
one lignocellulosic
polymer detected.
[0018] The present invention also relates to a method of determining a
physical property of pulp
or a paper product. A lignocellulosic polymer detection probe can be contacted
with a pulp or a
paper product. The specific binding of the probe to the pulp or the paper
product can be detected.
The amount of at least one lignocellulosic polymer on a surface of the pulp or
the paper product can
be determined (e.g., calculated). The at least one physical property of the
pulp or paper product can
be determined based on the amount of the at least one lignocellulosic polymer
detected.
[0019] The present invention further relates to a polymer detection probe.
The probe can have,
for example, one or more characteristics or functions of the lignocellulosic
polymer probes
described herein, overlapping characteristics or functions, or different
characteristics or functions.
The polymer detection probe can include, for example, a binding module that
specifically binds to at
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least one polymer (e.g., oligosaccharides or other saccharide polymers) and a
reporter module that is
spectroscopically detectable. A complex is provided including the probe
specifically bound to a
material including at least one surface available polymer.
[0020] The present invention also relates to a method of detecting a
polymer (e.g.,
oligosaccharides or other saccharide polymers). A probe of the present
invention can be contacted
with a material. A property associated with the reporter module can be
measured to determine the
presence or absence of at least one polymer in the material based on specific
binding of the probe to
the at least one polymer. The detection or non-detection has a number of
applications as described
herein.
[0021] The materials and methods disclosed herein enable rapid and
simultaneous detection of
various lignocellulosic polymers at the surface of fibers used in pulp and
paper, for example,
cellulose and hemicellulose using fusion proteins. Signal patterns can be
correlated to various
changes in pulp and paper properties. The methods enable prediction of various
treatments impact
on paper properties. Such prediction allows for rapid and accurate choice of
treatment, dosage,
and/or other conditions for a given target, for example, conditions leading to
a paper with higher
burst index, a pulp with higher drainage rate, and/or the optimal xylanase
concentration for
bleaching boost. The methods enable optimal use of chemical, enzymatic, or
physical treatments
alike. The methods can be used in any industry based on wood biomass.
[0022] Further, the present invention has many applications. The effect of
any relevant
chemical, physical, or biological treatment that has an impact on fiber
polymer exposure or
distribution or hydrolysis on pulp and paper properties can be determined or
predicted. For
instance, an enzymatic sequence for optimizing pulp refining can be determined
or predicted.
Xylanase treatment for boosting bleaching can be optimized. Degradation of
hemicelluloses when
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hydrolyzing biomass can be monitored. Production conditions for purified
cellulosic materials, for
example, nanocellulose or filaments, can be optimized by monitoring the
presence of amorphous
cellulose versus crystalline cellulose using the methods of the present
invention.
[0023] It is to be understood that both the foregoing general description
and the following
detailed description are exemplary and explanatory only and intended to
provide a further
explanation of the present invention, as claimed.
BRIEF DESCRIPTION OF DRAWINGS
[0024] FIG. 1 is a schematic depiction of an example of polymer detection
using the probes
of the present invention.
[0025] FIG. 2 is a schematic diagram of an expression vector of the pen 1A-
eGFP (eGFP
reporter module).
[0026] FIG. 3 is a schematic diagram of an expression vector of the pet11A-
mCheny
(mCherry reporter module).
[0027] FIG. 4 is a schematic diagram of an expression vector of the pet11A-
mOrange2
(mOrange2 reporter module).
[0028] FIG. 5 is a schematic diagram of an expression vector of the pet11A-
eCFP (eCFP
reporter module).
[0029] FIG. 6 is a SDS-PAGE example of purified fluorescence proteins. The
arrow
highlights the protein of interest.
[0030] FIG. 7 is an SDS-PAGE example of purified CBMs. The arrow highlights
the protein
of interest.
[0031] FIG. 8 is an example of a graph depicting emission spectra of
reporter modules
(eGFP and mCherry) and of a pulp handsheet disk after excitation at 549 nm.
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[0032] FIG. 9 is an example of emission spectra of FPs (eGFP and mCherry)
and a pulp
handsheet disk using an excitation wavelength of 488 nm.
[0033] FIG. 10 is an example of a schematic diagram of a pET11A-eGFP-CBM3a
expression vector (Probe 1).
[0034] FIG. 11 is an example of a schematic diagram of a pET11A-mCherry-
CBM4-1
expression vector (Probe 2a).
[0035] FIG. 12 is an example of a schematic diagram of a pET11A-mCherry-
CBM17
expression vector (Probe 2b).
[0036] FIG. 13 is an example of a schematic diagram of a pET11A-mOrange2-
CBM15
expression vector (Probe 3).
[0037] FIG. 14 is an example of a schematic diagram of a pET11A-eCFP-CBM27
expression vector (Probe 4).
[0038] FIG. 15. is a SDS-PAGE example analysis of Probes 1 to 4. From left
to right: Probe 1,
Probe 2a, Probe 2b (not used here has a higher affinity to amorphous cellulose
than Probe 2a),
Probe 3 and Probe 4. Shown on the left side of the last four gels is a
standard size ladder allowing
estimation of sizes for proteins migrated on the same gel.
[0039] FIGS. 16A-16D are examples of graphs of excitation (dashed) and
emission (full)
spectra of the FP-CBM proteins for probes 1, 2b, 3, and 4, respectively.
[0040] FIG. 17 is an example of a graph of binding saturation from a solid
state depletion
assay using Probe 1.
[0041] FIG. 18 is an example of a schematic diagram describing a probe-
binding experiment
on a paper disk.
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[0042] FIG. 19 is an example of a graph of fluorescence intensity (Fl) of
reporter module
(eGFP) alone bound to untreated and cellulase-treated pulp (after two levels
of refining).
[0043] FIG. 20 is an example of a graph of fluorescence intensity of Probe
1 bound to
untreated and cellulase-treated pulp (after two level of refining).
[0044] FIG. 21 is an example of a graph showing the impact of PFI refining
on the
quantification of crystalline cellulose on the surface of pulp 1 handsheets.
[0045] FIG. 22 is an example of a graph showing the impact of cellulase-
treatment on the
quantification of crystalline cellulose on the surface of pulp 1 handsheets.
[0046] FIG. 23 is an example of graph depicting quantification (ligimm2) of
Probe 1 bound
on the surface of xylanase-treated handsheets.
[0047] FIG. 24 is an example of a graph depicting quantification of
crystalline cellulose
( g/mm2) on the surface of PFI refined handsheet produced at two different
plants.
[0048] FIGS. 25A-25D are examples of graphs depicting correlations between
crystalline
cellulose quantification using Probe 1 ( g/mm2) and pulp 2/paper physical
properties as a
function of refining energy (PFI revolutions) including, respectively, tear
index (mN m2/g),
tensile index (N 'nig), internal bond strength (J/m2), an fibers mean length
(mm).
[0049] FIGS. 26A-26D are examples of graphs depicting correlations between
crystalline
cellulose quantification using Probe 1 ( g/mm2) and pulp 1/paper physical
properties as a
function of refining energy (PFI revolutions) including, respectively, tear
index (mN m2/g),
tensile index (N m/g), internal bond strength (J/m2), an fibers mean length
(mm).
[0050] FIG. 27 is an example of a graph depicting fluorescence intensity of
Probe 3 bound to
xylan at the surface of five different paper disks including, respectively,
UBKPR: Unbleached
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Kraft pulp (pulp 2), UBMP: unbleached mechanical pulp (pulp 5), BMP: bleached
mechanical
pulp (pulp 6), UBKP: unbleached Kraft pulp (pulp 3), and BKP: bleached Kraft
pulp (pulp 4).
[0051] FIG. 28 is an example of a graph depicting fluorescence intensity of
Probe 3 bound to
xylan at the surface of untreated and xylanase treated Pulp 4 (BKP) paper.
[0052] FIG. 29 is an example of a graph depicting fluorescence intensity of
Probe 4 (eCFP-
CBM27) bound to mannan at the surface of two different paper disks¨unbleached
Kraft pulp
(Pulp 3, UBKP) and bleached Kraft pulp (Pulp 4, BKP).
[0053] FIG. 30 is an example of a graph depicting fluorescence intensity of
Probe 4 bound to
mannan at the surface of mannanase-treated bleached Kraft pulp (Pulp 4, BKP)
paper.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0054] The present invention in part relates to a lignocellulosic polymer
detection probe
including a) a binding module that specifically binds to at least one
lignocellulosic polymer and
b) a reporter module that is spectroscopically detectable. Any suitable
binding module and
reporter module can be employed. A given probe can vary with respect to the
number and/or
kind of binding module and reporter module. A probe can contain a single
binding module and a
single reporter module. A probe can contain more binding modules than reporter
modules, more
reporter modules than binding modules, or an equal number of binding modules
and reporter
modules. A probe can contain from one to three, one to five, one to ten, or
more binding
modules and/or reporter modules. Each binding module can specifically bind a
particular
lignocellulosic polymer or a particular subset of lignocellulosic polymers.
[0055] The lignocellulosic polymer detection probe can include at least one
probe
polypeptide. All or part of a probe can be constructed from a polypeptide(s).
Polypeptides are
understood to contain proteins including the standard twenty amino acids
modified in whole or
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part. One or more non-standard amino acids can replace or be used in addition
to one or more
standard amino acids. The polypeptides can be constructed by chemical
synthesis or using
molecular biological expression systems whether in vitro or in vivo. Any
suitable expression
system can be employed, for example, prokaryotic expression systems,
eukaryotic expression
systems, plasmid based expression systems, chromosomally integrated expression
systems, or
any combination thereof. Modification can be done synthetically, via post-
translation
modification in an expression system, or both. Any suitable modification can
be employed, for
example, phosphorylation, sulfonation, fluorination, acetylation, addition of
carbohydrate
groups, addition of lipid groups, addition of nucleic acids, addition of
polynucleotides, addition
of other amino acids, addition of other polypeptides, addition of synthetic
organic molecules,
addition of inorganic groups, or any combination thereof
[0056] The binding module can be or include a binding module polypeptide.
The reporter
module can be or include a reporter module polypeptide. The binding module and
reporter module
polypeptide can be directly or indirectly connected to each other. For
example, the binding module
polypeptide can be fused directly to the reporter module polypeptide. The
binding module
polypeptide can be linked to the reporter polypeptide covalently, for example,
via a linker
polypeptide. The linker can be of any suitable length, for example, at least
one amino acid, from 2
to about 2,000 amino acids (residues), from about 5 to about 1,000 amino
acids, from about 10 to
about 500 amino acids, from about 25 to about 250 amino acids, from about 50
to about 100 amino
acids, or more than 2,000 amino acids. The linker can include a molecule other
than or in addition
to a polypeptide, for example, a carbohydrate, a polynucleotide, a lipid, a
synthetic organic small
molecule, a non-naturally occurring polymer, a metal, or any combination
thereof For example, the
binding module and reporter module can be cross-linked using formaldehyde,
glutaraldehyde, or
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both. The connection or attachment between the binding module and the reporter
module can be an
ionic bonding, hydrogen bonding, hydrophobic interactions, hydrophilic
interactions, solvent-
excluding interactions, or any combination thereof The connection or
attachment can include at
least a covalent bond to ensure that the reporter module is tethered or
otherwise stably attached to
the binding module.
[0057] The probe polypeptide can be or include, for example, an amino acid
sequence of any
one of SEQ ID NOS: 2, 6, 8, 10, and 12, or a combination thereof; encoded by a
nucleic acid
sequence of SEQ ID NOS: 1, 5, 7, 9, and 11, respectively. The binding module
polypeptide can
be or include, for example, an amino acid sequence of any one of SEQ ID NOS:
14, 16, 18, 20,
and 22; encoded by a nucleic acid sequence of 13, 15, 17, 19, and 21,
respectively. The reporter
module polypeptide can be or include, for example, an amino acid sequence of
any one of SEQ
ID NOS: 24, 26, 28, and 30; encoded by a nucleic acid sequence of 23, 25, 27,
and 29,
respectively. Histidine tag sequences can be omitted from the sequences for
the probes, binding
modules, and/or reporting modules, for example, if purification methods do not
employ a nickel
column.
[0058] The probes of the present invention are advantageous as they can
function
independent of, without including, or without using an antibody or a
polypeptide including an
antigen-binding fragment of an antibody. Neither the binding module
polypeptide nor the
reporter module need be or include an antibody or a fragment thereof Either
the binding module
polypeptide or the reporter module can be or include an antibody or a fragment
thereof Both the
binding module polypeptide and the reporter module can be or include an
antibody or a fragment
thereof Even if a binding module is not an antibody or antigen-binding
fragment thereof, the
binding module can still bind a target with the specificity of an antibody.
The binding of the
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binding module to its target lignocellulosic can occur under conditions
similar to or different
from the binding of an antibody to its antigen. Binding can be performed under
any suitable
conditions that increase specific binding of a binding module to its desired
target(s), and
decrease, minimize, or prevent non-specific binding of the binding module to
non-targets.
Binding can be assisted by the presence of one or more additional factors, for
example, probe
concentration, ions, for example calcium ions, ionic strength, pH, and/or
temperature, and the
like. Both, neither, or just one of the binding module and reporter module can
be or include a
polypeptide of any kind. Binding modules and/or reporter modules can be or
include
nucleotides, carbohydrates, lipids, synthetic organic groups, and/or inorganic
groups instead of
or in addition to polypeptides. For example, polynucleotides, polysaccharides,
fatty acids, esters,
sterols, and/or non-naturally occurring polymers can be used. Binding modules
and/or reporter
modules and/or other portions of a probe can contain any suitable type or
number of molecules,
for example, molecules described herein.
[0059] Any suitable reporter module or combination of reporter modules can
be used in the
probes and methods of the present invention. For example, the reporter module
in whole or part can
be fluorescent. The reporter module can have any suitable fluorescent
excitation and emission
spectra. The reporter module can have a unique excitation spectrum and/or
excitation peak
maximum. For example, the reporter module can have a fluorescence excitation
peak (maximum)
of, for example, lower than 300 nm, from about 300 nm to about 750 nm, from
about 350 nm to
about 700 nm, from about 400 nm to about 650 nm, from about 350 nm to about
400 nm, from
about 400 nm to about 450 nm, from about 450 nm to about 500 nm, from about
500 nm to about
550 nm, from about 550 nm to about 600 nm, from about 600 nm to about 650 nm,
from about 650
nm to about 700 nm, greater than 700 nm or any intervening range (for example,
a 1-3 nm, 5 nm, 10
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nm, or 25 nm range) or value. The reporter module can have a unique emission
spectrum ( 1 nm,
or 3 nm, or 5 nm) and/or emission peak maximum ( 1 nm, or 3 nm, or 5
nm). For
example, the reporter module can have a fluorescence emission peak (maximum)
of less than 350
nm, from about 350 nm to about 800 nm, from about 400 nm to about 750 nm, from
about 450 nm
to about 700 nm, from about 350 nm to about 400 nm, from about 400 nm to about
450 nm, from
about 450 nm to about 500 nm, from about 500 nm to about 550 nm, from about
550 nm to about
600 nm, from about 600 nm to about 650 nm, from about 650 nm to about 700 nm,
from about 700
nm to about 750 nm, greater than 750 nm, or any intervening range (for
example, a 1-3 nm, 5 nm,
nm, or 25 nm range) or value.
[0060] The reporter module can include any number or type of fluorescent
moieties. For
example, the fluorescent moiety can be or include a polypeptide, a
polynucleotide, a polysaccharide,
a small organic molecule, a metal, a coordination complex, or any combination
thereof. For
example, the reporter module can be or include a fluorescent protein or a
combination of fluorescent
proteins. The fluorescent protein can be or include an ultraviolet fluorescent
protein, a blue
fluorescent protein, a cyan fluorescent protein, a green fluorescent protein,
a yellow fluorescent
protein, an orange fluorescent protein, a red fluorescent protein, a far-red
fluorescent protein, a near
infrared fluorescent protein, an infrared fluorescent protein, a sapphire-type
fluorescent protein, a
long Stokes shift fluorescent protein, a switchable fluorescent protein, or
any combination thereof.
The fluorescent protein can be or include, for example, Sirius, TagBFP,
mTagBFP2, Azurite,
EBFP2, rnKalamal, Sirius, Sapphire, T-Sapphire, ECFP, mAmetrine, Cerulean,
mCerulean3,
SCFP3A, CyPet, mTurguoise, mTurquoise2, monomeric Midoriishi-Cyan, Aquamarine,
eCFP,
TagCFP, mTFP1, AmCyanl, EGFP, Emerald, Superfolder GFP, monomeric Azami Green,
TurboGFP, TagGFP2, mUKG, mWasabi, Clover, inNeonGreen, eGFP, AcGFP1, ZGreen 1
,
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ZsYellowl, mBanana, EYFP, Topaz, Citrine, Venus, SYFP2, Ypet, IanRFP-deltaS83,
mPapayal,
TagYFP, mOrange, mOrange2, monomeric Kusabira-Orange, mK0k, mK02, mTangerine,
mNectarine, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, DsRed-Express2, DsRed-
Express,
tdTomato, DsRed-Monomer, DsRed-Monomer, DsRed2, AsRed2, mStrawbeny, mCherry,
HcRedl, FusionRed, mRaspberry, E2-Crimson, mPlum, HcRed-Tandem, mKate2,
mNeptune,
NirFP, TagRFP657, TagRFP675, iFP1.4, iRFP(iRFP713), iRFP670, iRFP682, iRFP702,
iRFP720,
iFP2.0, mIFP, mKeima Red, LSS-mKatel, LSS-mKate2, LSSmOrange, mBeRFP, PA-GFP,
PATag
RFP, Dendra2, Timer, PAmCherry, Kaede (green), Kaeda (red), KikGR1 (green),
KikGR1 (red),
PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2(red), PSmOrange,
Dropna, or
any combination thereof. Reporter modules other than fluorescent reporter
modules can be
employed in addition to or in the alternative to fluorescent reporter modules.
For example,
antibodies, antibody fragments, radioisotopes, dyes, synthetic organic
molecules, phosphorescent
molecules, enzymes, or the like, or any combination thereof can be used.
Reporter modules
described in Knox P.J. (2012) Methods in Enzymology, volume 510, 233-245,
which is
incorporated by reference herein in its entirety, can be used. The accessible
primary amines of
binding modules, for example, CBMs, can be labelled using a reactive dye that
contains a
tetrafluoropheneyl ester moiety (Invitrogen).
[0061] Any suitable binding module or combination of binding modules can be
used in the
probes and methods of the present invention. For example, the binding module
can be or include a
carbohydrate-binding module (CBM) including CBM1, CBM2, CBM3, CBM3a, CBM4,
CBM5,
CBM6, CBM7, CBM8, CBM9, CBM10, CBM11, CBM12, CBM13, CBM14, CBM15, CBM16,
CBM17, CBM18, CBM19, CBM20, CBM21, CBM22, CBM23, CBM24, CBM25, CBM26,
CBM27, CBM28, CBM29, CBM30, CBM31, CBM32, CBM33, CBM34, CBM35, CBM36,
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CBM37, CBM38, CBM39, CBM40, CBM41, CBM42, CBM43, CBM44, CBM45, CBM46,
CBM47, CBM48, CBM49, CBM50, CBM51, CBM52, CBM53, CBM54, CBM55, CBM56,
CBM57, CBM58, CBM59, CBM60, CBM61, CBM62, CBM63, CBM64, CBM65, CBM66,
CBM67, CBM68, CBM69, CBM70, CBM71, or a family member thereof, or any
combination
thereof. Binding specificities can be or include those of particular CBM
families or specific CBM
family members, for example, CBM1 (cellulose), CBM2 (cellulose), CBM3
(crystalline cellulose),
CBM4 (amorphous cellulose), CBM5 (chitin), CBM6 (cello-oligosaccharides,
laminarins,
xylooligosaccharides, beta1,4,-beta1,3-mixed linked glucans), CBM8
(cellulose), CBM9
(crystalline cellulose), CBM10 (cellulose), CBM11 (cellulose), CBM12 (chitin),
CBM13 (cellulose,
xylans, mannose), CBM14 (chitin), CBM15 (xylans and xylooligosaccharides),
CBM16 (cellulose
and glucomannans), CBM17 (amorphous cellulose), CBM18 (chitin), CBM19
(chitin), CBM20
(starch), CBM21 (glycogen), CBM22 (mixed f3-1,3/13-1,4-glucans), CBM23
(mannans), CBM24 (a-
1,3-glucan), CBM25 (alpha-glucooligosaccharides and granular starch), CBM 26
(starch), CBM27
(beta-1,4-mannooligosaccharides, carob galactomannan, and konjac glucomannans,
mannans),
CBM28 (amorphous cellulose, cellooligosaccharides, and 13-(1,3)(1,4)-glucans),
CBM29 (mannans
and glucomannans), CBM30 (cellulose), CBM31 (13-1,3-xylans), CBM32 (galactose,
lactose,
polygalacturonic acid, and13-D-galactosyl-1,4-13-D-N-acetylglucosamine), CBM33
(chitin), CBM34
(granular starch), CBM35 (xylans, mannans, and 13-galactans), CBM36 (xylans
and
xylooligosaccharides), CBM37 (xylans, chitin, microcrystalline cellulose, and
phosphoric-acid
swollen cellulose), CBM38 (inulin), CBM39 (13-1,3-glucan, lipopolysaccharide,
and lipoteichoic
acid) CBM48 (glycogen), CBM40 (sialic acid), CBM 41 (a-glucans, amylose,
amylopectin, and
pullulans), CBM42 (arabinofuranose and arabinoxylans), CBM43 (13-1,3-glucans),
CBM44
(cellulose and xyloglucans), CBM45 (starch), CBM46 (cellulose), CBM47
(fucose), CBM48
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(glycogens), CBM49 (crystalline cellulose), CBM50 (chitin and chitopentaose),
CBM51 (galactose
and A/B blood group antigens), CBM52 (13-1,3-glucans), CBM53 (starch), CBM54
(xylans),
CBM55 (chitin), CBM56 (f3-1,3-glucans), CBM58 (maltoheptaose), CBM59 (mannans,
xylans, and
cellulose), CBM60 (xylans), CBM61 (13-1,4-galactans), CBM62 (galactose,
xyloglucans,
arabinogalactans, and galactomannans), CBM63 (cellulose), CBM64 (cellulose),
CBM65
(xyloglucans), CBM66 (fi-uctans), CBM67 (L-rhamnose), CBM68 (maltotriose and
maltotetraose),
CBM69 (starch), CBM70 (hyaluronan), CBM71 (lactose and 13-D-galactosy1-1,443-D-
N-
acetylglucosamine), or any combination thereof. Any suitable carbohydrate
binding module can be
employed, for example, as described in Boraston et al., Biochem J., 382: 769-
781 (2004), Shoseyov
et al., Microbiology and Molecular Biology Reviews, 70(2): 283-295 (2006) or
Christiansen et al.,
FEBS Journal, 276:5006-5029 (2009). CBMs or other binding modules can be
synthetically or
genetically evolved or otherwise generated to bind to any desired target
whether carbohydrates,
other types of polymers, or non-polymer compounds. Techniques such as phage
display, for
example, can be used. For example, CBMs can be produced that bind to polymers,
for example, a
polyaryletherketone (PAEK), a polyether ether ketone (PEEK), a polyethylene, a
polypropylene, a
polystyrene, a polytetrfiuoroethylene, a polyvinylchloride, a polyamide, a
para-aramid, a
polyethylene terephthalate, a polyimide, a polycarbonate, a polypeptide, a
polynucleotide, a
glycoprotein, a protein, a phosphorylated protein, a modified protein, a
lipid, a surfactant, lecithin,
or a biosurfactant, or any combination thereof. The CBMs can be replaced by
antibodies and the
detection method can be tailored for antibodies. See, for example, Knox P.J.
(2012) Methods in
Enzymology, volume 510, 233-245, which is incorporated by reference herein in
its entirety.
[0062] A binding module can bind specifically to any desired polymer (e.g.,
lignocellulosic
polymer or combination thereof, or any oligosaccharide). For example, the
binding module can
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specifically bind to cellulose, hemicellulose, lignin, xylan, mannan,
glucuronoxylan, arabinoxylan,
glucomannan, xyloglucan, or any combination thereof or a linear fragment
thereof, or a branched
fragment thereof, or an oligomer thereof (for example, an oligosaccharide), or
a monomer and/or
macromer thereof, for example, glucose, D-glucose, mannose, xylose, galactose,
rhamnose,
arabinose, monolignol, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol,
p-hydroxyphenyl
phenylpropanoid, guaiacyl phenylpropanoid, syringyl phenylpropanoid, or a
combination thereof.
The binding module can bind to an amorphous or crystalline lignocellulosic
polymer. The binding
module can recognize both an amorphous and crystalline form of a particular
lignocellulosic
polymer or be specific to one or the other. For example, the binding module
can specifically bind to
crystallized cellulose (and not to amorphous cellulose). A binding module can
specifically bind to
amorphous cellulose (and not to crystallized cellulose).
100631 The lignocellulosic polymer detection probe can include a plurality
of lignocellulosic
polymer detection probes, each lignocellulosic polymer detection probe binding
solely or essentially
solely to a different lignocellulosic polymer. The plurality of probes can
include any number or
kind of probes. For example, a plurality can include at least two probes, at
least three probes, at
least four probes, at least five probes, at least ten probes, from two probes
to twenty probes, from
two probes to ten probes, or from two probes to four probes. A plurality of
probes can be provided
as a pre-formulated composition or combined from separate stock solutions of
individual probes.
The concentration, for example, weight percentage, of each probe in a
composition can be the same
or differ between types of probes based on the total weight of the
composition. For example, the
weight ratio of probes in a composition including four different types of
probes could be 1:1:1:1, or
some other ratio. The relative amounts, ratio, and/or concentration of probes
can be adjusted based
on the lignocellulosic polymer profile of a particular biomass.
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[0064] A probe composition can include various other components, for
example, one or more
stabilizers, one or more preservatives, one or more emulsifiers, one or more
thickeners, one or more
diluents, or any combination thereof.
[0065] Each lignocellulosic polymer detection probe can include a different
reporter module.
For example, each reporter module can have a different (or unique)
fluorescence signature. Any
combination of fluorescent reporter modules can be used, for example, one or
more of eGFP,
mCherry, mOrange2, and eCFP. Any combination of binding modules can be used,
for example,
one or more of CBM3a, CBM4-1, CBM15, and CBM27. A plurality of probes can be
provided
such that each binding module is paired with a unique reporter module and vice
versa. For
example, the lignocellulosic polymer detection probe can include eGFP-CBM3a,
mCherry-CBM4-
1, mOrange2-CBM15, eCFP-CBM27, or any combination thereof.
[0066] The lignocellulosic or any polymer detection probe of the present
invention can be
detectable at a distinct wavelength ( 0.5 nm, 1 nm, 3 nm, 5 nm). Thus, a
combination of
different probes can be used and measured simultaneously, instead of using
different probes
separately or sequentially.
[0067] The present invention also relates to a complex including any probe
or combination
thereof bound to a pulp or paper product including at least one surface
available lignocellulosic
polymer. The present invention also relates to a pulp or paper product
including at least one surface
available lignocellulosic polymer and at least one probe bound thereto. The
pulp can be any grade
of pulp and a pulp at any stage of production of a paper or other biomass
product. The pulp can
include furnish. The pulp can include white water. The pulp can be or include
product waste, for
example, paper sludge. The pulp can be chemical pulp, mechanical pulp,
thermomechanical pulp or
chemi (thermo) mechanical pulp or a Kraft pulp. The pulp can be pulp from
hardwood, softwood,
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or both types, or can include textile fibers, agricultural plant pulp, or the
like. The pulp can be
beached, pre-bleached, or unbleached. The pulp can be refined or unrefined.
The paper product can
be an intermediate paper product, a sample paper product useful for testing,
or a fmished paper
product. Paper products can be, for example, printable or inkable paper
sheets, sheets for cardboard
construction, tissue paper, hygiene and personal care sheet or liner
materials, or the like.
100681 The present invention also is directed to various methods that
employ lignocellulosic or
any polymer detection probes. The methods can use any number or types of
probes, for example a
single probe or a plurality of probes. The present invention relates to a
method of detecting a
lignocellulosic polymer or any polymer. A probe can be contacted with a
biomass material. This
can be accomplished by introducing, adding, mixing, or otherwise combining the
probe with the
biomass material (e.g., pulp or paper product). Contacting can include and/or
result in binding of
the probe if its specific target lignocellulosic polymer or any polymer is
present. A property
associated with the reporter module can be measured to determine the presence
or absence of at
least one lignocellulosic polymer in the biomass material. For example, the
property measured can
be or include fluorescence. The method can also include calculating the amount
of the at least one
lignocellulosic polymer, determining the type of the at least one
lignocellulosic polymer, or both.
[0069] The biomass material measured can be or include any suitable biomass
material. The
biomass material can be a raw material, a partially processed material, or a
finished product. The
biomass material can be or include a wood biomass material. The wood biomass
material can be or
include pulp, furnish, white water, paper, a paper product, paper sludge, or
any combination thereof.
The method can include forming at least one handsheet from the wood biomass
product, wherein
the measuring is performed on the handsheet.
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[0070] The measuring can be performed before treatment, during treatment,
and/or after
treatment, or any combination thereof of the biomass material. The treatment
can include, for
example, an enzymatic treatment, bleaching, amorphogenesis, milling, or PFI
refining, or any
combination thereof. The treatment can be or include, for example, enzymatic
treatment with at
least one enzyme including a cellulase, a xylanase, a mannase, a lignase, or
any combination
thereof. The method can be or include performing at least one treatment of the
biomass material
based on the amount of the at least one lignocellulosic polymer measured, the
type of
lignocellulosic polymer measured, or both. The amount of lignocellulosic
polymer measured can
correlate negatively or positively with at least one physical property of the
biomass product. The at
least one physical property can include, for example, burst index, drainage
rate, tear index, tensile
index, internal bond strength, or any combination thereof. The at least one
physical property can
include, for example, density, elastic modulus, shear modulus, Young's
modulus, Poisson's ratio,
yielding stress, ultimate stress, fiber length, elongation, or the like.
[0071] The amount of lignocellulosic polymer measured, the type of
lignocellulosic polymer
measured or both can be used to determine the amount or type of treatment to
be applied to the
biomass material. For example, the method can include dosing at least one
enzyme based on the
amount of lignocellulosic polymer measured, the type of lignocellulosic
polymer measured or both.
For example, if the amount of crystalline cellulose is relatively low, a
greater amount of cellulase
can be added. If the amount of lignin is high, for example, the amount of
bleach can be
increased. A high amount of xylan measured can be addressed, for example, by
increasing the
amount of xylanase added to the pulp. For pre-bleaching, for example, a
measurement of high
mannan content can be addressed by increasing the amount of mannase added to
the pulp. The
method can include adjusting mill speed based on the amount of lignocellulosic
polymer measured,
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the type of lignocellulosic polymer measured or both. For example, if the
amount of crystalline
cellulose is relatively low, the intensity of refining (e.g., mill speed, in
number of rpms) can be
increased. The method can include adjusting total water content of the biomass
material based on
the amount of lignocellulosic polymer measured, the type of lignocellulosic
polymer measured or
both. For example, water can be added to the pulp if the amount of crystalline
cellulose is relatively
high and the amount of amorphous cellulose is relatively low. Thus, the method
can be used to
adjust the concentration of pulp or one or more components thereof.
[0072] The present invention further relates to a method of determining the
effectiveness of an
industrial treatment on pulp or a paper product. A lignocellulosic polymer
detection probe can be
contacted with or attached to a pulp or a paper product. The specific binding
of the probe to the
pulp or the paper product can be detected. The amount of at least one
lignocellulosic polymer on a
surface of the pulp or the paper product can be determined (e.g., calculated).
The effectiveness of
an industrial treatment on the pulp or paper product can be determined based
on the amount of the at
least one lignocellulosic polymer detected. The effectiveness of any suitable
industrial treatment
can be determined. The industrial treatment can be or include, for example, an
enzymatic treatment,
a chemical treatment, a physical treatment, or any combination thereof. The
method can be
performed before the industrial treatment, during the industrial treatment,
after the industrial
treatment, or any combination thereof. The polymer detection probe can be
detectable at a distinct
wavelength as part of the method. The method can be performed on paper waste,
for example,
paper sludge, and the industrial treatment can include a treatment for paper
waste.
[0073] The present invention also relates to a method of determining a
physical property of pulp
or a paper product. A lignocellulosic polymer detection probe can be contacted
with pulp or a paper
product. The specific binding of the probe to the pulp or the paper product
can be detected. The
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amount of at least one lignocellulosic polymer on a surface of the pulp or the
paper product can be
calculated. The at least one physical property of the pulp or paper product
can be determined based
on the amount of the at least one lignocellulosic polymer detected. The at
least one physical
property can be or include, for example, burst index, drainage rate, tear
index, tensile index, or
internal bond strength, or any combination thereof. The at least one physical
property can be or
include, for example, density, elastic modulus, shear modulus, Young's
modulus, Poisson's ratio,
yielding stress, ultimate stress, fiber length, or elongation, or the like.
[0074] The probes and methods of the present invention are applicable in
applications other
than pulp and paper processing. For example, the probes and methods can be
used for
environmental compliance and/or monitoring. The probes and methods can be
used, for example, in
the food industry to track the types and/or amounts of carbohydrates present
during various stages
of food production, and subsequently to detect food spoliation or other
conditions or properties.
The probes and methods can be used to track the presence and/or amounts of
microbes or other cell
types in any material. The probes and methods can be used to detect cell
markers such as
glycoproteins, for example, those characteristic of blood groups, cancers, or
pathogens. The probes
and methods can be used in medical contexts, for example, histology. The
probes and methods of
the present invention are applicable to any relevant sector of industrial
production, food
production/testing, agricultural applications, plastics, medicine,
diagnostics, microbiology,
biomass/biofuel, and/or petroleum production/testing, and the like.
[0075] A polymer detection probe, in general, is provided by the present
invention. The probe
can have, for example, one or more characteristics or functions of the
lignocellulosic polymer
probes described herein, overlapping characteristics or functions, or
different characteristics or
functions. The polymer detection probe can include, for example, a) a binding
module that
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specifically binds to at least one polymer and b) a reporter module that is
spectroscopically
detectable. The polymer detection probe can be or include, for example, a
probe polypeptide. The
reporter module can be, for example, fluorescent. The reporter module can be
or include a
fluorescent protein. The binding module can be a carbohydrate-binding module
(CBM). The
binding module can specifically bind to, for example, cellulose,
hemicellulose, lignin, xylan,
mannan, glucuronoxylan, arabinoxylan, glucomannan, xyloglucan, or any
combination thereof or a
linear fragment thereof, or a branched fragment thereof, or an oligomer
thereof, or a monomer
and/or macromer thereof, for example, glucose, D-glucose, mannose, xylose,
galactose, rhamnose,
arabinose, monolignol, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol,
p-hydroxyphenyl
phenylpropanoid, guaiacyl phenylpropanoid, or syringyl phenylpropanoid, or a
combination thereof.
The binding module can specifically bind to, for example, a glycoprotein,
carbohydrate, or both,
specific to a particular blood antigen, type, group, or subgroup.
100761 The binding module can specifically bind to any particular polymer
(e.g.,
oligosaccharide or other saccharide polymer or other polymer). The polymer can
be naturally
occurring or synthetic. The binding module can specifically bind to, for
example, a
polyaryletherketone (PAEK), a polyether ether ketone (PEEK), a polyethylene, a
polypropylene, a
polystyrene, a polytetrfluoroethylene, a polyvinylchloride, a polyamide, a
para-aramid, a
polyethylene terephthalate, a polyimide, a polycarbonate, a polypeptide, a
polynucleotide, a
glycoprotein, a protein, a phosphorylated protein, a modified protein, a
lipid, lecithin, a surfactant,
or a biosurfactant, or any combination thereof or a material including or
containing one or more of
these polymers. The polymer detection probe can include a plurality of polymer
detection probes,
each polymer detection probe specifically binding to a different polymer. Each
polymer detection
probe can include a different reporter module. Each reporter module can have a
different
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fluorescence signature. The polymer detection probe can be detectable at a
distinct wavelength (
0.2 nm, 0.5 nm, 1 nm, 3 nm, 5nm). A complex is provided including the
probe specifically
bound to a material including at least one surface available polymer. The
manner in which the
lignocellulosic polymer probe is designed and used (as described herein) can
equally apply in
general to the broader polymer probes of the present invention for any polymer
and for the detection
of that polymer that specifically binds to the probe.
[0077] A method of detecting a polymer (such as the classes and specific
ones described earlier)
is provided. A probe of the present invention can be contacted with a
material. A property
associated with the reporter module can be measured to determine the presence
or absence of at
least one polymer in the material based on specific binding of the probe to
the at least one polymer.
The property measured can be, for example, fluorescence. The method can
include calculating the
amount of the at least one polymer, determining the type of the at least one
polymer, or both. The
material can be or include a biological sample, for example, a cancer biopsy,
cells, a tissue sample, a
microbiological sample, or a blood sample. The presence or absence of a cancer
and/or an identity
of cancer type can be determined, for example, by using a binding module
specific to a cancer cell
surface marker, such as a glycoprotein. The presence or absence of a
beneficial or pathogenic
microorganism and/or an identify of a microorganism such as a virus or
bacterium can be
determined, for example, by using a binding module specific to a microorganism
cell surface
marker, such as a cell wall polysaccharide or a cell surface glycoprotein. At
least one of a blood
antigen, type, group, or subgroup of the blood sample can be determined. For
example, types A, B,
AB, and 0 can be distinguished in the ABO blood group system. An Rh antigen,
such as the D
antigen, can be detected in the Rh blood group system to determine whether a
blood sample is Rh
positive or negative. A plurality of probes, for example, different kinds of
probes, can be used in
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the method. Thus, the ABO group and Rh factor identities of a blood sample,
for example, can be
determined simultaneously.
[0078] The present invention will be further clarified by the following
examples, which are
intended to be exemplary of the present invention.
EXAMPLES
[0079] The following examples demonstrate the utility of the present
invention and its
surprising advantages over existing techniques. Predictive methods, based on
the use of CBMs
fused to various fluorescent proteins, were created. Using molecular biology
techniques and
constructs, four different fusion proteins were created. Each of these
"probes" is constructed of a
specific binding module (the CBM moiety) and a reporter module (the
fluorescent protein).
Each probe emits fluorescence at a distinct wavelength, allowing for
unambiguous detection of
the polymer specifically bound by the binding module. FIG. 1 is a schematic
diagram of
polymer detection using a probe.
[0080] After production and characterization of the probes, they are
demonstrated as
specifically binding to relevant polymers, and change binding in response to
change in surface
availability of such polymers. Accordingly, these probes demonstrate that the
present invention
enables the detection of changes, and that the signal generate by the probes
can be correlated
with pulp and paper properties measured independently, with industry-standard
methods. The
method can be used to predict the impact of any industrial treatment on
properties that depend on
exposure of polymers. As demonstrated by these examples, the present invention
can also be
used for monitoring surface polymers at any stage for any process involving
lignocellulosic
biomass.
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[0081] Table 1 lists CBMs, binding targets, and associated fluorophores.
The emission
wavelengths are separated by several nanometers, enabling detection of
individual probes even
when mixed with other probes (spectral deconvolution).
Table 1
Probe # CBM Target Fluorescent protein tag
(Ex/Em)
1 CBM3a Crystalline cellulose eGFP (488/507)
2a CBM4-1 Amorphous cellulose mCherry (587/610)
2b CBM17 Amorphous cellulose mCherry (587/610)
3 CBM15 Xylan mOrange2 (549/565)
4 CBM27 Mannan eCFP (434/477)
[0082] Probe 1 detects, for example, crystalline cellulose (CBM3a-eGFP) and
fluoresces at 507
nm. Probe 2a detects, for example, amorphous cellulose (CBM4-1-mCherry) and
emits
fluorescence at 610 nm. Probe 2b (CBM17-mCherry) also detects amorphous
cellulose, but with a
higher affinity compared to Probe 2a. CBM15 fused to mOrange2 detects xylan
(Probe 3) and is
visible at 565 nm. Probe 4 includes CBM27 fused to eCFP and emits light at 477
nm. The probes
emission maxima are separated, and can be detectable even when probes are
mixed with others
described here.
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Example 1
[0083] This example demonstrates the production of reporter modules
(fluorescent proteins) in
accordance with the present invention. Four reporter modules (eCFP, eGFP,
mCherry and
mOrange2) were cloned in pET11 vector and expressed in prokaryotic systems.
FIGS. 2-5 show the
genetic maps of the respective reporter modules (fluorescent proteins) cloned
into the pET11 vector.
Nucleic acid and amino acid sequences for the reporter modules are shown in
SEQ ID NOS: 23-30,
respectively. These vector constructs are used for labeling binding modules
from cloned genes with
the reporter modules. Single fluorescent proteins (reporter modules) are used
to measure the
background (non-specific) binding to the pulp polymers.
[0084] Proteins were purified using the following methodology. E. coil
BL21(DE3) GOLD
pLysS cells (ThermoFisher Scientific) bearing the selected expression plasmid
(reporter module,
binding module or complete Probe) were grown at 37 C in Luria-Bertani broth
containing 100
jig/ml of ampicillin. Induction of recombinant protein expression was
performed by the addition of
500 uM IPTG (ThermoFisher Scientific) to mid-log-phase cells (0.D. 600nm of
0.6-0.8) and the
subsequent incubation for 18 hours at 25 C. Cells were afterward harvested
and kept at -80 C.
Thawed cell pellets were resuspended in 50 mM NaPO4 pH 8 containing 300 mM
NaCl, 2 mM
imidazole, and 1 mM PMSF, and then lysed using six cycles of 60 seconds of
sonication (Branson
Ultrasonics Corporation) at 200 W. Clarification of the lysate was achieved by
centrifugation at
10,000 g for 30 minutes at 4 C.
[0085] The protein of interest was then purified by affinity chromatography
over a HisPrep FF
16/10 column (GE Healthcare Life Sciences) equilibrated in 50 mM NaPO4 pH 8.0
buffer
containing 300 mM NaCl and 10 mM imidazole. Following a ten column volumes
buffer washes,
the desired protein was eluted following a ten column volumes gradient of
imidazole (10 to 100
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mM) in 50 mM NaPO4 pH 8.0 buffer containing 300 mM NaCl. A final purification
step was
performed over a SUPERDEX 200 HR 16/50 column (GE Healthcare Life Sciences) in
50 mM
Tris-HC1 pH 7.5 buffer containing 300 mM NaC1 to insure its hom*ogenous purify.
The purified
probe was then dialyzed in a 20 Tris-HC1 pH 7.5 buffer containing 20 mM NaC1
and 5 mM CaC12 at
4 C and concentrated using a 10k MACROSEP Advance centrifugal device (Pall
Corporation).
Concentrated protein solutions were stored at -80 C using flash freezing.
Protein purity was
verified by SDS-PAGE. The amount of protein was quantified by the Bradford
method.
100861 The reporter modules were successfully produced and purified by
affinity
chromatography (FIG. 6). The yield of the production was around 25 mg of
protein/ L of culture.
FIG. 6 is a SDS-PAGE of purified fluorescence proteins; the arrow highlights
the protein of interest.
Table 2 shows the properties of the purified reporter modules (here,
fluorescent proteins).
Table 2
Protein Amino Acids (#) Mol. Wt. (Da) pI
eGFP 247 27878.1 5.99
mCheny 244 27658.8 6.06
mOrange2 244 27756.1 6.72
eCFP 247 27842.0 5.99
Example 2
[0087] This example demonstrates production of binding modules (CBMs) in
accordance with
the present invention. Five CBM genes (CBM 3a, 4-1, 15, 17, and 27) were
cloned in pET11 vector
and expressed in prokaryotic systems. The CBMs were produced and purified by
affinity
chromatography (FIG. 7). The yield of the production of the CBM was around 10
mg of protein/ L
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of culture. FIG. 7 is a SDS-PAGE of purified CBMs; the arrow highlights the
protein of interest.
Table 3 displays the properties of the purified binding modules (here, CBMs).
Table 3
Protein Amino Acids (#) Mol. Wt. (Da)
CBM3 a 206 22375.1 7.58
CBM4-1 161 16528.7 3.88
CBM17 221 23926.9 5.46
CBM15 171 17949.4 4.31
CBM27 185 21242.6 5.57
Example 3
100881 This example demonstrates the fluorescence emission by reporter
modules in
accordance with the present invention. Emission spectra of pulp and reporter
modules (eGFP,
mCherry and mOrange2) were measured. The spectra were acquired at 23 C and the
proteins
were diluted in a 20 rnM Tris-HC1 pH 7.5 buffer containing 20 mM NaC1 and 5 mM
CaC12. The
spectra in FIGS. 8 and 9 show that depending on the excitation wavelength (488
nm vs 549 nm), the
change in fluorescence intensity (Fl) (eGFP vs mCherry) can be measured
independent of pulp
auto-fluorescence or other probes. FIG. 8 shows the emission spectra of
reporter modules (eGFP
and mCherry) and of a pulp handsheet disk after excitation at 549 nm. With
this excitation
wavelength, mCherry fluorescence dominates over the other substrates signal.
FIG. 9 shows
emission spectra of FPs (eGFP and mCherry) and a pulp handsheet disk using an
excitation
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wavelength of 488 nm. At this excitation wavelength, the pulp and mCherry
fluorescence is much
weaker than eGFP.
Example 4
[0089] This example demonstrates the production of complete probes (binding
module-reporter
module). The genes that were used for production of reporter modules and
binding modules were
fused to generate the probes. FIGS. 10-14 show the pET11 vectors and the
genetic map covering
the fusion sequence. The resulting molecules (probes) include a reporter
module at the N-terminus,
followed by the binding module. FIG. 10 shows the vector and map for pET11A-
eGFP-CBM3a
expression vector (Probe 1). FIG. 11 shows the vector and map for pET11A-
mCherry-CBM4-1
expression vector (Probe 2a). FIG. 12 shows the vector and map for pET11A-
mCherry-CBM17
expression vector (Probe 2b). FIG. 13 shows the vector and map for pET11A-
mOrange2-CBM15
expression vector (Probe 3). FIG. 14 shows the vector and map for pET11A-eCFP-
CBM27
expression vector (Probe 4). The nucleotide and amino acid sequences for Probe
1 are included in
the sequence listing as SEQ ID NOS: 1 and 2 respectively. The nucleotide and
amino acid
sequences for the linker (joining eGFP and CBM3a) in Probe 1 are included in
the sequence listing
as SEQ ID NOS: 3 and 4 respectively. The nucleotide and amino acid sequences
for Probe 2a are
included in the sequence listing as SEQ ID NOS: 5 and 6 respectively. The
nucleotide and amino
acid sequences for Probe 2b are included in the sequence listing as SEQ ID
NOS: 7 and 8
respectively. The nucleotide and amino acid sequences for Probe 3 are included
in the sequence
listing as SEQ ID NOS: 9 and 10 respectively. The nucleotide and amino acid
sequences for Probe
4 are included in the sequence listing as SEQ ID NOS: 11 and 12 respectively.
The new molecules
(Probes) start with an N-terminal reporter module (fluorescent protein)
followed by the
appropriate binding module (CBM). The sequence linking the reporter to the
binding module is
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composed of a glycine, except for Probe 1, wherein the linking sequence is SEQ
ID NO: 3
encoded by SEQ ID NO: 4 (including a thrombin cleavage site).
[0090] The vectors encoding the probes were transformed in E. coli BL21-
Gold (DE3) pLysS
competent cells. Transformed cells were selected on LB-agar with ampicillin
(100 gimp. E. coli
cells harboring probe vectors were cultured in Luria-Bertani (LB) broth
containing ampicillin (100
tig/mL) at 27 C to mid exponential phase (A600 nm = 0.5). Recombinant protein
expression was
induced by the addition of 0.5 mM IPTG and further incubated for 16 hours at
27 C (agitation 200
rpm). Induced cells were centrifuged at 4 C for 30 min at 4000g and the
pellet were store at -80
C. Cells were suspended in 50 mM sodium phosphate buffer (pH 8.0) and
disrupted by sonication.
Cells debris was removed by centrifugation at 15,000 rpm for 15 min. The 6xHis
tagged protein
was purified under native conditions using Ni-NTA nickel affinity resin
(Qiagen) according to
manufacturer specification at pH 8 using imidazole for elution. A second
passage was utilized to
increase the purity of the protein preparation. In order to remove salts and
imidazole, purified
probes were dialyzed in a buffer containing 20 mM Tris-HC1 pH 7.5, 20 mM NaC1
and 5 mM
CaCl2 for 24 hours at 4 C. Protein concentration was determined using a
Bradford protein assay.
[0091] FIG. 15 shows a SDS PAGE analysis of each probe. The arrows indicate
the position of
the probe fused proteins. The size of each probe corresponds to the size
expected on the basis of its
amino acid residue content. Shown on the left side of the last four gels is a
standard size ladder
allowing estimation of sizes for proteins migrated on the same gel. Table 4
shows the properties of
the purified Probes (reporter module-binding module).
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Table 4
Protein Amino Acids (#) Mol. Wt. (Da) pI
eGFP -CBM3 a 415 46260.9 5.91
mCherry-CBM4-1 397 43158.4 4.67
mCherry-CBM17 457 50556.6 5.49
mOrange2-CBM15 407 44676.4 5.10
eCFP-CBM27 424 48055.5 5.59
Example 5
[0092] This example demonstrates spectroscopic characterization of probes
after production in
accordance with the present invention. In order to verify that the fusion of
the reporter modules to
the binding modules did not prevent native folding, a spectroscopic analysis
was carried out. The
spectra were acquired at 23 C and the proteins were diluted in a 20 mM Tris-
HC1 pH 7.5 buffer
containing 20 mM NaC1 and 5 mM CaC12. The absorption and emission spectra
recorded for
Probe 1, Probe 2b, Probe 3 and Probe 4 are shown in FIGS. 16A-16D. They are
comparable with
spectra reported in the literature and strongly suggest that the reporter
modules are not affected by
the presence of the binding modules. In FIGS. 16A-D, excitation spectra are
shown as dashed lines
and emission spectra as full lines for the FP-CBM proteins. These results
strongly indicate that the
reporter modules (fluorescent proteins) are properly folded after their
recovery from bacteria and
that the close proximity of the binding module has no appreciable impact on
its reporting capacity.
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Example 6
[0093] This example demonstrates binding of the probes to model compounds
in accordance
with the present invention. A solid state depletion assay was performed
involving Probe 1 and
AVICEL or WHATMAN paper as the substrate. AVICEL is a commercial substrate
made of
crystalline cellulose available from FMC Corporation. WHATMAN paper is an
amorphous
cellulose filter paper available from GE Healthcare Life Sciences. FIG. 17
shows the results of a
solid state depletion assay in which binding saturation of Probe 1 to AVICEL
is apparent.
[0094] Affinity (Ka) of Probe 1 for AVICEL was measured using a modified
version of a solid
state depletion assay in order to ascertain that the fusion of the reporter
module (eGFP) with the
binding module (CBM3a) did not negatively affect the folding and thus the
affinity of CBM3a for
AVICEL. FIG. 17 shows that Probe 1 has an affinity constant (Ka) of 8 1.1M for
AVICEL, a value
similar to that reported for the commercial construct for crystalline
cellulose (7.7 p,M). This result
confirms that the binding module (CBM3a) of Probe 1 is well folded and that it
binds to crystalline
cellulose with a high affinity regardless of the close proximity of the
reporter module (eGFP).
[0095] Binding isotherms of eGFP-CBM3a to AVICEL (filled circle) and
WHATMAN paper
(open circle) after a 1 hour incubation at 23 C of the various cellulose
support with eGFP-CBM3a
(1001.ig/well) in a 20 mM Tris-1-1C1 pH 7.5 buffer containing 20 mM NaCl and 5
mM CaCl2. The
affinity constant (Ka) was calculated from nonlinear regression of
[Pbound = NoKa[Pfree] / (1+Kafree]). In this example, Ka is equal to 8 [iM for
AVICEL and 0.083 for
WHATMAN paper.
Example 7
100961 Example 7 sets forth parameters for a typical probe binding
experiment used in the
following examples. Materials and solutions included filtered buffer (20 mM
Tris-CL pH 7.5 +
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20 mM NaC1 + 5 mM CaC12; agitation at room temperature for 1 hour), 6% milk
(fresh) in buffer
(dissolve 1.2 g of milk into 20 ml of buffer; Centrifuge 2 min, 100g, RT), 3%
milk (fresh) in
buffer, TWEEN 0.05% in buffer, pulp-derived handsheet (paper disk: 3 mm
diameter) glued onto
the bottom of black microplate shining face down. Fluorescence acquisition
included endpoint
and area scanning 3x3 500 p.m; excitation wavelength (reporter module-
dependent) (9 mm) /
emission wavelength (reporter module-dependent) (9 mm); gain 50; 75 and 100;
and top (4.5
mm) detection. The reaction volume was 200 1. The fluorescent (fusion) probe
(or fiber
polymer) treatment method FPTM protocol was consistent with that described in
Knox P.J.
(2012) Methods in Enzymology, volume 510, 233-245, which is incorporated by
reference
herein in its entirety. A similar procedure is described in Ding et al. (2006)
BioTechniques 41,
435-443, which is incorporated by reference herein in its entirety.
[0097] The fluorescence of the untreated paper disks was measured. The
handsheet was
incubated in 200 pi of milk 3%, one hour at room temperature with a slow
agitation. Wash 3
(200 pp x 5 min was performed with buffer at room temperature with agitation.
The
fluorescence of the blocked paper disks was measured. 200 pl of probe/milk 3%
was added and
incubated at room temperature for one hour with a slow agitation. The
supernatant was removed
and total fluorescence was measured. Wash 3 (200 IA) x 5 min with buffer was
performed at
room temperature with agitation. The residual fluorescence was measured. Wash
3 (200 pl) x 5
min was performed with TWEEN 0.05% at room temperature with agitation.
Residual
fluorescence was measured. Black 96 wells microplate (Costar, cat # 3631) were
employed. A
SYNERGY Mx program was run (BioTek Instruments, Inc., Winooski, Vermont).
[0098] FIG. 18 is a schematic diagram describing the probe binding
experiments on a paper
disk. The various pulps used in the following examples are described in Table
5 (NA: Not
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available; "*"=Pulp chemical composition (wt%) measured at CRML). Pulps used
were from
North American paper plants.
Table 5: Chemical Composition and Available Characteristics of Different
Pulps.
Pulp 1 Pulp 2 Pulp 3 Pulp 4 Pulp 5 Pulp 6
Plant 1 Plant 2 Plant 3 Plant 3 Plant 3 Plant 3
Pulp origin
High yield
Kraft Kraft Kraft Mechanical Mechanical
Kraft
Unrefined Unrefmed Unrefmed Unrefined Unrefined Unrefined
Pulp
Unbleached Unbleached Unbleached bleached Unbleached bleached
Furnish: Furnish: Furnish: Furnish:
Furnish: Furnish:
softwood softwood softwood softwood softwood
softwood
(resinous)
(resinous) (resinous) (resinous)
(resinous) (resinous)
Pulp
Consistency 5.16 4.39 2.81 4.37 3.77 4.17
(wt%)
Pulp
9 7.15 9.28 7.11 4.48 4.72
Initial pH
Kappa
27-29 20-25 NA NA NA NA
number
Cellulose* 83.91 89.49 86.7 89.7 46 46.6
Hemicellulose* 9.54 7.29 16 16.9 21.7 22.2
Lignin* 6.7 3.2 4.4 1.9 29 29
Example 8
10099] This example demonstrates the comparison of fluorescence from a
commercial
"binding-reporter" probe with the fluorescence from a reporter module alone at
the surface of
paper in accordance with the present invention. The specific detection of
crystalline cellulose on
the surface of small disks of pulp 1 (¨ 0.5 mg) was achieved by binding
commercial eGFP-
CBM3a (NZYTech company). The non-specific signal provided by the binding of
the reporter
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module on the small disks was measured after incubation with the reporter
module alone. The
assay was performed into 96 wells microplate. Excess of Probe 1 (or reporter
module GFP) was
removed by washing using Tween 20 (0.05%).
[0100] The fluorescence from the reporter module (alone or as component of
the Probe) was
measured at a wavelength of 511 nm (excitation wavelength 488 nm) using a
SYNERGY Mx
microplate reader (BioTek Instruments, Inc.). The fluorescence intensities are
presented here as
a function of refining intensity and cellulase treatments. FIG. 19 shows
fluorescence intensity of
reporter module (eGFP) alone bound to untreated and cellulase-treated pulp
(after two levels of
refining). The non-specific fluorescent signal obtained with the reporter
module alone was very
low compared to Probe 1.
[0101] FIG. 20 depicts fluorescence intensity of Probe 1 bound to untreated
and cellulases
treated pulp (after two level of refining). FIG. 20 shows the detection of
crystalline cellulose at
the surface of cellulase-treated handsheet using Probe 1. Cellulase enzyme
treatments of the old
pulp increased the fluorescent signal compared to untreated pulp at all levels
of refining, which
indicates that the cellulase treatments of the pulp uncovered additional
crystalline cellulose
regions. Accordingly, using Probe 1 of the present invention, the impact of
cellulase treatments
on pulp can be detected without using any pulp and paper classical tests.
Example 9
[0102] This example demonstrates the detection of changes in surface
crystalline cellulose
after refining and cellulase treatments in accordance with the present
invention. The method is
based on the fluorescence intensity associated to the recognition of
crystalline cellulose by the
CBM3a-eGFP (Probe 1) on the surface of handsheets. Using a standardized Probe
1
fluorescence curve, the measured fluorescence intensity can be converted into
a quantity (ug)
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that is then divided by the surface (mm2) of the handsheets. The results
(lig/mm2) enable the
quantification of the crystalline cellulose on the surface of the handsheets.
[0103] Quantification of crystalline cellulose on the surface of pulp 1
handsheets was
performed on small disks of ¨ 0.5 mg. The handsheets at basis weight 60 g/m2
were produced
from previously treated (cellulases at 0.05 %, lh, 50 C, pH 7) and untreated
pulp 1. The specific
detection of crystalline cellulose on the surface of pulp 1 handsheets was
achieved with Probe 1
(250 1.ig/ml, 1 h, room temperature, pH 7.4) in a 24 wells microplate (Gourlay
et al., 2012). After
three washing steps, the green residual fluorescence was measured at 511 nm
(excitation
wavelength: 488 nm) using a Synergy Mx microplate reader (Bioteck). The
fluorescence
intensities were averaged and presented as percentages, as a function of PFI
revolutions (FIG.
21) or cellulase treatments (FIG. 22).
[0104] FIG. 21 depicts the impact of PFI refining on the quantification of
crystalline
cellulose on the surface of pulp 1 handsheets. The unrefined fluorescence
intensity and
corresponding CSF value were set as reference points (100%). FIG. 21 shows
that the residual
fluorescence, which should indicate the amount of Probe 1-tagged crystalline
cellulose at the
surface of pulp 1 handsheets, increased from 2.7 % to 28.6 % as the PFI
revolutions increased
from 1500 to 6000. This decrease is not linear and was correlated with the
decrease in CSF
(from 87.9 ml to 71.1 ml), which is an indicator of the degree of hydration of
pulp. Accordingly,
the amorphogenesis process (water swelling of the pulp), which is associated
to PFI refining, is
evidently responsible for the recorded decrystallization or loss of
crystalline cellulose at the
surface of pulp 1 handsheets.
[0105] FIG. 22 depicts the impact of cellulase treatment on the
quantification of crystalline
cellulose on the surface of pulp 1 handsheets. The untreated fluorescence
intensities at 0, 3000,
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and 4500 revolutions were set as the reference values (100%). FIG. 22 shows
that all cellulase
treatments increased the detection of eGFP-tagged crystalline celluloses.
These results indicate
that enzymes expose previously buried crystalline celluloses at the surface of
pulp 1 handsheets
compared to untreated handsheets.
[0106] The highest increase recorded was for the treatment of pulp 1 with
cellulase C6
without refining. Cellulase Cl and C2 treatments led to the lowest increase of
crystalline
cellulose on unrefined and refined pulp 1 at 3000 revolutions (vs untreated)
compared to the
other enzyme samples. However, the use of more intense refining (4500 revs) on
treated pulp 1
with cellulase sample Cl, C2, and C3 lead to an increase of crystalline
celluloses. These
observations might be explained by the generations of microfibrils after
refining and
consequently an increase exposition of crystalline cellulose at the surface of
the fibers. These
results suggest that cellulase treatments of pulp 1 followed by mechanical
treatment expose a
larger quantity of crystalline celluloses. In opposition to all other enzymes,
refining had no
impact on C4-treated handsheet.
Example 10
[0107] This example demonstrates the prediction of the presence or absence
of changes in
surface crystalline cellulose after refining and xylanase treatments in
accordance with the present
invention. Digestion of the xylan polymers by xylanases can uncover
crystalline cellulose and
increase its detection on handsheet surfaces when compared with the untreated
controls. Probe 1
was used to detect changes in crystalline cellulose at the surface of paper
after xylanase
treatments. FIG. 23 depicts the quantification (p,g/mm2) of Probe 1 bound on
the surface of
xylanase-treated handsheets from plant 2 refined at 0, 3000 and 4500 PFI
revolutions. Pulp 2
was used for sheet preparation. The excitation wavelength was 488 nm. No
xylanase treatments
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used here had a significant impact on fluorescence detected. The quantity of
crystalline cellulose
measured here suggests that xylanase preparations used had no impact on
cellulose fiber surface.
This result is in complete agreement with the analysis of paper physical
properties and fiber
morphologies after xylanase treatments. The probe 1 FPTM results reveal the
absence of
xylanase impact in a much less time than involved for traditional TAPPI
methods, thus enabling
a fast, high throughput protocol.
Example 11
[0108] This example demonstrates the detection of crystalline cellulose
after refining in
accordance with the present invention. Correlations were determined between
Probe 1 detection
and paper physical properties using two different pulps. Two pulps (Pulp 1 and
Pulp 2) were
refined and analyzed by Probe 1 and by standard TAPPI methods. The measurement
of
crystalline cellulose on handsheet disks (60 g/m2) was performed by incubating
the paper disks
with 250 g/ml of Probe 1. Probe 1 fluorescence was used for recognition of
crystalline
cellulose on the surface of handsheets after refining (various levels of
energy). Using a
standardized curve, the measured fluorescence intensity is converted into a
quantity (lig) of
crystalline cellulose, which is then divided by the surface (mm2) of the
handsheets. The results
(gg/mm2) enable the quantification of the crystalline cellulose on the surface
of the handsheets.
[0109] FIG. 24 depicts the quantification of crystalline cellulose (i.tg /
mm2) on the surface of
PFI refined handsheet (Pulp 1 (plant 1; left bar) vs Pulp 2 (plant 2; right
bar). These FPTM results
support the concept that pulp refining partially decrystallizes cellulose. In
the case of the Pulp 2
handsheets, at least three decrystallization events (or steps) are observed: 0-
2000, 3000-4500 and at
6000 PFI revolutions. This effect is less apparent than when using more than
3000 revs.
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101101 FIGS. 25A-25D depict correlations between crystalline cellulose
quantification using
Probe 1 (n/mm2) and Pulp 2/paper physical properties as a function of refming
energy (PFI
revolutions). FIG. 25A depicts tear index (mN m2/g), FIG. 25B depicts tensile
index (N m/g), FIG.
25C depicts internal bond strength (J/m2), and FIG. 25D depicts fibers mean
length (mm). FIGS.
25A-25D show the correlations between Probe 1 signal and the corresponding
paper's physical
properties for the Pulp 2 handsheets. An inverse correlation is observed with
the tensile index and
the internal bond strength (Figure 25B-25C). In contrast, the tear index is
directly correlated with
the crystalline cellulose quantification (Figure 25A). These correlations can
be used for predicting
the impact of various refining treatments on final paper properties in a very
short time (1 hour vs
days) compared to TAPPI pulp and paper analyses. The internal bond and fiber
length were further
modified by refining past 3000 revs, at variance with tear and tensile indices
that stabilized under
similar conditions.
[0111] The changes in properties and crystalline cellulose detected after
treatment of pulp 1 are
shown in FIGS. 26A-26D. Correlations between crystalline cellulose
quantification using Probe 1
( g/mm2) and pulp 1/paper physical properties as a function of refining energy
(PFI revolutions) are
depicted. FIG. 26A depicts tear index (mN m2/g), FIG. 26B depicts tensile
index (N m/g), FIG.
26C depicts internal bond strength (J/m2), and FIG. 26D depicts fibers mean
length (mm). The
amount of cellulose detected by Probe 1 decreased as refining energy increased
in general, with the
exception of the value associated with 1500 revs. Except for this particular
value, all panels suggest
the same correlations as those observed with pulp 2 (FIGS. 25A-25B). The
different content in
lignin of either pulp did not appear to affect the overall correlations.
Correlations were positive for
tear index, and reciprocal for internal bond and tensile index.
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[0112] The four properties monitored continued to change when refining was
increased past
3000 revs. Thus, applying the method to different pulp may result in subtle
changes in correlations
at high refining energy. Independent of the physical properties and the pulp
studied here, the impact
of refining on crystalline cellulose was observed after a minimal degradation
of fiber (i.e. when
treatment exceeded 1500 revs). The impact of refining past 3000 revs is no
longer associated with
changes in crystalline cellulose at the surface (it is not changed by further
refining). Consequently,
to improve and develop pulp/paper physical properties, the refining intensity
should minimally
degrade the crystalline matrix of the pulp.
[0113] The results demonstrate that the method is a diagnostic indicator
that can be taken into
account when determining the refining conditions for a given set of
properties. Its simplicity and
high sensibility enable a fast and efficient diagnostic of the best refining
conditions used for a set of
physical properties. The results show that the method can be used to predict
the impact of a
treatment on pulp and paper properties for tear, tensile and internal bond
strength, without
performing actual measurements of those properties on handsheets.
Example 12
[0114] This example demonstrates the detection of xylan on the surface of
paper disks in
accordance with the present invention. Pulp 5 (unbleached mechanical pulp),
Pulp 6 (bleached
mechanical pulp, Pulp 3 (unbleached Kraft pulp), Pulp 4 (bleached Kraft pulp),
and Pulp 2
(unbleached Kraft pulp) were used in this example (as described above in Table
5). The assay
was performed in 96 well-microplates with each well containing a small
handsheet disk (60
g/m2). The specific detection of xylan on the surface of such disks made from
different pulps
was achieved by binding Probe 3 (CBM15-mOrange2). The fluorescence intensity
was
measured after incubating the paper disks with 200 tl solution of Probe 3. The
excess Probe 3,
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as well as non-specific binding of the same adduct to any other moieties, was
removed by
washing three times with a TWEEN 20 solution (0.05%). Fluorescence intensity
from bound
Probe 3 was measured at a wavelength of 569 nm (excitation wavelength 549 urn)
using a
SYNERGY Mx microplate reader.
[0115] FIG. 27 depicts fluorescence intensity of Probe 3 bound to xylan at
the surface of five
different paper disks¨UBKPR Unbleached Kraft pulp (Pulp 2), UBMP unbleached
mechanical
pulp (Pulp 5), BMP bleached mechanical pulp (Pulp 6), UBKP unbleached Kraft
pulp (Pulp 3); and
BKP bleached Kraft pulp (Pulp 4). These results show that Probe 3 has a higher
binding affinity
towards BKP derived disks compared to the other paper disks. These results may
be attributed to
the lignin content on the surface of the paper disks. In Kraft pulping,
chemicals and heat are used to
dissolve the lignin, the binding agent that covers cellulose and
hemicellulose. The lignin content of
unbleached chemical pulp is approximatively 3-5% and bleaching process removes
practically all of
the remaining lignin, taking it towards 0% of the total content. Mechanical
pulping methods
preserve most of the wood component so that the lignin content is similar to
that of wood (20-28%).
Bleached Kraft paper that contains the lowest amount of lignin has been
detected by the probe much
more efficiently than the other paper disks. This can be ascribed by the
increased exposure of xylan
on the surface of bleached fibers. This analysis shows that Probe 3 detect
xylan on the surface of
the paper disks and that it can discriminate between paper surfaces made from
different pulps.
Example 13
[0116] This example demonstrates the detection of xylan removal after
enzymatic treatments
in accordance with the present invention. The ability to detect xylan was also
tested for pulp
where xylan content was changed at the fiber surface. In order to demonstrate
binding of Probe
3 to xylan unambiguously, paper disks (from bleached Kraft pulp (BKP)) were
treated with a
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commercial xylanase prior detection assay. Xylanase was used at two doses: 0.1
U per paper
disk and 0.4 U per paper disk. Results are shown in FIG. 28. FIG. 28 depicts
fluorescence
intensity of Probe 3 bound to xylan at the surface of untreated and xylanase
treated Pulp 4 (BI(P)
paper. Treatment duration is indicated on the x axis. Untreated indicates Pulp
4 paper disks
without xylanase treatment. "0/N" indicates paper treated with xylanase
overnight at room
temperature. Binding of Probe 3 was reduced by almost 80% after xylanase
treatment,
establishing that the method detects variations in polymers at the surface of
fibers.
Example 14
[0117] This example demonstrates the detection of mannan on the surface of
paper disks in
accordance with the present invention. A high content of mannan (and
redeposition on the
surface of cellulose fiber during the Kraft pulping) may inhibit the bleaching
process.
Accordingly, mannanase, in addition with xylanase, is used for prebleaching.
In order to
optimize mannanase treatment, it is useful to determine mannan content before
and after
prebleaching.
[0118] Two different grades of pulp (Pulp 3: unbleached Kraft pulp (UBKP)
and pulp 4:
bleached Kraft pulp (BKP)) were investigated with respect to their mannan
content. The assay
was performed in 96 well-microplates with each well containing a small
handsheet disk (60
g/m2). The specific detection of mannan on the surface of such disks made from
different pulps
was achieved by binding the eCFP-CBM27 protein fusion, also named Probe 4. The
fluorescence intensity was measured after incubating the paper disks with 200
1.1.1 solution of
Probe 4. The excess Probe 4, as well as non-specific binding of the same
adduct to any other
moieties, was removed by washing 3 times with a TWEEN 20 solution (0.05%).
Fluorescence
intensity from bound Probe 4 was measured at a wavelength of 477 nm
(excitation wavelength
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434 nm) using a SYNERGY Mx microplate reader. The fluorescence intensities of
the bound
probes are presented in FIG. 29.
101191 FIG. 29 depicts fluorescence intensity of Probe 4 (eCFP-CBM27) bound
to mannan at
the surface of two different paper disks: unbleached Kraft pulp (Pulp 3, UBKP)
and bleached
Kraft pulp (Pulp 4, BKP). These results show that Probe 4 has a higher binding
affinity towards
Pulp 4 (BKP) derived disks compared to the pulp 3 (UBKP) paper disks. These
results may be
attributed to the redeposit of the hemicelluloses and to the lignin content on
the surface of the
paper disks of pulp 4 (BKP). In Kraft pulping, chemicals and heat are used to
dissolve lignin,
the binding agent that covers cellulose and hemicellulose. Thus the lignin
content drops from 3-
5% down to nearly zero. So, mannan in bleached Kraft paper (that contains the
lowest amount
of lignin) has been detected by the probe much more efficiently than the other
paper disks. This
result reflects the increased exposure of mannan on the surface of bleached
fibers.
Example 15
[0120] This example demonstrates the detection of mannan removal after
enzymatic
treatment in accordance with the present invention. The ability to detect
mannan at the surface
of fibers was tested. In order to show the binding of Probe 4 to mannan
unambiguously, paper
disks (from bleached Kraft pulp (Pulp 4, BKP)) were treated with a commercial
mannanase
(MEGAZYME-E-BMANN) enzyme prior to detection assay. Results are shown in FIG.
30.
FIG. 30 depicts fluorescence intensity of Probe 4 bound to mannan at the
surface of mannanase-
treated bleached Kraft pulp (Pulp 4, BKP) paper. Binding of Probe 4 was
reduced by almost
44% after a 1 hour treatment with mannanase. These results clearly demonstrate
that the method
detects variations in polymers at the surface of fibers.
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[01211 The present invention includes the following
aspects/embodiments/features in any
order and/or in any combination:
1. A lignocellulosic polymer detection probe comprising a) a binding module
that
specifically binds to at least one lignocellulosic polymer and b) a reporter
module that is
spectroscopically detectable.
2. The probe of any preceding or following embodiment/feature/aspect, wherein
the
lignocellulosic polymer detection probe comprises a probe polypeptide.
3. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module is a binding module polypeptide and the reporter module is a
reporter module
polypeptide.
4. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module polypeptide is fused directly to the reporter module
polypeptide.
5. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module polypeptide is linked to the reporter polypeptide via a linker
polypeptide.
6. The probe of any preceding or following embodiment/feature/aspect, wherein
the probe
polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 2, 6,
8, 10, and 12.
7. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module polypeptide comprises an amino acid sequence of any one of SEQ
ID NOS: 14, 16,
18, 20, and 22 and the reporter module polypeptide comprises an amino acid
sequence of any one
of SEQ ID NOS: 24, 26,28, and 30.
8. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module polypeptide is not an antibody or a fragment thereof.
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9. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module is not an antibody or a fragment thereof.
10. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module is not a polypeptide.
11. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module is fluorescent.
12. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module has a fluorescence excitation peak (maximum) of from about 350
nm to about 700
nm.
13. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module has a fluorescence emission peak (maximum) of from about 400
nm to about 750
nm.
14. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module comprises a fluorescent protein.
15. The probe of any preceding or following embodiment/feature/aspect, wherein
the
fluorescent protein comprises an ultraviolet fluorescent protein, a blue
fluorescent protein, a cyan
fluorescent protein, a green fluorescent protein, a yellow fluorescent
protein, an orange fluorescent
protein, a red fluorescent protein, a far-red fluorescent protein, a near
infrared fluorescent protein, an
infrared fluorescent protein, a sapphire-type fluorescent protein, a long
Stokes shift fluorescent
protein, a switchable fluorescent protein, or any combination thereof.
16. The probe of any preceding or following embodiment/feature/aspect, wherein
the
fluorescent protein comprises Sirius, TagBFP, mTagBFP2, Azurite, EBFP2,
mKalamal, Sirius,
Sapphire, T-Sapphire, ECFP, mAmetine, Cerulean, mCerulean3, SCFP3A, CyPet,
mTurguoise,
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mTurquoise2, monomeric Midoriishi-Cyan, Aquamarine, eCFP, TagCFP, mTFP1,
AmCyanl,
EGFP, Emerald, Superfolder GFP, monomeric Azami Green, TurboGFP, TagGFP2,
mUKG,
mWasabi, Clover, mNeonGreen, eGFP, AcGFP1, ZGreenl, ZsYellowl, mBanana, EYFP,
Topaz,
Citrine, Venus, SYFP2, Ypet, IanRFP-deltaS83, mPapayal, TagYFP, mOrange,
mOrange2,
monomeric Kusabira-Orange, mK0k, mK02, mTangerine, mNectarine, TagRFP, TagRFP-
T,
mApple, tnRuby, mRuby2, DsRed-Express2, DsRed-Express, tdTomato, DsRed-
Monomer, DsRed-
Monomer, DsRed2, AsRed2, mStrawberry, mCherry, HcRedl, FusionRed, mRaspberry,
E2-
Crimson, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, TagRFP675,
iFP1.4,
iRFP(iRFP713), iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, mKeima Red,
LSS-
mKatel, LSS-mKate2, LSSmOrange, mBeRFP, PA-GFP, PATag RFP, Dendra2, Timer,
PAmCherry, Kaede (green), Kaeda (red), KikGR1 (green), KikGR1 (red), PS-CFP2,
mEos2
(green), mEos2 (red), mEos3.2 (green), mEos3.2(red), PSmOrange, Dropna, or any
combination
thereof.
17. The probe of any preceding or following embodiment/feature/aspect, wherein
binding
module is a carbohydrate-binding module (CBM) comprising CBM1, CBM2, CBM3,
CBM3a
CBM4, CBM5, CBM6, CBM9, CBM10, CBM11, CBM12, CBM14, CBM15, CBM17, CBM18,
CBM19, CBM20, CBM21, CBM25, CBM27, CBM28, CBM33, CBM48, CBM49, or any
combination thereof.
18. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module specifically binds to cellulose, hemicellulose, lignin, xylan,
mannan, or any
combination thereof.
19. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module specifically binds to crystallized cellulose.
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20. The probe of any preceding or following embodiment/feature/aspect, wherein
the
lignocellulosic polymer detection probe comprises a plurality of
lignocellulosic polymer detection
probes, each lignocellulosic polymer detection probe specifically binding to a
different
lignocellulosic polymer.
21. The probe of any preceding or following embodiment/feature/aspect, wherein
each
lignocellulosic polymer detection probe comprises a different reporter module.
22. The probe of any preceding or following embodiment/feature/aspect, wherein
each
reporter module has a different fluorescence signature.
23. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter modules comprise one or more of eGFP, mCheiTy, mOrange2, and eCFP.
24. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding modules comprise one or more of CBM3a, CBM4-1, CBM15, and CBM27.
25. The probe of any preceding or following embodiment/feature/aspect, wherein
the
lignocellulosic polymer detection probe comprises eGFP-CBM3a, mCherry-CBM4-1,
mOrange2-
CBM15, eCFP-CBM27, or any combination thereof.
26. The probe of any preceding or following embodiment/feature/aspect, wherein
the
lignocellulosic polymer detection probe is detectable at a distinct
wavelength.
27. A complex comprising the probe of any preceding or following
embodiment/feature/aspect specifically bound to a pulp or paper product
comprising at least one
surface available lignocellulosic polymer.
28. A pulp or paper product comprising at least one surface available
lignocellulosic
polymer and at least one probe of any preceding or following
embodiment/feature/aspect
specifically bound thereto.
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29. A method of detecting a lignocellulosic polymer, the method comprising:
contacting the probe of any one of any preceding or following
embodiment/feature/aspect
with a biomass material; and
measuring a property associated with the reporter module to determine the
presence or
absence of at least one lignocellulosic polymer in the biomass material based
on specific binding of
the probe to the at least one lignocellulosic polymer.
30. The method of any preceding or following embodiment/feature/aspect,
wherein the
property measured is fluorescence.
31. The method of any preceding or following embodiment/feature/aspect,
further
comprising calculating the amount of the at least one lignocellulosic polymer,
determining the type
of the at least one lignocellulosic polymer, or both.
32. The method of any preceding or following embodiment/feature/aspect,
wherein the
biomass material comprises a wood biomass material.
33. The method of any preceding or following embodiment/feature/aspect,
wherein the
wood biomass material is pulp, furnish, paper, or any combination thereof
34. The method of any preceding or following embodiment/feature/aspect,
further
comprising forming at least one handsheet from the wood biomass product,
wherein the measuring
is performed on the handsheet.
35. The method of any preceding or following embodiment/feature/aspect,
wherein the
measuring is performed before treatment, during treatment, or after treatment,
or any combination
thereof
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36. The methpd of any preceding or following embodiment/feature/aspect,
wherein the
treatment comprises an enzymatic treatment, bleaching, amorphogenesis,
milling, or PFI refining, or
any combination thereof.
37. The method of any preceding or following embodiment/feature/aspect,
wherein the
treatment comprises enzymatic treatment with at least one enzyme comprising a
cellulase, a
xylanase, a mannase, a lignase, or any combination thereof.
38. The method of any preceding or following embodiment/feature/aspect,
further
comprising performing at least one treatment of the biomass material based on
the amount of the at
least one lignocellulosic polymer measured, the type of lignocellulosic
polymer measured, or both.
39. The method of any preceding or following embodiment/feature/aspect,
wherein the
amount of lignocellulosic polymer measured correlates negatively or positively
with at least one
physical property of the biomass product.
40. The method of any preceding or following embodiment/feature/aspect,
wherein the at
least one physical property comprises burst index, drainage rate, tear index,
tensile index, or internal
bond strength, or any combination thereof.
41. The method of any preceding or following embodiment/feature/aspect,
further
comprising dosing at least one enzyme based on the amount of lignocellulosic
polymer measured,
the type of lignocellulosic polymer measured, or both.
42. The method of any preceding or following embodiment/feature/aspect,
further
comprising adjusting mill speed based on the amount of lignocellulo sic
polymer measured, the type
of lignocellulosic polymer measured, or both.
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43. The method of any preceding or following embodiment/feature/aspect,
further
comprising adjusting total water content of the biomass material based on the
amount of
lignocellulosic polymer measured, the type of lignocellulosic polymer
measured, or both.
44. The method of any preceding or following embodiment/feature/aspect,
wherein the
probe comprises a plurality of probes.
45. A method of determining the effectiveness of an industrial treatment on
pulp or a paper
product comprising:
contacting the lignocellulosic polymer detection probe of any one of any
preceding or
following embodiment/feature/aspect with pulp or a paper product;
detecting the specific binding of the probe to the pulp or the paper product;
calculating the amount of at least one lignocellulosic polymer on a surface of
the pulp or the
paper product; and
determining the effectiveness of an industrial treatment on the pulp or paper
product based
on the amount of the at least one lignocellulosic polymer detected.
46. The method of any preceding or following embodiment/feature/aspect,
wherein the
industrial treatment comprises an enzymatic treatment, a chemical treatment,
or a physical
treatment, or any combination thereof.
47. The method of any preceding or following embodiment/feature/aspect,
wherein the
method is performed before the industrial treatment, during the industrial
treatment, or after the
industrial treatment, or any combination thereof
48. The method of any preceding or following embodiment/feature/aspect,
wherein the
lignocellulosic polymer detection probe is detectable at a distinct
wavelength.
49. A method of determining a physical property of pulp or a paper product
comprising:
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contacting the lignocellulosic polymer detection probe of any preceding or
following
embodiment/feature/aspect with pulp or a paper product;
detecting the specific binding of the probe to the pulp or the paper product;
calculating the amount of at least one lignocellulosic polymer on a surface of
the pulp or the
paper product; and
determining at least one physical property of the pulp or paper product based
on the amount
of the at least one lignocellulosic polymer detected.
50. The method of any preceding or following embodiment/feature/aspect,
wherein the at
least one physical property comprises burst index, drainage rate, tear index,
tensile index, or internal
bond strength, or any combination thereof.
51. A polymer detection probe comprising a) a binding module that specifically
binds to
at least one polymer and b) a reporter module that is spectroscopically
detectable.
52. The probe of any preceding or following embodiment/feature/aspect, wherein
the
polymer detection probe comprises a probe polypeptide.
53. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module is fluorescent.
54. The probe of any preceding or following embodiment/feature/aspect, wherein
the
reporter module comprises a fluorescent protein.
55. The probe of any preceding or following embodiment/feature/aspect, wherein
the
fluorescent protein comprises an ultraviolet fluorescent protein, a blue
fluorescent protein, a cyan
fluorescent protein, a green fluorescent protein, a yellow fluorescent
protein, an orange
fluorescent protein, a red fluorescent protein, a far-red fluorescent protein,
a near infrared
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fluorescent protein, an infrared fluorescent protein, a sapphire-type
fluorescent protein, a long
Stokes shift fluorescent protein, a switchable fluorescent protein, or any
combination thereof.
56. The probe of any preceding or following embodiment/feature/aspect, wherein
binding
module is a carbohydrate-binding module (CBM).
57. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module specifically binds to cellulose, hemicellulose, lignin, xylan,
marman,
glucuronoxylan, arabinoxylan, glucomannan, xyloglucan, or any combination
thereof or a linear
fragment thereof, or a branched fragment thereof, or an oligomer thereof, or a
monomer and/or
macromer thereof, for example, glucose, D-glucose, mannose, xylose, galactose,
rharnnose,
arabinose, monolignol, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol,
p-hydroxyphenyl
phenylpropanoid, guaiacyl phenylpropanoid, or syringyl phenylpropanoid, or a
combination
thereof.
58. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module specifically binds to a glycoprotein, carbohydrate, or both,
specific to a
particular blood antigen, type, group, or subgroup.
59. The probe of any preceding or following embodiment/feature/aspect, wherein
the
binding module specifically binds to a polyaryletherketone (PAEK), a polyether
ether ketone
(PEEK), a polyethylene, a polypropylene, a polystyrene, a
polytetrfluoroethylene, a
polyvinylchloride, a polyamide, a para-aramid, a polyethylene terephthalate, a
polyimide, a
polycarbonate, a polypeptide, a polynucleotide, a glycoprotein, a protein, a
phosphorylated
protein, a modified protein, a lipid, a surfactant, lecithin, or a
biosurfactant, or any combination
thereof.
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60. The probe of any preceding or following embodiment/feature/aspect, wherein
the
polymer detection probe comprises a plurality of polymer detection probes,
each polymer
detection probe specifically binding to a different polymer.
61. The probe of any preceding or following embodiment/feature/aspect, wherein
each
polymer detection probe comprises a different reporter module.
62. The probe of any preceding or following embodiment/feature/aspect, wherein
each
reporter module has a different fluorescence signature.
63. The probe of any preceding or following embodiment/feature/aspect, wherein
the
polymer detection probe is detectable at a distinct wavelength.
64. A complex comprising the probe of any preceding or following
embodiment/feature/aspect specifically bound to a material comprising at least
one surface
available polymer.
65. A method of detecting a polymer, the method comprising:
contacting the probe of any preceding or following embodiment/feature/aspect
with a
material; and
measuring a property associated with the reporter module to determine the
presence or
absence of at least one polymer in the material based on specific binding of
the probe to the at least
one polymer.
66. The method of any preceding or following embodiment/feature/aspect,
wherein the
property measured is fluorescence.
67. The method of any preceding or following embodiment/feature/aspect,
further
comprising calculating the amount of the at least one polymer, determining the
type of the at
least one polymer, or both.
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68. The method of any preceding or following embodiment/feature/aspect,
wherein the
material comprises a blood sample.
69. The method of any preceding or following embodiment/feature/aspect,
further
comprising determining at least one of a blood antigen, type, group, and
subgroup of the blood
sample.
70. The method of any preceding or following embodiment/feature/aspect,
wherein the
probe comprises a plurality of probes.
71. The probe of any preceding or following embodiment/feature/aspect, wherein
said
polymer is at least one saccharide polymer.
72. The probe of any preceding or following embodiment/feature/aspect, wherein
said
polymer is at least one oligosaccharide polymer.
[0122] The present invention can include any combination of these various
features or
embodiments above and/or below as set forth in sentences and/or paragraphs.
Any combination
of disclosed features herein is considered part of the present invention and
no limitation is
intended with respect to combinable features.
101231 The following references are incorporated by reference herein in
their entireties:
Linder, M. et al. (1996) J Biol Chem 271, 21268-21272.
McLean, B. W. et al. (2002) J Biol Chem 277, 50245-50254.
Hilden, L. et al. (2003) Biotechnology letters 25, 553-558.
Lehtio, J. et al. (2003) Proc Natl Acad Sci U S A 100, 484-489.
Czjzek et al., J Biol Chem. 2001 Dec 21;276(51):48580-7. Epub 2001 Oct 22.
Ding, S.-Y. et al. (2006) BioTechniques 41, 435-443.
McCartney, L. et al. (2006) Proc Natl Acad Sci U S A 103, 4765-4770.
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Hong, J. et al. (2007) Langmuir : the ACS journal of surfaces and colloids 23,
12535-
12540.
Herve, C. et al. (2010) Proc Nat! Acad Sci U S A 107, 15293-15298.
Gourlay, K. etal. (2012) Biotechnology for biofuels 5, 51.
Knox, J. P. (2012) Methods in enzymology 510, 233-245.
Ruel, K. et al. (2012) Plant science: an international journal of experimental
plant
biology 193-194, 48-61.
Luis, A. S. et al. (2013) J. Biol. Chem. 288, 4799-4809
Zhang, M. etal. (2013) Physical chemistry chemical physics : PCCP 15, 6508-
6515.
Mou et al. (2013) Bioresources 8, 2325-2336.
Machado et al., (2009) Cellulose 16, 817-824.
Kim et al. (2010) Planta 232, 817-824.
Kim et al. (2012) Planta 236, 35-50.
µSirolc5ret al. (2012) Carbohyd. Polym. 89, 213-221.
Gao et al., (2014) Biotechnology for biofuels 7, 24.
Filonova et al. (2007) Biomacromolecules 8, 91-97.
http://www.cazy.org/Carbohydrate-Binding-Modules.html on March 30th, 2015
5,856,201; 5,837,814; 5,738,984; 5,719,044; 5,670,623; 6,174,700; 5,962,289;
7,361,487; 5,928,917
Boraston et al., Biochem J., 382: 769-781 (2004)
Shoseyov et al., Microbiology and Molecular Biology Reviews, 70(2): 283-295
(2006)
Christiansen et al., FEBS Journal, 276:5006-5029 (2009).
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[0124] Applicants specifically incorporate the entire contents of all cited
references in this
disclosure. Further, when an amount, concentration, or other value or
parameter is given as either a
range, preferred range, or a list of upper preferable values and lower
preferable values, this is to be
understood as specifically disclosing all ranges formed from any pair of any
upper range limit or
preferred value and any lower range limit or preferred value, regardless of
whether ranges are
separately disclosed. Where a range of numerical values is recited herein,
unless otherwise stated,
the range is intended to include the endpoints thereof, and all integers and
fractions within the range.
It is not intended that the scope of the invention be limited to the specific
values recited when
defining a range.
[0125] Other embodiments of the present invention will be apparent to those
skilled in the art
from consideration of the present specification and practice of the present
invention disclosed
herein. It is intended that the present specification and examples be
considered as exemplary
only with a true scope and spirit of the invention being indicated by the
following claims and
equivalents thereof.
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SEQUENCE LISTING
SEQ ID NO: 1 (nucleic acid) for eGFP-CBM3a (Probe 1). The underlined text
represents the
thrombin cleavage site which link eGFP to CBM3a.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGCTGTTCACCGG
GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG
TGTC CGGC GAGGGC GAGGGCGATGC CAC CTAC GGCAAGCTGAC CCTGAAGTTCATC
TGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTAC
GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAG
TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG
CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG
GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG
CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG
CC GAC CACTACCAGCAGAACAC CC CCATCGGCGAC GGCCC CGTGCTGCTGC CCGAC
AACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA
TCACATGGTCCTGCTGGAGTTC GTGACC GCC GC CGGGATCACTCTC GGCATGGAC GA
GCTGTACAAAAGTTCCGGTCTGGTGCCGCGTGGTAGCACACCGGTATCAGGCAATTT
GAAGGTTGAATTCTACAACAGCAATCCTTCAGATACTACTAACTCAATCAATCCTCA
GTTCAAGGTTACTAATACCGGAAGCAGTGCAATTGATTTGTCCAAACTCACATTGAG
ATATTATTATACAGTAGACGGACAGAAAGATCAGACCTTCTGGTGTGACCATGCTGC
AATAATCGGCAGTAACGGCAGCTACAACGGAATTACTTCAAATGTAAAAGGAACAT
TTGTAAAAATGAGTTCCTCAACAAATAACGCAGACACCTACCTTGAAATTAGCTTTA
CAGGCGGAACTCTTGAACCGGGTGCACATGTTCAGATACAAGGTAGATTTGCAAAG
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AATGACTGGAGTAACTATACACAGTCAAATGACTACTCATTCAAGTCTGCTTCACAG
TTTGTTGAATGGGATCAGGTAACAGCATACTTGAACGGTGTTCTTGTATGGGGTAAA
GAATAA
SEQ ID NO: 2 (amino acid) for eGFP-CBM3a (Probe 1). The underlined text
represents the
thrombin cleavage site which link eGFP to CBM3a.
MGHHHHHHGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYK
TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFK
IRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVT
AAGITLGMDELYKS S GLVPRGSTPV S GNLKVEFYN SNP SDTTNSINPQFKVTNTGS SAID
LSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYL
EIS FTGGTLEPGAHVQIQ GRFAKNDWSNYTQ SNDYSFKSAS QFVEWDQVTAYLNGVLV
WGKE
SEQ ID NO: 3 for Probe 1 linker.
AGTTCCGGTCTGGTGCCGCGTGGTAGC
SEQ ID NO: 4 (amino acid) for Probe 1 linker.
SSGLVPRGS
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SEQ ID NO: 5 (nucleic acid) for mCheiTy-CBM4-1 (Probe 2a). The underlined text
represents
the glycine which link mCherry to CBM4-1.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGGATAACATGGC
CATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCC
ACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGAC
CGCCAAGCTGAAGGTGACCAAGGGTGGC CC CCTGCCCTTC GCCTGGGACATCCTGTC
CC CTCAGTTCATGTAC GGCTC CAAGGC CTAC GTGAAGCACCCCGCC GACATC C CCGA
CTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGA
GGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCA
TCTACAAGGTGAAGCTGC GC GGCACCAACTTCC C CTC CGACGGCC C C GTAATGCAG
AAGAAGACCATGGGCTGGGAGGC CTC CTCCGAGCGGATGTACC C CGAGGACGGC GC
CCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGAC
GCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTA
CAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGG
AACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTAC
AAGGGTGCGTCGCCGATCGGGGAGGGAACGTTCGACGACGGGCCCGAGGGGTGGGT
CGCGTACGGCACCGACGGCCCCCTCGACACGAGCACGGGCGCGCTGTGCGTCGCCG
TGCCGGCCGGCTCCGCGCAGTACGGCGTCGGCGTCGTGCTCAACGGCGTCGCGATC
GAGGAAGGGACCAC CTACACGCTCCGGTACAC C GC GACGGCCTCGACCGACGTCAC
C GTGCGGGCGCTC GTCGGGCAGAACGGCGCC C CCTAC GGCACCGTGCTC GACAC GA
GCCCGGCCCTGACGTCCGAGCCGCGGCAGGTGACCGAGACGTTCACGGCCTCGGCG
ACGTACC CCGCGACACC CGCC GCCGACGACCCCGAGGGGCAGATC GCCTTC CAGCT
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CGGCGGGTTCAGCGCCGACGCGTGGACGTTCTGCCTCGACGACGTCGCGCTCGACTC
CGAGGTCGAGCTCTAA
SEQ ID NO: 6 (amino acid) for mCherry-CBM4-1 (Probe 2a). The underlined text
represents
the glycine which link mCherry to CBM4-1.
MGHHHHHHGVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEI
KQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
RHSTGGMDELYKGASPIGEGTFDDGPEGWVAYGTDGPLDTSTGALCVAVPAGSAQYG
VGVVLNGVAIEEGTTYTLRYTATASTDVTVRALVGQNGAPYGTVLDTSPALTSEPRQVT
ETFTASATYPATPAADDPEGQIAFQLGGFSADAWTFCLDDVALD SEVEL
SEQ ID NO: 7 (nucleic acid) for mCherry-CBM17 (Probe 2b). The underlined text
represents
the glycine codon that link mCherry to CBM17.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGGATAACATGGC
CATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCC
ACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGAC
CGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTC
CCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGA
CTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGA
GGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCA
TCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAG
AAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGC
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CCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGAC
GCTGAGGTCAAGACCACCTACAAGGCCAAGAAGC CC GTGCAGCTGC CC GGC GC CTA
CAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGG
AACAGTACGAAC GCGC CGAGGGCCGCCACTC CAC CGGCGGCATGGACGAGCTGTAC
AAGGGTTTATGGGCAGATAATGAATTAACCACTTCAGGTCAATATGTACGTGCTCGT
ATTAAGGGAGCTTATTATGCTACACCAGTTGATCCTGTAACAAACCAACCAACAGCA
CC GAAAGACTTTTCTTCAGGCTTTTGGGAC TTTAATGACGGTACTACACAAGGTTTT
GGTGTAAATCCAGATAGTCCAATAACTGCTATTAATGTTGAAAATGCTAACAATGCT
TTAAAAATCAGCAATCTTAATAGTAAGGGTAGTAATGATTTATCTGAAGGAAACTTT
TGGGCCAATGTCCGCATTTCAGCTGATATCTGGGGACAAAGTATAAATATATATGGA
GACACAAAACTAACAATGGATGTTATAGCTCCAACACCTGTAAATGTATCAATCGCA
GCTATCCCACAAAGTAGTACTCACGGTTGGGGAAATCCTACAAGAGCTATACGTGTT
TGGACAAACAACTTTGTAGCACAAACTGATGGAACCTATAAAGCAACTTTGACCATT
TCTACAAACGATAGTCCAAATTTCAATACTATAGCTACAGATGCTGCTGATAGTGTA
GTAACAAATATGATTCTATTTGTTGGTTCAAATTCAGATAATATTTCTTTAGACAATA
TAAAGTTTACTAAATAAGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTT
GGCTGCTGC CAC CGCTGAGCAATAACTAGCATAAC CC CTTGGGGCCTCTAA
SEQ ID NO: 8 (amino acid) for mCherry-CBM17 (Probe 2b). The underlined text
represents the
glycine codon that link mCherry to CBM17.
MGHHHHHHGVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDIL SPQFMYGSKAYVKHPADIPDYLKLS FPEGFKWERVMNFEDGG
VVTVTQDSSLQDGEFIYKVKLRGTNFP SDGPVMQKKTMGWEASSERMYPEDGALKGEI
KQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
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RHSTGGMDELYKGLWADNELTTSGQYVRARIKGAYYATPVDPVTNQPTAPKDFSSGF
WDFNDGTTQGFGVNPDSPITAINVENANNALKISNLNSKGSNDLSEGNFWANVRISADI
WGQSINIYGDTKLTMDVIAPTPVNVSIAAIPQS STHGWGNPTRAIRVWTNNFVAQTDGT
YKATLTISTNDSPNFNTIATDAADSVVTNMILFVGSNSDNISLDNIKFTK
SEQ ID NO: 9 (nucleic acid) for mOrange2-CBM15 (Probe 3). The underlined text
represents
the glycine codon that link mOrange2 to CBM15.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGAATAACATGGC
CATCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCC
ACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCTTTCAGACC
GCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCC
CCTCATTTCACCTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGAC
TACTTCAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTACGAG
GACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCAT
CTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTGATGCAGA
AGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGTGCC
CTGAAGGGCAAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACACCTC
CGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACA
TCGTCGACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAA
CAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAA
GGGTGTCGCTGCCAGCGAGGGCAATGTTGTTATAGAGGTGGACATGGCAAATGGCT
GGAGAGGCAACGCATCAGGCAGTACCAGCCATTCCGGTATTACCTACAGTGCCGAT
GGCGTTACCTTTGCCGCACTGGGCGATGGCGTGGGCGCTGTTTTTGATATTGCCCGA
CCAACCACACTGGAAGATGCTGTGATAGCAATGGTTGTTAATGTCAGCGCTGAATTT
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AAGGCCAGTGAAGCCAACTTGCAGATATTTGCCCAGTTAAAAGAAGACTGGTCAAA
GGGCGAATGGGATTGTCTGGCGGCCAGCAGCGAACTCACTGCGGATACTGACCTAA
CCCTGACCTGCACCATTGATGAAGACGACGATAAATTCAACCAAACGGCGCGCGAT
GTGCAAGTCGGTATCCAGGCCAAGGGAACACCCGCCGGAACTATCACCATTAAAAG
CGTCACCATTACACTCGCACAGGAAGCCTATTCAGCCAATTAA
SEQ ID NO: 10 (amino acid) for mOrange2-CBM15 (Probe 3). The underlined text
represents
the glycine codon that link mOrange2 to CBM15.
MGHHHHHHGVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTAK
LKVTKGGPLPFAWDILSPHFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNYEDGG
VVTVTQDS SLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGKI
KMRLKLKDGGHYTSEVKTTYKAI(KPVQLP GAYIVDIKLD ITS HNEDYTIVEQYERAEGR
HSTGGMDELYKGVAASEGNVVIEVDMANGWRGNASGSTSHSGITYSADGVTFAALGD
GVGAVFDIARPTTLEDAVIAMVVNVSAEFKASEANLQIFAQLKEDWSKGEWDCLAASS
ELTADTDLTLTCTIDEDDDKFNQTARDVQVGIQAKGTPAGTITIKS VTITLAQEAYSAN
SEQ ID NO: 11 (nucleic acid) for eCFP-CBM27 (Probe 4). The underlined text
represents the
glycine codon that link eCFP to CBM27.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGCTGTTCACCGG
GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG
TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC
TGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGG
GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAG
TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG
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CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG
GAGTACAACTACATCAGCCACAACGTCTATATCACC GC CGACAAGCAGAAGAAC GG
CATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG
CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC
AACCACTACCTGAGCACC CAGTCC GC CCTGAGCAAAGACC CCAAC GAGAAGCGCGA
TCACATGGTC CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGAC GA
GCTGTACAAGGGTAACGAAGCACGGTACGTGCTCGCAGAGGAAGTTGATTTTTCCTC
TCCAGAAGAGGTGAAAAACTGGTGGAACAGCGGAACCTGGCAGGCAGAGTTCGGGT
CACCTGACATTGAATGGAACGGTGAGGTGGGAAATGGAGCACTGCAGCTGAACGTG
AAACTGCCCGGAAAGAGCGACTGGGAAGAAGTGAGAGTAGCAAGGAAGTTCGAAA
GACTCTCAGAATGTGAGATCCTCGAGTACGACATCTACATTCCAAACGTCGAGGGAC
TCAAGGGAAGGTTGAGGCCGTACGCGGTTCTGAACCCCGGCTGGGTGAAGATAGGC
CTCGACATGAACAACGCGAACGTGGAAAGTGCGGAGATCATCACTTTCGGCGGAAA
AGAGTACAGAAGATTCCATGTAAGAATTGAGTTCGACAGAACAGCGGGGGTGAAAG
AACTTCACATAGGAGTTGTCGGTGATCATCTGAGGTACGATGGACCGATTTTCATCG
ATAATGTGAGACTTTATAAAAGAACAGGAGGTATGTAA
SEQ ID NO: 12 (amino acid) for eCFP-CBM27 (Probe 4). The underlined text
represents the
glycine codon that link eCFP to CBM27.
MGHHHHHHGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTWGVQCF SRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYK
TRAEVKFEGDTINNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKI
RHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLS TQ SALSKDPNEKRDHMVLLEFVTA
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AGITLGMDELYKGNEARYVLAEEVDFS SPEEVKNWWNS GTWQAEFGSPDIEWNGEVG
NGALQLNVKLP GKSDWEEVRVARKFERL SECEILEYDIYIPNVEGLKGRLRPYAVLNPG
WVKIGLDMNNANVESAEHTFGGKEYRRFHVRIEFDRTAGVKELHIGVVGDHLRYDGPIF
IDNVRLYKRTGGM
SEQ ID NO: 13 (nucleic acid) for CBM3a.
ATGGGTCATCACCATCACCATCACGGTACACCGACCAAGGGAGCAACACCAACAAA
TACAGCTACGC C GACAAAATCAGCTAC GGCTAC GCC CACCAGGC CATC GGTACC GA
CAAACACACCGACAAACACACCGGCAAATACACCGGTATCAGGCAATTTGAAGGTT
GAATTCTACAACAGCAATCCTTCAGATACTACTAACTCAATCAATCCTCAGTTCAAG
GTTACTAATACCGGAAGCAGTGCAATTGATTTGTCCAAACTCACATTGAGATATTAT
TATACAGTAGACGGACAGAAAGATCAGACCTTCTGGTGTGACCATGCTGCAATAAT
CGGCAGTAACGGCAGCTACAACGGAATTACTTCAAATGTAAAAGGAACATTTGTAA
AAATGAGTTCCTCAACAAATAACGCAGACACCTACCTTGAAATTAGCTTTACAGGCG
GAACTCTTGAACCGGGTGCACATGTTCAGATACAAGGTAGATTTGCAAAGAATGAC
TGGAGTAACTATACACAGTCAAATGACTACTCATTCAAGTCTGCTTCACAGTTTGTT
GAATGGGATCAGGTAACAGCATACTTGAACGGTGTTCTTGTATGGGGTAAAGAATA
A
SEQ ID NO: 14 (amino acid) for CBM3a.
MGHHHHHHGTPTKGATPTNTATPTKSATATPTRP SVPTNTPTNTPANTPVSGNLKVEFY
N SNP SDTTNSINPQFKVTNTGS S AID L SKLTLRYYYTVDGQKDQTF WCDHAAIIGSNGS Y
NGITSNVKGTFVKMS S S TNNAD TYLEI S F TG GTLEP GAHV Q IQ GRFAKND W SNYTQ SND
YSFKSASQFVEWDQVTAYLNGVLVWGKE
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SEQ ID NO: 15 (nucleic acid) for CBM4-1.
ATGGGTCATCACCATCACCATCACGGTGCGTCGCCGATCGGGGAGGGAACGTTCGA
CGACGGGCCCGAGGGGTGGGTCGCGTACGGCACCGACGGCCCCCTCGACACGAGCA
CGGGC GCGCTGTGC GTC GC CGTGC CGGCCGGCTCCGC GCAGTAC GGC GTC GGCGTC
GTGCTCAACGGCGTCGCGATC GAGGAAGGGAC CAC CTACACGCTC C GGTACACCGC
GACGGCCTCGACCGACGTCACCGTGCGGGCGCTCGTCGGGCAGAACGGCGCCCCCT
AC GGCAC CGTGCTC GACACGAGCC C GGCCCTGACGTC C GAGCCGCGGCAGGTGACC
GAGACGTTCACGGC CTCGGCGACGTACCCCGCGACACC CGCC GC CGACGAC CC C GA
GGGGCAGATCGCCTTCCAGCTCGGCGGGTTCAGCGCCGACGCGTGGACGTTCTGCCT
CGACGACGTCGCGCTCGACTCCGAGGTCGAGCTCTAA
SEQ ID NO: 16 (amino acid) for CBM4-1.
MGHHHHHHGASPIGEGTFDDGPEGWVAYGTDGPLDTSTGALCVAVPAGSAQYGVGVV
LNGVAIEEGTTYTLRYTATASTDVTVRALVGQNGAPYGTVLDTSPALTSEPRQVTETFT
ASATYPATPAADDPEGQIAFQLGGFSADAWTFCLDDVALDSEVEL
SEQ ID NO: 17 (nucleic acid) for CBM17.
ATGGGTCATCAC CATCACCATCACGGTTTATGGGCAGATAATGAATTAAC CACTTCA
GGTCAATATGTAC GTGCTC GTATTAAGGGAGCTTATTATGCTACACCAGTTGATC CT
GTAACAAACCAACCAACAGCACCGAAAGACTTTTCTTCAGGCTTTTGGGACTTTAAT
GACGGTACTACACAAGGTTTTGGTGTAAATCCAGATAGTCCAATAACTGCTATTAAT
GTTGAAAATGCTAACAATGCTTTAAAAATCAGCAATCTTAATAGTAAGGGTAGTAAT
GATTTATCTGAAGGAAACTTTTGGGCCAATGTCCGCATTTCAGCTGATATCTGGGGA
CAAAGTATAAATATATATGGAGACACAAAACTAACAATGGATGTTATAGCTCCAAC
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ACCTGTAAATGTATCAATCGCAGCTATCCCACAAAGTAGTACTCACGGTTGGGGAAA
TCCTACAAGAGCTATACGTGTTTGGACAAACAACTTTGTAGCACAAACTGATGGAAC
CTATAAAGCAACTTTGACCATTTCTACAAACGATAGTCCAAATTTCAATACTATAGC
TACAGATGCTGCTGATAGTGTAGTAACAAATATGATTCTATTTGTTGGTTCAAATTC
AGATAATATTTCTTTAGACAATATAAAGTTTACTAAATAA
SEQ ID NO: 18 (amino acid) for CBM17.
MGHHHHHHGLWADNELTTS GQYVRARIKGAYYATPVDPVTNQPTAPKDFSSGFWDFN
DGTTQGFGVNPD SPITAINVENANNALKISNLN SKGSNDLSEGNFWANVRISADIWGQ SI
NIYGDTKLTMDVIAPTPVNVSIAAIPQS STHGWGNPTRAIRVWTNNFVAQTDGTYKATL
TISTNDSPNFNTIATDAADSVVTNMILFVGSNSDNISLDNIKFTK
SEQ ID NO: 19 (nucleic acid) for CBM15.
ATGGGTCATCACCATCACCATCACGGTGTCGCTGCCAGCGAGGGCAATGTTGTTATA
GAGGTGGACATGGCAAATGGCTGGAGAGGCAACGCATCAGGCAGTACCAGCCATTC
CGGTATTACCTACAGTGCCGATGGCGTTACCTTTGCCGCACTGGGCGATGGCGTGGG
CGCTGTTTTTGATATTGCCCGACCAACCACACTGGAAGATGCTGTGATAGCAATGGT
TGTTAATGTCAGCGCTGAATTTAAGGCCAGTGAAGCCAACTTGCAGATATTTGCCCA
GTTAAAAGAAGACTGGTCAAAGGGCGAATGGGATTGTCTGGCGGCCAGCAGCGAAC
TCACTGCGGATACTGACCTAACCCTGACCTGCACCATTGATGAAGACGACGATAAAT
TCAACCAAACGGCGCGCGATGTACAAGTCGGTATCCAGGCCAAGGGAACACCCGCC
GGAACTATCACCATTAAAAGCGTCACCATTACACTCGCACAGGAAGCCTATTCAGCC
AATTAA
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SEQ ID NO: 20 (amino acid) for CBM15.
MGHHHHHHGVAASEGNVVIEVDMANGWRGNAS GS TS H S GITYSADGVTFAALGDGV
GAVFDIARPTTLEDAVIAMVVNV SAEFKAS EANLQIFAQLKEDWSKGEWDCLAAS SELT
ADTDLTLTCTIDEDDDKFNQTARDVQVGIQAKGTPAGTITIKSVTITLAQEAYSAN
SEQ ID NO: 21 (nucleic acid) for CBM27.
ATGGGTCATCACCATCACCATCACGGTAACGAAGCACGGTACGTGCTCGCAGAGGA
AGTTGATTTTTCCTCTCCAGAAGAGGTGAAAAACTGGTGGAACAGCGGAACCTGGC
AGGCAGAGTTCGGGTCACCTGACATTGAATGGAACGGTGAGGTGGGAAATGGAGCA
CTGCAGCTGAACGTGAAACTGCCCGGAAAGAGCGACTGGGAAGAAGTGAGAGTAG
CAAGGAAGTTCGAAAGACTCTCAGAATGTGAGATCCTCGAGTACGACATCTACATTC
CAAACGTCGAGGGACTCAAGGGAAGGTTGAGGCCGTACGCGGTTCTGAACCCCGGC
TGGGTGAAGATAGGCCTCGACATGAACAACGCGAACGTGGAAAGTGCGGAGATCAT
CACTTTCGGCGGAAAAGAGTACAGAAGATTCCATGTAAGAATTGAGTTCGACAGAA
CAGCGGGGGTGAAAGAACTTCACATAGGAGTTGTCGGTGATCATCTGAGGTACGAT
GGACCGATTTTCATCGATAATGTGAGACTTTATAAAAGAACAGGAGGTATGTAA
SEQ ID NO: 22 (amino acid) for CBM27.
MGHHHHHHGNEARYVLAEEVDFSSPEEVKNWWNS GTWQAEFGSPDIEWNGEVGNGA
LQLNVKLPGKSDWEEVRVARKFERL SECEILEYDIYIPNVEGLKGRLRPYAVLNPGWVK
IGLDMNNANVESADITFGGKEYRRFHVRIEFDRTAGVKELHIGVVGDHLRYDGPIFIDN
VRLYKRTGGM
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SEQ ID NO: 23 (nucleic acid) for eGFP.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGCTGTTCACCGG
GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG
TGTCCGGCGAGGGC GAGGGCGATGC CAC CTAC GGCAAGCTGAC CCTGAAGTTCATC
TGCACCACC GGCAAGCTGC CC GTGCCCTGGCCCACC CTCGTGACCAC CCTGACCTAC
GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAG
TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG
CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG
GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG
CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG
C CGAC CACTACCAGCAGAACACC CCCATC GGCGACGGC CC C GTGCTGCTGC CCGAC
AACCACTACCTGAGCACC CAGTCC GC CCTGAGCAAAGACC CCAACGAGAAGCGCGA
TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA
GCTGTACAAGTAA
SEQ ID NO: 24 (amino acid) for eGFP.
MGHHHHHHGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYK
TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFK
IRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLS TQ SALS KDPNEKRDHMVLLEFVT
AAGITLGMDELYK
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SEQ ID NO: 25 (nucleic acid) for mCherry.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGGATAACATGGC
CATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCC
AC GAGTTCGAGATC GAGGGCGAGGGCGAGGGCCGC CCCTACGAGGGCAC CCAGAC
C GCCAAGCTGAAGGTGAC CAAGGGTGGC CC C CTGCCCTTC GCCTGGGACATCCTGTC
CC CTCAGTTCATGTACGGCTCCAAGGC CTACGTGAAGCACC CCGC CGACATCCC CGA
CTACTTGAAGCTGTC CTTCC CC GAGGGCTTCAAGTGGGAGCGC GTGATGAACTTC GA
GGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCA
TCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAG
AAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACC CC GAGGAC GGC GC
CCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGAC
GCTGAGGTCAAGACCAC CTACAAGGCCAAGAAGC CC GTGCAGCTGC CC GGCGCCTA
CAACGTCAACATCAAGTTGGACATCACCTCC CACAAC GAGGACTACAC CATC GTGG
AACAGTACGAAC GC GC C GAGGGCC GC CACTC CACC GGCGGCATGGACGAGCTGTAC
AAGTAA
SEQ ID NO: 26 (amino acid) for mCherry.
MGHHHHHHGVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDIL SPQFMYGSKAYVKHPADIPDYLKL SFPEGFKWERVMNFEDGG
VVTVTQDS SLQDGEFIYKVKLRGTNFP SDGPVMQKKTMGWEAS SERMYPEDGALKGEI
KQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
RHSTGGMDELYK
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SEQ ID NO: 27 (nucleic acid) for mOrange2.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGAATAACATGGC
CATCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCC
AC GAGTTCGAGATC GAGGGCGAGGGC GAGGGCCGC CC CTAC GAGGGCTTTCAGAC C
GCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCC
CCTCATTTCACCTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGAC
TACTTCAAGCTGTCCTTCCC CGAGGGCTTCAAGTGGGAGC GCGTGATGAACTAC GAG
GACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCAT
CTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTGATGCAGA
AGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGTGCC
CTGAAGGGCAAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACACCTC
CGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACA
TCGTCGACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAA
CAGTACGAAC GC GC CGAGGGCCGC CACTCCAC CGGC GGCATGGAC GAGCTGTACAA
GTAA
SEQ ID NO: 28 (amino acid) for mOrange2.
MGHHHHHHGVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTAK
LKVTKGGPLPFAWDIL SPHFTYGSKAYVKHPADIPDYFKL S FPEGFKWERVMNYEDGG
VVTVTQDS S LQDGEFIYKVKLRGTNFP SD GPVMQKKTMGWEAS SERMYPEDGALKGKI
KMRLKLKDGGHYTSEVKTTYKAKKPVQLP GAYIVD IKLDITSHNEDYTIVEQYERAEGR
HSTGGMDELYK
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SEQ ID NO: 29 (nucleic acid) for eCFP.
ATGGGTCATCACCATCACCATCACGGTGTGAGCAAGGGCGAGGAGCTGTTCACCGG
GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG
TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC
TGCAC CAC C GGCAAGCTGCC CGTGCC CTGGC C CAC CCTCGTGAC CACC CTGAC CTGG
GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAG
TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG
CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TC GAGCTGAAGGGCATC GACTTCAAGGAGGAC GGCAACATCCTGGGGCACAAGCTG
GAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGG
CATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG
C CGAC CACTACCAGCAGAACACC CC CATC GGC GACGGCCC CGTGCTGCTGC CCGAC
AACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA
TCACATGGTC CTGCTGGAGTTC GTGACC GCC GC CGGGATCACTCTC GGCATGGACGA
GCTGTACAAGTAA
SEQ ID NO: 30 (amino acid) for eCFP.
MGHHHHHHGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYK
TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKI
RHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLS TQSALSKDPNEKRDHMVLLEFVTA
AGITLGMDELYK
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