U.S. patent application number 14/032692 was filed with the patent office on 2014-09-04 for plant wall degradative compounds and systems.
The applicant listed for this patent is Nathan A. Ekborg, Michael Howard, Steven Wayne Hutcheson, Larry Edmund Taylor, Ronald M. Weiner. Invention is credited to Nathan A. Ekborg, Michael Howard, Steven Wayne Hutcheson, Larry Edmund Taylor, Ronald M. Weiner.
Application Number | 20140248688 14/032692 |
Document ID | / |
Family ID | 36387151 |
Filed Date | 2014-09-04 |
United States Patent
Application |
20140248688 |
Kind Code |
A1 |
Taylor; Larry Edmund ; et
al. |
September 4, 2014 |
PLANT WALL DEGRADATIVE COMPOUNDS AND SYSTEMS
Abstract
The present invention relates to cell wall degradative systems,
in particular to systems containing enzymes that bind to and/or
depolymerize cellulose. These systems have a number of
applications.
Inventors: |
Taylor; Larry Edmund;
(Lakewood, CO) ; Weiner; Ronald M.; (Potomac,
MD) ; Hutcheson; Steven Wayne; (Columbia, MD)
; Ekborg; Nathan A.; (Beverly, MA) ; Howard;
Michael; (Annapolis, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Taylor; Larry Edmund
Weiner; Ronald M.
Hutcheson; Steven Wayne
Ekborg; Nathan A.
Howard; Michael |
Lakewood
Potomac
Columbia
Beverly
Annapolis |
CO
MD
MD
MA
MD |
US
US
US
US
US |
|
|
Family ID: |
36387151 |
Appl. No.: |
14/032692 |
Filed: |
September 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13465714 |
May 7, 2012 |
8541563 |
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14032692 |
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12099653 |
Apr 8, 2008 |
8173787 |
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13465714 |
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11121154 |
May 4, 2005 |
7365180 |
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12099653 |
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60567971 |
May 4, 2004 |
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Current U.S.
Class: |
435/252.33 ;
435/320.1; 536/23.2 |
Current CPC
Class: |
C12N 9/2437 20130101;
C12N 9/248 20130101; A01N 37/46 20130101; C12Y 302/01004 20130101;
C12N 9/2488 20130101; C12N 9/2402 20130101; A01N 63/10 20200101;
C12P 19/14 20130101 |
Class at
Publication: |
435/252.33 ;
536/23.2; 435/320.1 |
International
Class: |
C12N 9/42 20060101
C12N009/42 |
Claims
1-6. (canceled)
7. A chimeric gene comprising at least one polynucleotide encoding
a polypeptide comprising an amino acid sequence of at least one of
the peptides listed in FIGS. 4-10; wherein the gene is operably
linked to regulatory sequences that allow expression of the amino
acid sequence in a host cell.
8. The chimeric gene of claim 7 contained in a host cell.
9. The chimeric gene of claim 8, wherein the host cell is an
Escherichia coli cell.
10. A vector comprising the chimeric gene of claim 7.
11. A vector comprising at least one polynucleotide encoding a
polypeptide comprising an amino acid sequence of at least one of
the peptides listed in FIGS. 4-10.
12. (canceled)
13. A host cell comprising the isolated polynucleotide of claim
7.
14. The host cell of claim 13, wherein the cell is an Escherichia
coli cell.
15-31. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 12/099,653, filed Apr. 8, 2008, which claims
priority to U.S. divisional patent application Ser. No. 11/121,154,
filed May 4, 2005, issued as U.S. Pat. No. 7,365,180, on Apr. 29,
2008, which claims priority to U.S. Provisional Application No.
60/567,971, filed May 4, 2004, the contents of which are
incorporated herein, in their entirety, by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is generally directed to degradative enzymes
and systems. In particular, the present invention is directed to
plant cell wall degrading enzymes and associated proteins found in
Microbulbifer degradans and systems containing such enzymes and/or
proteins.
[0004] 2. Background of the Invention
[0005] Cellulases and related enzymes have been utilized in food,
beer, wine, animal feeds, textile production and laundering, pulp
and paper industry, and agricultural industries. Various such uses
are described in the paper "Cellulases and related enzymes in
biotechnology" by M.K. Bhat (Biotechnical Advances 18 (2000)
355-383), the subject matter of which is hereby incorporated by
reference in its entirety.
[0006] Saccharophaagus degradans strain 2-40 (herein referred to as
"S. degradans 2-40" or "2-40") is a representative of an emerging
group of marine bacteria that degrade complex polysaccharides (CP).
S. degradans has been deposited at the American Type Culture
Collection and bears accession number ATCC 43961. S. degradans
2-40, formerly known and referred to synonomously herein as
Microbulbifer degradans strain 2-40 ("M. degradans 2-40"), is a
marine y-proteobacterium that was isolated from decaying Sparina
altemiflora, a salt marsh cord grass in the Chesapeake Bay
watershed. Consistent with its isolation from decaying plant
matter, S. degradans strain 2-40 is able to degrade many complex
polysaccharides, including cellulose, pectin, xylan, and chitin,
which are common components of the cell walls of higher plants. S.
degradans strain 2-40 is also able to depolymerize algal cell wall
components, such as agar, agarose, and laminarin, as well as
protein, starch, pullulan, and alginic acid. In addition to
degrading this plethora of polymers, S. degradans strain 2-40 can
utilize each of the polysaccharides as the sole carbon source.
Therefore, S. degradans strain 2-40 is not only an excellent model
of microbial degradation of insoluble complex polysaccharides
(ICPs) but can also be used as a paradigm for complete metabolism
of these ICPs. ICPs are polymerized saccharides that are used for
form and structure in animals and plants. They are insoluble in
water and therefore are difficult to break down.
[0007] Microbulbifer degradans strain 2-40 requires at least 1% sea
salts for growth and will tolerate salt concentrations as high as
10%. It is a highly pleomorphic, Gram-negative bacterium that is
aerobic, generally rod-shaped, and motile by means of a single
polar flagellum. Previous work has determined that 2-40 can degrade
at least 10 different carbohydrate polymers (CP), including agar,
chitin, alginic acid, carboxymethylcellulose (CMC), 6-glucan,
laminarin, pectin, pullulan, starch and xylan (Ensor, Stotz et al.
1999). In addition, it has been shown to synthesize a true
tyrosinase (Kelley, Coyne et al. 1990). 16S rDNA analysis shows
that 2-40 is a member of the gamma-subclass of the phylum
Proteobacteria, related to Microbulbifer hydrolvyicus (Gonzalez and
Weiner 2000) and to Teridinibacter sp., (Distel. Morrill et al.
2002) cellulolytic nitrogen-fixing bacteria that are symbionts of
shipworms.
[0008] The agarase, chitinase and alginase systems have been
generally characterized. Zymogram activity gels indicate that all
three systems are comprised of multiple depolymerases and multiple
lines of evidence suggest that at least some of these depolymerases
are attached to the cell surface (Stotz 1994: Whitehead 1997;
Chakravorty 1998). Activity assays reveal that the majority of 2-40
enzyme activity resides with the cell fraction during logarithmic
growth on CP, while in later growth phases the bulk of the activity
is found in the supernatant and cell-bound activity decreases
dramatically (Stotz 1994). Growth on CP is also accompanied by
dramatic alterations in cell morphology. Glucose-grown cultures of
2-40 are relatively uniform in cell size and shape, with generally
smooth and featureless cell surfaces. However, when grown on
agarose, alginate, or chitin, 2-40 cells exhibit novel surface
structures and features.
[0009] These exo- and extra-cellular structures (ES) include small
protuberances, ger bleb-like structures that appear to be released
from the cell, fine fimbrae or pili, and a network of fibril-like
appendages which may be tubules of some kind. Immunoelectron
microscopy has shown that agarases, alginases and/or chitinases are
localized in at least some types of 2-40 ES. The surface topology
and pattern of immunolocalization of 2-40 enzymes to surface
protuberances are very similar to what is seen with cellulolytic
members of the genus Clostridium.
[0010] There exists a need to identify enzyme systems that use
cellulose as a substrate, express the genes encoding the proteins
using suitable vectors, identify and isolate the amino acid
products (enzymes and non-enzymatic products), and use these
products as well as organisms containing these genes for purposes
such as those described in the Bhat paper.
SUMMARY OF THE INVENTION
[0011] One aspect of the present invention is directed to systems
of plant wallactive carbohydrases and related proteins.
[0012] A further aspect of the invention is directed to a method
for the degradation of substances comprising cellulose. The method
involves contacting the cellulose containing substances with one or
more compounds obtained from Saccharophagus degradans strain
2-40.
[0013] Another aspect of the present invention is directed to
groups of enzymes that catalyze reactions involving cellulose.
[0014] Another aspect of the present invention is directed to
polynucleotides that encode polypeptides with cellulose degrading
or cellulose binding activity.
[0015] A further aspect of the invention is directed to chimeric
genes and vectors comprising genes that encode polypeptides with
cellulose depolymerase activity.
[0016] A further aspect of the invention is directed to a method
for the identification of a nucleotide sequence encoding a
polypeptide comprising any one of the following activities from S.
degradans: cellulose depolymerase, or cellulose binding. An S.
degradans genomic library can be constructed in E. coli and
screened for the desired activity. Transformed E. coli cells with
specific activity are created and isolated.
[0017] Further aspects of the invention are directed to utilization
of the cellulose degrading substances in food, beer, wine, animal
feeds, textile production and laundering, pulp and paper industry,
and agricultural industries.
[0018] Other aspects, features, and advantages of the invention
will become apparent from the following detailed description, which
when taken in conjunction with the accompanying figures, which are
part of this disclosure, and which illustrate by way of example the
principles of this invention.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1A shows the chemical formula of cellulose;
[0020] FIG. 1B illustrates the physical structure of cellulose;
[0021] FIG. 2A illustrates the degradation of cellulose
fibrils;
[0022] FIG. 2B shows the chemical representation of cellulose
degradation tocellobiose and glucose;
[0023] FIG. 3 shows SDS-PAGE and Zymogram analysis of 2-40
culturesupernatants;
[0024] FIG. 4 lists the predicted cellulases of S. degradans 2-40
the sequences from FIGS. 4-10 are disclosed as SEQ ID) NOs 1-214,
respectively in order of appearance, 1--Acronyms, cel=cellulase,
ced=cellodextrinase, bql=6-glucosidase, cep=cellobiose/cellodextrin
phosphorylase; 2--Protein identified by tandem mass spectrometry in
supernatant concentrates. Growth substrates: av=avicel, aq=agarose,
al=alginate cm=CMC, xn=xylan: 3--MW and amino acid count calculated
using the protParam (protein parameters) tool at the Expasy website
based on the DOE/JGI gene model amino acid sequence translations
and includes any predicted signal peptide; 4--Predictions of
function and GH, GT and CBM module determination according to CAZy
ModO analysis by B. Henrissat, AFMB-CRNS; Da.sup.88ers (t) indicate
lack of a secretion signal sequence; 5-Nonstandard module
abbreviations, LPB=lipobox motif, PSL=polyserine linker.
EPR=glutamic acid-proline rich region, PLP=phospholipase-like
domain, number in parentheses indicates the length of the indicated
feature in amino acid residues; 6--Refseq accession number of gene
amino acid sequence from the Entrez protein database;
[0025] FIG. 5 lists the predicted xylanases, xylosidases and
related accessories of M. degradans 2-40;
[0026] FIG. 6 lists the predicted pectinases and related
accessories of S. degradans 2-40, 1--Acronyms, pel=pectate lyase,
pes=pectin methylesterase, rql=rhamnogalacturonan lyase; 2--MW and
amino acid count calculated using the protParam (protein
parameters) tool at the Expasy website based on the DOE/JGI gene
model amino acid sequence translations and includes any predicted
signal peptide; 3-Predictions of function and GH, GT, PL, CE and
CBM module determination according to CAZy ModO analysis by B.
Henrissat, AFMB-CRNS; 4--Module abbreviations, CE=carbohydrate
esterase, FN3=fibronectin type3-like domain, LPB=lipobox motif.
PL=pectate lyase, PSR=polyserine region, EPR=glutamic acid-proline
rich region, number in parentheses indicates the length of the
indicated feature in amino acid residues; 5--Refseq accession
number of gene model amino acid sequence from the Entrez Pubmed
database:
[0027] FIG. 7 lists the arabinanases and arabinogalactanases of S.
degradans 2-40;
[0028] FIG. 8 lists the mannanases of S. degradans 2-40;
[0029] FIG. 9 lists the laminarinases of S. Degradans 2-40,
Superscripts: 1-Acronyms, lam=laminarinase; 2--MW and amino acid
count calculated using the protParam (protein parameters) tool at
the Expasy website based on the DOE/JGI gene model amino acid
sequence translations and includes any predicted signal peptide;
3-Predictions of function and GH, GT, PL, CE and CBM module
determination according to CAZy Mod( ) analysis by B. Henrissat,
AFMB-CRNS; 4--Module abbreviations: TSP3=thrombospondin type3
repeats, COG3488=thiol-oxidoreductase like domain of unknown
function (Interestingly, a similar domain is found in cbm32A: see
table 7), PSD=polyserine domain. TMR=predicted transmembrane
region, FN3=tibronectin type3 like domain, EPR=qlutamic
acid-proline rich region, CADG=cadherin-like calcium binding motif,
number in parentheses indicates the length of the indicated feature
in amino acid residues; 5--Refseq accession number can be used to
retrieve the gene model amino acid sequence from the Entrez Pubmed
database:
[0030] FIG. 10 lists selected carbohydrate-binding module proteins
of S. degradans 2-40; and
[0031] FIG. 11 lists the recombinant proteins of S. degradans 2-40
and a comparison of predicted vs. observed molecular weights
thereof.
DETAILED DESCRIPTION
[0032] Analysis of the genome sequence of S. degradans 2-40 reveals
an abundance of genes coding for enzymes that are predicted to
degrade plant-derived carbohydrates. To date, 2-40 is the only
sequenced marine bacterium with apparently complete cellulase and
xylanase systems, as well as a number of other systems containing
plant-wall active carbohydrases.
[0033] Thus it appears that 2-40 can play a significant role in the
marine carbon cycle, functioning as a "super-degrader" that
mediates the breakdown of CP from various algal, plantal, and
invertebrate sources. The remarkable enzymatic diversity, novel
surface features (ES), and the apparent localization of
carbohydrases to ES make S. degradans 2-40 an intriguing organism
in which to study the cell biology of CP metabolism and surface
enzyme attachment.
[0034] It has now been discovered that 2-40 has a complete
complement of enzymes, suitably positioned, to degrade plant cell
walls. This has been accomplished by the following approaches: a)
annotation and genomic analysis of 2-40 plant-wall active enzyme
systems, b) identification of enzymes and other proteins which
contain domains or motifs that may be involved in surface enzyme
display, c) the development of testable models based on identified
protein motifs, and d) cloning and expression of selected proteins
for the production of antibody probes to allow testing of proposed
models of surface enzyme display using immunoelectron
microscopy.
[0035] These efforts have been greatly facilitated by the recent
sequencing of the genome of 2-40, allowing a strategy where genes
which code for proteins with potential involvement in surface
attachment may be identified based on sequence homology with
modules or domains known to function in surface attachment and/or
adhesion.
[0036] Enzymatic and non-enzymatic ORFs with compelling sequence
elements are identified using BLAST and other amino acid sequence
alignment and analysis tools. Genes of interest can be cloned into
E. coli, expressed with in-frame polyhistidine affinity tag fusions
and purified by nickel ion chromatography, thus providing the means
of identifying and producing recombinant 2-40 proteins for study
and antibody probe production.
[0037] The genome sequence of 2-40 was recently obtained in
conjunction with the Department of Energy's Joint Genome Initiative
(JGI). The finished draft sequence dated Jan. 19, 2005 comprises
5.1 Mbp contained in a single contiguous sequence. Automated
annotation of open reading frames (ORFs) was performed by the
computational genomics division of the Oak Ridge National
Laboratory (ORNL), and the annotated sequence is available on the
World Wide Web.
[0038] The initial genome annotation has revealed a variety of
carbohydrases, including a number of agarases, alginases and
chitinases. Remarkably, the genome also contains an abundance of
enzymes with predicted roles in the degradation of plant cell wall
polymers, including a number of ORFs with homology to cellulases,
xylanases, pectinases, and other glucanases and glucosidases. In
all, over 180 open reading frames with a probable role in
carbohydrate catabolism were identified in the draft genome.
[0039] To begin to define the cellulase, xylanase and pectinase
systems of 2-40, genes were initially classified as belonging to
one of those systems by BLAST homology. Ambiguous ORFs were
tentatively assigned to the class of the best known hit. Other
tools used to refine this tentative classification include Pfam
(Protein families database of alignments and HMMs and SMART (Simple
Modular Architecture Research Tool) which use multiple alignments
and hidden Markov models (statistical models of sequence consensus
homology) to identify discreet modular domains within a protein
sequence. These analyses were relatively successful; however, a
number of ORFs remained difficult to classify based on sequence
homology alone.
[0040] Enzymes have traditionally been classified by substrate
specificity and reaction products. In the pre-genomic era, function
was regarded as the most amenable (and perhaps most useful) basis
for comparing enzymes and assays for various enzymatic activities
have been well-developed for many years, resulting in the familiar
EC classification scheme. Cellulases and other O-Glycosyl
hydrolases, which act upon glycosidic bonds between two
carbohydrate moieties (or a carbohydrate and non-carbohydrate
moiety--as occurs in nitrophenol-glycoside derivatives) are
designated as EC 3.2.1.--, with the final number indicating the
exact type of bond cleaved. According to this scheme an endo-acting
cellulase (1,4-8-endoglucanase) is designated EC 3.2.1.4.
[0041] With the advent of widespread genome sequencing projects and
the ease of determining the nucleotide sequence of cloned genes,
ever-increasing amounts of sequence data have facilitated analyses
and comparison of related genes and proteins on an unprecedented
scale. This is particularly true for carbohydrases; it has become
clear that classification of such enzymes according to reaction
specificity, as is seen in the E.C. nomenclature scheme, is limited
by the inability to convey sequence similarity. Additionally, a
growing number of carbohydrases have been crystallized and their
3-D structures solved.
[0042] One of the major revelations of carbohydrase sequence and
structure analyses is that there are discreet families of enzymes
with related sequence, which contain conserved three-dimensional
folds that can be predicted based on their amino acid sequence.
Further, it has been shown that enzymes with the same
three-dimensional fold exhibit the same stereospecificity of
hydrolysis, even when they catalyze different reactions (Henrissat,
Teeri et al. 1998: Coutinho and Henrissat 1999).
[0043] These findings form the basis of a sequence-based
classification of carbohydrase modules which is available in the
form of an internet database, the Carbohydrate-Active enZYme server
(CAZy)(Coutinho and Henrissat 1999; Coutinho and Henrissat
1999).
[0044] CAZy defines four major classes of carbohydrases, based on
the type of reaction catalyzed: Glycosyl Hydrolases (GH's),
Glycosyltansferases (GT's), Polysaccharide Lyases (PL's), and
Carbohydrate Esterases (CE's). GH's cleave glycosidic bonds through
hydrolysis. This class includes many familiar polysaccharidases
such as cellulases, xylanases, and agarases. GT's generally
function in polysaccharide synthesis, catalyzing the formation of
new glycosidic bonds through the transfer of a sugar molecule from
an activated carrier molecule, such as uridine diphosphate (UDP),
to an acceptor molecule. While GT's often function in biosynthesis,
there are examples where the mechanism is exploited for bond
cleavage, as occurs in the phosphorolytic cleavage of cellobiose
and cellodextrins (Lou, Dawson et al. 1996). PL's use a
6-elimination mechanism to mediate bond cleavage and are commonly
involved in alginate and pectin depolymerization. CE's generally
act as deacetylases on O- or N--substituted polysaccharides. Common
examples include xylan and chitin deacetylases. Sequence-based
families are designated by number within each class, as is seen
with GH5: glycosyl hydrolase family 5. Members of GHS hydrolyze
6-1,4 bonds in a retaining fashion, using a double-displacement
mechanism which results in retention of the original bond
stereospecificity. Retention or inversion of anomeric configuration
is a general characteristic of a given GH family (Henrissat and
Bairoch 1993; Coutinho and Henrissat 1999). Many examples of
endocellulases, xylanases and mannanases belonging to GHS have been
reported, illustrating the variety of substrate specificity
possible within a GH family. Also, GH5s are predominantly
endohydrolases--cleaving chains of their respective substrates at
random locations internal to the polymer chains. While true for
GH5, this generalization does not hold for many other GH families.
In addition to carbohydrases, the CAZy server defines numerous
families of Carbohydrate Binding Modules (CBM). As with catalytic
modules. CBM families are designated based on amino acid sequence
similarity and conserved three-dimensional folds.
[0045] The CAZyme structural families have been incorporated into a
new classification and nomenclature scheme, developed by Bernard
Henrissat and colleagues (Henrissat. Teeri et al. 1998).
Traditional gene/protein nomenclature assigns an acronym indicating
general function and order of discovery; in this scheme an
organism's cellulose genes are designated celA, celB, etc.,
regardless of their actual mechanism of action on cellulose. Some
researchers have attempted to convey more information by naming
cellulases as endoglucanases (engA, engB) or cellobiohydrolases
(cbhA, cbhB), however this requires determination of function in
vitro and still fails to convey relatedness of protein sequence and
structure. CAZyme nomenclature retains the familiar acronym to
indicate the functional system a gene belongs to and incorporates
the family number designation. Capital letters after the family
number indicate the order of report within a given organism system.
An example is provided by two endoglucanases, CenA and CenB, of
Cellulomonas fimi. In the old nomenclature nothing can be deduced
from the names except order of discovery.
[0046] Naming them Cel6A and Cel9A, respectively, makes it
immediately clear that these two cellulases are unrelated in
sequence, and so belong to different GH families (where Cel stands
for cellulase, and 9 for glycosyl hydrolase family nine). While
this scheme does not distinguish between endo and exo--activity,
these designations are not absolute and can be included in
discussion of an enzyme when relevant (i.e. the cellobiohydrolase
Cel6A, the endoxylanase Xyn 10B). Catalytic modules take precedence
in naming carbohydrases; since many (or even most) carbohydrases
contain at least one CBM, they are named for their enzymatic
module. If more than one catalytic domain is present, they are
named in order from N-terminus to C-terminus, i.e. cel9A-cel48A
contains a GH9 at the amino-terminus and a GH48 at the
carboxyterminus. Both domains act against cellulose. There are,
however, many examples of CBM modules occurring on proteins with no
predicted carbohydrase module. In the absence of some other
predicted functional domain (like a protease) these proteins are
named for the CBM module family. If there are multiple CBM families
present, then naming is again from amino to carboxy end, i.e.
cbm2D-cbm 10 A (Henrissat, Teeri et al. 1998). This nomenclature
has been widely accepted and will be used in the naming of all 2-40
plant-wall active carbohydrases and related proteins considered as
part of this study.
[0047] The cell walls of higher plants are comprised of a variety
of carbohydrate polymer (CP) components. These CP interact through
covalent and non-covalent means, providing the structural integrity
plants required to form rigid cell walls and resist turgor
pressure. The major CP found in plants is cellulose, which forms
the structural backbone of the cell wall. See FIG. 1A. During
cellulose biosynthesis, chains of poly-13-1,4-D-glucose self
associate through hydrogen bonding and hydrophobic interactions to
form cellulose microfibrils which further self-associate to form
larger fibrils. Cellulose microfibrils are somewhat irregular and
contain regions of varying crystallinity. The degree of
crystallinity of cellulose fibrils depends on how tightly ordered
the hydrogen bonding is between its component cellulose chains.
Areas with less-ordered bonding, and therefore more accessible
glucose chains, are referred to as amorphous regions (FIG. 1B). The
relative crystallinity and fibril diameter are characteristic of
the biological source of the cellulose (Beguin and Aubert 1994;
Tomme, Warren et al. 1995; Lynd, Weimer et al. 2002). The
irregularity of cellulose fibrils results in a great variety of
altered bond angles and steric effects which hinder enzymatic
access and subsequent degradation.
[0048] The general model for cellulose depolymerization to glucose
involves a minimum of three distinct enzymatic activities (See
FIGS. 2A and 2B). Endoglucanases cleave cellulose chains internally
to generate shorter chains and increase the number of accessible
ends, which are acted upon by exoglucanases. These exoglucanases
are specific for either reducing ends or non-reducing ends and
frequently liberate cellobiose, the dimer of cellulose
(cellobiohydrolases). The accumulating cellobiose is cleaved to
glucose by cellobiases ((3-1,4-glucosidases). In many systems an
additional type of enzyme is present: cellodextrinases are
6-1,4-glucosidases which cleave glucose monomers from cellulose
oligomers, but not from cellobiose. Because of the variable
crystallinity and structural complexity of cellulose, and the
enzymatic activities required for is degradation, organisms with
"complete" cellulase systems synthesize a variety of endo and/or
exo-acting 6-1,4-glucanases.
[0049] For example, Cellulomonas fimi and Thermomonospora fusca
have each been shown to synthesize six cellulases while Clostridium
thermocellum has as many as 15 or more (Tomme, Warren et al. 1995).
Presumably, the variations in the shape of the substrate-binding
pockets and/or active sites of these numerous cellulases facilitate
complete cellulose degradation (Warren 1996). Organisms with
complete cellulase systems are believed to be capable of
efficiently using plant biomass as a carbon and energy source while
mediating cellulose degradation. The ecological and evolutionary
role of incomplete cellulose systems is less clear, although it is
believed that many of these function as members of consortia (such
as ruminal communities) which may collectively achieve total or
near-total cellulose hydrolysis (Ljungdahl and Eriksson 1985;
Tomme, Warren et al. 1995).
[0050] In the plant cell wall, microfibrils of cellulose are
embedded in a matrix of hemicelluloses (including xylans, arabinans
and mannans), pectins (galacturonans and galactans), and various
6-1,3 and 6-1,4 glucans. These matrix polymers are often
substituted with arabinose, galactose and/or xylose residues,
yielding arabinoxylans, galactomannans and xyloglucans--to name a
few (Tomme, Warren et al. 1995; Warren 1996; Kosugi, Murashima et
al. 2002: Lynd, Weimer et al. 2002). The complexity and sheer
number of different glycosyl bonds presented by these
non-cellulosic CP requires specific enzyme systems which often
rival cellulase systems in enzyme count and complexity. Because of
its heterogeneity, plant cell wall degradation often requires
consortia of microorganisms (Ljungdahl and Eriksson 1985; Tomme,
Warren et al. 1995).
[0051] Objectives--M degradans synthesizes complete multi-enzyme
systems that degrade the major structural polymers of plant cell
walls. A) define cellulase and xylanase systems, determining the
activities of genes for which function cannot be predicted by
sequence homology; and B) genomic identification and annotation of
other plant-degrading enzyme systems by sequence homology (i.e.
pectinases, laminarinases, etc.).
[0052] Experimental Results
[0053] I: Genomic, Proteomic and Functional Analyses of 2-40
Plant-Wall Activeenzymes
[0054] From the ORNL annotation it is clear that the 2-40 genome
contains numerous enzymes with predicted activity against plant
cell wall polymers. This is particularly surprising since 2-40 is
an estuarine bacterium with several complex enzyme systems that
degrade common marine polysaccharides such as agar; alginate, and
chitin. Defining multienzyme systems based on automated annotations
is complicated by the presence of poorly conserved domains and/or
novel combinations of domains. There are many examples of this in
the plant-wall active enzymes of 2-40. Accordingly, the ORNL
annotations of carbohydrase ORFs were manually reviewed with
emphasis on the modular composition and then assigned to general
groups based on the substrate they were likely to be involved with
(i.e. cellulose or xylan degradation). These genomic sequence
analyses resulted in a pool of about 25 potential cellulases, 11
xylanases and 17 pectinases.
[0055] When sequence homology is well-conserved, highly accurate
predictions of function are possible. Therefore, to verify the
presence of functioning cellulase and xylanase systems in M
degradans, zymograms and enzyme activity assays were performed as
discussed below. Also, attempts were made to identify enzymes from
2-40 culture supernatants using Mass Spectrometry based
proteomics.
[0056] Next, more sophisticated genomic analyses were used to
predict function where possible and to identify ORFs which require
functional characterization to determine their roles, if any, in
the cellulase and xylanase systems. ORFs which belong to other
plant wall-active enzyme systems were tentatively classified based
on the sequence analyses and functional predictions of B.
Henrissat.
[0057] To gain insight into the induction and expression of 2-40
cellulases and xylanases, specific activities were determined for
avicel and xylan-grown cells and supernatants by dinitrosalicylic
acid reducing-sugar assays (DNSA assays), as discussed in the
Experimental Protocols section at the end of this proposal.
Xylanase activity was measured for avicel-grown cultures, and vice
versa, in order to investigate possible co-induction of activity by
these two substrates which occur together in the plant cell
wall.
[0058] Growth on either avicel or xylan yields enzymatic activity
against both substrates, suggesting co-induction of the cellulase
and xylanase systems. As with other 2-40 carbohydrase systems,
highest levels of activity were induced by the homologous
substrate. The results also reveal some key differences in the
expression of these two systems. When grown on avicel, cellulase
activity is cell-associated in early growth and accumulates
significantly in late-stage supernatants. Cell and supernatant
fractions exhibit low levels of xylanase activity that remain
roughly equal throughout all growth phases. In contrast,
xylan-grown cultures exhibit the majority of xylanase and cellulase
activity in the cellular fraction throughout the growth cycle.
Cellulase activity does not accumulate in the supernatant and
xylanase activity accumulates modestly, but still remains below the
cell-bound activity.
[0059] Enzyme activity gels (zymograms) of avicel and xylan grown
cell pellets and culture supernatants were analyzed to visualize
and identify expressed cellulases and xylanases. The zymograms
revealed five xylanolytic bands in xylan-grown supernatants (FIG.
3), four of which correspond well with the calculated MW of
predicted xylanases (xyl/arb43G-xyn1 OD: 129.6 kDa, xyn10 E: 75.2
kDa, xyn 1 OC: 42.3 kDa, and xyn11A: 30.4 kDa: see Table 2).
Avicel-grown cultures showed eight active bands with MWs ranging
from 30-150 kDa in CMC zymograms. CMC is generally able substrate
for endocellulase activity. These zymograms clearly demonstrate
that 2-40 synthesizes a number of endocellulases of varied size
during growth on avicel--indicative of a functioning multienzyme
cellulase system. Together, the CMC and xylan zymograms confirm the
results of the genomic analyses and the inducible expression of
multienzyme cellulase and xylanase systems in M degradans 2-40.
[0060] To identify individual cellulases and xylanases produced
during growth on CP, culture supernatants were subjected to
proteomic analysis using reversed-phase high-performance liquid
chromatography (RP-HPLC) coupled with tandem Mass Spectrometry
(MS/MS). The power resulting from separating the peptides on the
RPHPLC column prior to electrospray ionization and MS/MS analysis
allows the identification of a great number of proteins from
complex samples (Smith, Loo et al. 1990; Shevchenko, Wilm et al.
1996; Jonsson. Aissouni et al. 2001). These analyses confidently
identified over 100 different non-enzymatic proteins and a number
of carbohydrases, including a xylanase, two xylosidases, a
cellulase, and two cellodextrinases. An agarase was identified
during additional analyses of agarose-grown supernatant.
[0061] Gel-slice digestion, extraction, and MS/MS analyses
performed at the Stanford University Mass Spectrometry facility
identified two annotated cellulases from an avicel-grown
supernatant sample. One, designated cel5H, has a predicted MW of 67
kDa and was identified from a band with an apparent MW of 75 kDa.
The other, cel9B, has a predicted MW of 89 kDa, but an apparent MW
of 120 kDa. The discrepancy between the predicted and apparent MW
of cel9B is consistent with similar instances where certain 2-40
proteins, cloned and expressed in E coli, exhibit apparent MWs
which are 30-40% higher than their predicted MW.
[0062] The amino acid translations of all gene models in the 2-40
draft genome were analyzed on the CAZy ModO (Carbohydrase Active
enZyme Modular Organization) server at AFMB-CRNS. This analysis
identified all gene models that contain a catalytic module (GH, GT,
PL, or CE) and/or a CBM. In all, the genome contains 222 gene
models containing CAZy domains, most of which have modular
architecture. Of these, 117 contain a GH module, 39 have GTs, 29
PLs, and 17 CE. Many of these carry one or more CBM from various
families, that contain a CBM but no predicted carbohydrase
domain.
[0063] Detailed comparisons of 2-40 module sequences to those in
the ModO database allowed specific predictions of function for
modules where the sequence of the active site is highly conserved.
For example, Cel9B (from the gel slice MS/MS) contains a GH9 module
which is predicted to function as an endocellulase, a CBM2 and a
CBM 10 module.
[0064] When catalytic module sequences are less conserved, only a
general mechanism can be predicted. This is the case with gly5M
which contains a GH5 predicted to be either a 1,3 or 1,4
glucanase--sequence analysis cannot be certain which, and so the
acronym designation "gly" for glycanase.
[0065] The results of this detailed evaluation and analysis were
used to assign genes to cellulase, xylanase, pectinase,
laminarinase, arabinanase and mannanase systems. Each system was
also assigned the relevant accessory enzymes, i.e. cellobiases
belong to the cellulase system and xylosidases belong to the
xylanase system. Genes with less-conserved GH modules which have
the most potential to function as cellulases, xylanases or
accessories were identified and designated as needing demonstration
of function.
[0066] The results of the ORNL annotation, follow-up annotation
analyses, proteomic (mass spectrometry) analyses, CAZyme modular
analyses and functional predictions have been incorporated into
FIGS. 4-11, which contain tables that summarize the predicted plant
wall active carbohydrases and selected CBM only genes of 2-40.
[0067] The genes chosen for cloning and functional analysis include
the carbohydrases gly3C, gly5K, gly5M, gly9C, and gly43M. Because
the active site of gly5L is highly homologous to that of gly5K, its
activity is inferred from the results obtained from gly5K. Four of
the 20 "CBM only" proteins, cbm2A, cbm2B, cbm2C and cbm2D-cbm10A
are included in activity assays to investigate their predicted lack
of enzymatic function. These four contain CBM2 modules that are
predicted to bind to crystalline cellulose. This predicted affinity
is the reason for their inclusion in activity assays; those
proteins that bind to cellulose are most likely to contain
cellulase or xylanase modules which were not detected by sequence
analysis. With CBM only proteins, a lack of detected enzyme
activity will confirm the absence of a catalytic domain (CD).
[0068] In order to define the complete cellulase and xylanase
systems of M degradans, those enzymes which may belong to the
systems but cannot be confidently assigned based on sequence
homology will be expressed, purified and assayed for activity as
described in the Experimental Protocols. To date, gly3C, gly5K,
gly5M, gly9C and gly43M, as well as cbm2A, cbm2B, cbm2C and
cbm2D-cbm10A, have been cloned into expression strains as pETBlue2
(Novagen) constructs. This vector places expression under the
control an inducible T7 lac promoter and incorporates a C-terminal
6.times.Histidine tag, allowing purification of the recombinant
protein by nickel ion affinity. Successful cloning and expression
of these proteins was confirmed by western blots using
a-HisTag.RTM. monoclonal antibody (Novagen). All expressed proteins
have apparent MWs which are close to, or larger, than their
predicted MW (Table 8) except for Cbm2DCbm10A which appears to be
unstable; two separate attempts to clone and express this protein
have resulted in HisTag.RTM. containing bands which occur near the
dye front in western blots, suggesting proteolytic degradation of
this gene product. An additional enzyme, Cel5A, has been cloned and
expressed for use as an endocellulase positive control in activity
assays. Cel5A has a predicted MW of 129 kDa, contains two GH5
modules, and is highly active in HE-cellulose zymograms.
[0069] The major criteria for assigning function will be the
substrate acted upon, and the type of activity detected. As such,
the various enzyme activity assays will focus on providing a
qualitative demonstration of function rather than on rigorously
quantifying relative activity levels. The assays required are
dictated by the substrate being tested, and are discussed in more
detail in Experimental Protocols. For cellulose it is important to
distinguish between 13-1,4-endoglucanase (endocellulase),
13-1,4-exoglucanase (cellobiohydrolase), and 13-1,4-glucosidase
(cellobiase) activities. This will be accomplished using zymograms
to assay for endocellulase, DNSA reducing-sugar assays for
cellobiohydrolase, and p-nitrophenol-13-1,4-cellobioside
(pnp-cellobiose) for cellobiase activity. The combined results from
all three assays will allow definition of function as follows: a
positive zymogram indicates endocellulase activity, a negative
zymogram combined with a positive DNSA assay and a negative
pnp-cellobiose assay indicates an exocellulase, while a negative
zymogram and DNSA with a positive pnp-cellobiose result will imply
that the enzyme is a cellobiase.
[0070] Xylanase (13-1,4-xylanase), laminarinase (13-1,3-glucanase),
and mixed glucanase (13-1,3(4)-glucanase) activity will be
determined by xylan, laminarin and barley glucan zymograms,
respectively. Unlike cellulose, there do not appear to be any
reports of "xylobiohydrolases" or other exo-acting enzymes which
specifically cleave dimers from these substrates. Thus zymograms
will suffice for demonstrating depolymerase (endo) activity and
pnp-derivatives will detect monosaccharide (exo) cleavage. The
pnp-derivatives used in this study will include
pnp-a-L-arabinofuranoside,-a-L-arabinopyranoside,-13-L-arabinopyranoside,-
-P-D-cellobioside, -a-D-xylopyranoside and -p-D-xylopyranoside.
These substrates were chosen based on the possible activities of
the domains in question. The assays will allow determination of
function for any a- and p-arabinosidases, P-cellobiases,
P-xyiosidases, bifunctional aarabinosidase/p-xylosidascs, and
a-xylosidases--which cleave a-linked xylose substituents from
xyloglucans. The pnp-derivative assays will be run in 96-well
microtiter plates using a standard curve of p-nitrophenol
concentrations, as discussed in Experimental Protocols.
[0071] The combination of assays for 13-1,4-, P-1,3-, and
13-1,3(4)-glucanase activities, as well as for 13-1,4-xylanase and
the various exo-glycosidase activities should clearly resolve the
function of the ambiguous carbohydrases. Proteins with demonstrated
activity will be assigned to the appropriate enzyme system.
[0072] Experimental Protocols
[0073] Zymograms
[0074] All activity gels were prepared as standard SDS-PAGE gels
with the appropriate CP substrate incorporated directly into the
separating gel. Zymograms are cast with 8% polyacrylamide
concentration and the substrate dissolved in dH.sub.2O and/or gel
buffer solution to give a final concentration of 0.1%
(HE-cellulose), 0.15% (barley 13-glucan), or 0.2% (xylan). Gels are
run under discontinuous conditions according to the procedure of
Laemmli (Laemmli 1970) with the exception of an 8 minute treatment
at 95.degree. C. in sample buffer containing a final concentration
of 2% SDS and 100 mM dithiothreitol (DTT). After electrophoresis,
gels are incubated at room temperature for 1 hour in 80 ml of a
renaturing buffer of 20 mM PIPES buffer pH 6.8 which contains 2.5%
Triton X-100, 2 mM DTT and 2.5 mM CaCl.sub.2. The calcium was
included to assist the refolding of potential calcium-binding
domains such as the tsp3s of Lam 16A.
[0075] After the 1 hour equilibration, gels were placed in a fresh
80 ml portion of renaturing buffer and held overnight at 4.degree.
C. with gentle rocking. The next morning gels were equilibrated in
80 ml of 20 mM PIPES pH6.8 for 1 hour at room temperature,
transferred to a clean container, covered with the minimal amount
of PIPES buffer and incubated at 37.degree. C. for 4 hours.
Following incubation gels were stained for 30 minutes with a
solution of either 0.25% Congo red in dH.sub.2O (HE-cellulose,
p-glucan and xylan) or 0.01% Toluidine blue in 7% acetic acid. Gels
were destained with 1M NaCl for Congo red and dH.sub.2O for
Toluidine blue until clear bands were visible against a stained
background.
[0076] Nelson-Somogyi Reducing-Sugar Assays
[0077] Purified proteins were assayed for activity using a
modification of the Nelson-Somogyi reducing sugar method adapted
for 96-well microtiter plates, using 50 ul reaction volumes (Green,
Clausen et al. 1989). Test substrates included avicel, CMC,
phosphoric-acid swollen cellulose (PASC), Barley glucan, laminarin,
and xylan dissolved at 1% in 20 mM PIPES pH 6.8 (Barley glucan and
laminarin. 0.5%). Barley glucan, laminarin and xylan assays were
incubated 2 hours at 37.degree. C.; avicel, CMC and PASC assays
were incubated 36 hours at 37.degree. C. Samples were assayed in
triplicate, corrected for blank values, and levels estimated from a
standard curve. Protein concentration of enzyme assay samples was
measured in triplicate using the Pierce BCA protein assay according
to the manufacture's instructions. Enzymatic activity was
calculated, with one unit (U) defined as 11.1M of reducing sugar
released/minute and reported as specific activity in U/mg
protein.
[0078] Exoglycosidase Activity Assays: Pnp-Derivatives
[0079] Purified proteins were assayed for activity against pNp
derivatives of
.alpha.-L-arabinofuranoside,-.alpha.-L-arabinopyranoside,
-.beta.-L-arabinopyranoside,-.beta.-D-cellobioside,-.alpha.-D-glucopyrano-
side,-.beta.-D-glucopyranoside,-.alpha.-D-xylopyranoside and
-.beta.-Dxylopyranoside, 25 .mu.l of enzyme solution was added to
1251 .mu.l of 5 mM substrate solution in 20 mM PIPES pH 6.8,
incubated for 30 min at 37.degree. C. and A.sub.405 was determined.
After correcting for blank reactions, readings were compared to a
p-nitrophenol standard curve and reported as specific activities in
U/mg protein, with one unit (U) defined as 1 .mu.lmol p-Np/min.
[0080] Mass Spectrometry and Proteomic Analyses
[0081] Stationary-phase supernatants from avicel, CMC, and
xylan-grown cultures were concentrated to 25.times. by centrifugal
ultrafiltration using microcon or centricon devices (Millipore).
Sample protein concentrations were determined by the BCA protein
assay. Samples were exchanged into 100 mM Tris buffer, pH 8.5,
which also contained 8M urea and 10 mM DTT. Samples were incubated
2 hours at 37.degree. C. with shaking to denature the proteins and
reduce disulfide bonds. After reduction, 1M iodoacetate was added
to a final concentration of 50 mM and the reaction was incubated 30
minutes at 25.degree. C. in the dark. This step alkylates the
reduced cysteine residues, thereby preventing reformation of
disulfide bonds. The samples are then exchanged into 50 mM Tris, 1
mM Ca01.sub.2, pH 8.5 using microcon devices. The denatured,
reduced, and alkylated sample is digested into peptide fragments
using proteomics-grade trypsin (Promega) at a 1:50 enzyme (trypsin)
to substrate (supernatant) ratio. Typical digestion reactions were
around 1501 total volume. Digestions were incubated overnight at
37.degree. C., stopped by addition of 99% formic acid to a final
concentration of 1% and analyzed by RPHPLC-MS/MS at the UMCP
College of Life Sciences CORE Mass Spectrometry facility.
[0082] Peptide fragments were loaded onto a Waters 2960 HPLC fitted
with a 12 cm microbore column containing 018 as the adsorbent and
eluted with a linear gradient of increasing acetonitrile
(CH.sub.3CN) concentration into an electrospray ionization
apparatus. The electrospray apparatus ionized and injected the
peptides into a Finnagin LCQ tandem Mass Spectrometer. Automated
operating software controlled the solvent gradient and continually
scanned the eluted peptides. The program identifies each of the
three most abundant ion species in a survey scan, isolates each of
them in the Mass Spectrometer's ion trap and fragments them by
inducing collisions with helium molecules. The resulting
sub-fragment masses are recorded for further analysis by peptide
analysis packages like SEQUEST and MASCOT. After the three subscan
and collision cycles have completed, the MS takes another survey
scan and the cycle repeats until the end of the run, usually about
three hours. The raw MS reads are used by the analysis software to
generate peptide fragment sequences, which were compared to amino
acid sequence translations of all gene models in the 2-40 draft
genome. Peptide identity matches were evaluated using accepted
thresholds of statistical significance which are specific for each
program.
[0083] Cloning and Expression of 2-40 Proteins in E coli
[0084] The basic cloning and expression system uses pETBlue2
(Novagen) as the vector, E coli DH5a (invitrogen) as the cloning
strain, and E coli BL-21(DE3) Tuner cells (Novagen) for protein
expression strain. This system allows the cloning of toxic or
otherwise difficult genes because the vector places expression
under the control of a T7 lac promoter-which is lacking in the
cloning strain DH5a, thereby abolishing even low-level expression
during plasmid screening and propagation. After the blue/white
screen, plasmids are purified from DH5a and transformed into the
expression host (Tuners). The Tuner strain has the T7 lac promoter,
allowing IPTG-inducible expression of the vector-coded protein and
lacks the Lon and Omp proteases.
[0085] The nucleotide sequences of gene models were obtained from
the DOEJGI's Microbulbifer degradans genome web server and entered
into the PrimerQuest.TM. design tool provided on Integrated DNA
Technologies web page. The design parameters were Optimum T.sub.m
60.degree. C., Optimum Primer Size 2 Ont. Optimum GC %=50, and the
product size ranges were chosen so that the primers were selected
within the first and last 100 nucleotides of each ORF in order to
clone as much of the gene as reasonably possible. The cloning and
expression vector, pETBlue2, provides a C-terminal
6.times.Histidine fusion as well as the start and stop codon for
protein expression. Thus, careful attention to the frame of the
vector and insert sequences is required when adding 5' restriction
sites to the PCR primers. The resulting "tailed primers" were
between 26 to 3 Ont long, and their sequences were verified by
"virtual cloning" analysis using the PDRAW software package. This
program allows vector and insert DNA sequences to be cut with
standard restriction enzymes and ligated together. The amino acid
translations of the resulting sequences were examined to detect any
frame shifts introduced by errors in primer design. Following this
verification, the primers were purchased from Invitrogen
(Frederick. MD).
[0086] PCR reactions contained 10 pMol of forward and reverse
primers, 111.1 of 10 mM DNTPs, 1.5.sub.111 of 100 mM MgCl.sub.2,
and 1[11 Proof Pro.RTM. Pfu Polymerase in a 50111 reaction with 0.5
pl of 2-40 genomic DNA as the template. PCRs conditions used
standard parameters for tailed primers and Pfu DNA polymerase. PCR
products were cleaned up with the QIAGEN QIAquick PCR Cleanup kit
and viewed in 0.8% agarose gels. Following cleanup and confirmation
of size. PCR products and pETBlue2 are digested with appropriate
restriction enzymes, usually Ascl and C/a/ at 37.degree. C. for 1
to 4 hours, cleaned up using the QIAquick kit, and visualized in
agarose gels. Clean digestions are ligated using T4 DNA ligase for
at least 2 hours in the dark at room temperature. Ligations are
then transformed into E coli DH5a by electroporation. Transformants
are incubated one hour at 37.degree. C. in non-selective media, and
then plated onto LB agar containing ampicillin and X-gal. As
pETBlue2 carries an Amp.sup.r gene and inserts are cloned into the
lacZ ORF, white colonies contain the insert sequence. White
colonies are picked with toothpicks and patched onto a new
LB/Amp/X-gal plate, with three of the patched colonies also being
used to inoculate 3 ml overnight broths. Plasmids are prepped from
broths which correspond to patched colonies which remained white
after overnight outgrowth. These plasmid preps are then singly
digested with an appropriate restriction enzyme and visualized by
agarose electrophoresis for size confirmation.
[0087] The plasmids are then heat-shock transformed into the Tuner
strain, which carries a chromosomal chloramphenicol resistance gene
(Cm'). The Transformants are incubated 1 hour at 37.degree. C. in
non-selective rescue medium, plated on LB agar with Amp and Cm
(Tuner medium) and incubated overnight at 37.degree. C. Any
colonies thus selected should contain the vector and insert. This
is confirmed by patching three colonies onto a Tuner medium plate
and inoculating corresponding 3 ml overnight broths. The next
morning the broths are used to inoculate 25 ml broths which are
grown to an OD.sub.600of around 0.6 (2-3 hours). At this point a 1
ml aliquot is removed from the culture, pelleted and resuspended in
1/10 volume 1.times.SDS-PAGE treatment buffer. This pre-induced
sample is frozen at -20.degree. C. for later use in western blots.
The remaining broth is then amended to 1 mM IPTG and incubated 4
hours at 37.degree. C. Induced pellet samples are collected at
hourly intervals. These samples and the pre-induced control are run
in standard SDS-PAGE gels and electroblotted onto PVDF membrane.
The membranes are then processed as western blots using a 1/5000
dilution of monoclonal mouse cc-HisTag0 primary antibodies followed
by HRP-conjugated goat a-mouse IgG secondary antibodies. Bands are
visualized colorimetrically using BioRad's Opti-4CN substrate kit.
Presence of His tagged bands in the induced samples, but not in
uninduced controls, confirms successful expression and comparison
of bands from the hourly time points are used to optimize induction
parameters in later, larger-scale purifications.
[0088] Production and Purification of Recombinant Proteins
[0089] Expression strains are grown to an OD.sub.600 of 0.6 to 0.8
in 500 ml or 1 liter broths of tuner medium. At this point a
non-induced sample is collected and the remaining culture induced
by addition of 100 mM IPTG to a final concentration of 1 mM.
Induction is carried out for four hours at 37.degree. C. or for 16
hours at 25.degree. C. Culture pellets are harvested and frozen
overnight at -20.degree. C. for storage and to aid cell lysis.
Pellets are then thawed on ice for 10 minutes and transferred to
pre-weighed falcon tubes and weighed. The cells are then rocked for
1 hour at 25.degree. C. in 4 ml of lysis buffer (8M Urea, 100 mM
NaH.sub.2PO.sub.4, 25 mM Tris, pH 8.0) per gram wet pellet weight.
The lysates are centrifuged for 30 minutes at 15,000 g to pellet
cell debris. The cleared lysate (supernatant) is pipetted into a
clean falcon tube, where 1 ml of QIAGEN 50% Nickel-NTA resin is
added for each 4 ml cleared lysate. This mixture is gently agitated
for 1 hour at room temperature to facilitate binding between the
Ni.sup.+2 ions on the resin and the His tags of the recombinant
protein. After binding, the slurry is loaded into a disposable mini
column and the flow thru (depleted lysate) is collected and saved
for later evaluation. The resin is washed twice with lysis buffer
that has been adjusted to pH 7.0; the volume of each of these
washes is equal to the original volume of cleared lysate. The flow
thru of these two washes is also saved for later analysis in
western blots to evaluate purification efficiency.
[0090] At this point the columns contain relatively purified
recombinant proteins which are immobilized by the His tags at their
C-terminus. This is an ideal situation for refolding, so the column
is moved to a 4.degree. C. room and a series of renaturation
buffers with decreasing urea concentrations are passed through the
column. The renaturation buffers contain varying amounts of urea in
25 mM Tris pH 7.4, 500 mM NaCl, and 20% glycerol. This buffer is
prepared as stock solutions containing 6M, 4M, 2M and 1M urea.
Aliquots of these can be easily mixed to obtain 5M and 3M urea
concentrations thus providing a descending series of urea
concentrations in 1M steps. One volume (the original lysate volume)
of 6M buffer is passed through the column, followed by one volume
of 5M buffer, continuing on to the 1M buffer--which is repeated
once to ensure equilibration of the column at 1M urea. At this
point the refolded proteins are eluted in 8 fractions of
1/10.sup.th original volume using 1M urea, 25 mM Tris pH 7.4, 500
mM NaCl, 20% glycerol containing 250 mM imidazole. The imidazole
disrupts the Nickel ion-His tag interaction, thereby releasing the
protein from the column.
[0091] Western blots are used to evaluate the amount of His tagged
protein in the depleted lysate, the two washes, and the eluted
fractions. If there is an abundance of recombinant protein in the
depleted lysate and/or washes it is possible to repeat the process
and "scavenge" more protein. Eluate fractions that contain the
protein of interest are pooled and then concentrated and exchanged
into storage buffer (20 mM Tris pH 7.4, 10 mM NaCl, 10% glycerol)
using centricon centrifugal ultrafiltration devices (Millipore).
The enzyme preparations are then aliquoted and frozen at
-80.degree. C. for use in activity assays.
[0092] In various embodiments of this invention, the cellulose
degrading enzymes, related proteins and systems containing thereof,
of this invention, for example including one or more enzymes or
cellulose-binding proteins, have a number of uses. Many possible
uses of the cellulases of the present invention are the same as
described for other cellulases in the paper "Cellulases and related
enzymes in biotechnology" by M. K. Bhat (Biotechnical Advances 18
(2000) 355-383), the subject matter of which is hereby incorporated
by reference in its entirety. For examples, the cellulases and
systems thereof of this invention can be utilized in food, beer,
wine, animal feeds, textile production and laundering, pulp and
paper industry, and agricultural industries.
[0093] In one embodiment, these systems can be used to degrade
cellulose to produce short chain peptides for use in medicine.
[0094] In other embodiments, these systems are used to break down
cellulose in the extraction and/or clarification of fruit and
vegetable juices, in the production and preservation of fruit
nectars and purees, in altering the texture, flavor and other
sensory properties of food, in the extraction of olive oil, in
improving the quality of bakery products, in brewing beer and
making wine, in preparing monogastic and ruminant feeds, in textile
and laundry technologies including "fading" denim material,
defribillation of lyocell, washing garments and the like, preparing
paper and pulp products, and in agricultural uses.
[0095] In some embodiments of this invention, cellulose may be used
to absorb environmental pollutants and waste spills. The cellulose
may then be degraded by the cellulase degrading systems of the
present invention. Bacteria that can metabolize environmental
pollutants and can degrade cellulose may be used in bioreactors
that degrade toxic materials. Such a bioreactor would be
advantageous since there would be no need to add additional
nutrients to maintain the bacteria--they would use cellulose as a
carbon source.
[0096] In some embodiments of this invention, cellulose degrading
enzyme systems can be supplied in dry form, in buffers, as pastes,
paints, micelles, etc. Cellulose degrading enzyme systems can also
comprise additional components such as metal ions, chelators,
detergents, organic ions, inorganic ions, additional proteins such
as biotin and albumin.
[0097] In some embodiments of this invention, the cellulose
degrading systems of this invention could be applied directly to
the cellulose material. For example, a system containing one, some
or all of the compounds listed in FIGS. 4-11 could be directly
applied to a plant or other cellulose containing item such that the
system would degrade the plant or other cellulose containing item.
As another example, 2-40 could be grown on the plant or other
cellulose containing item, which would allow the 2-40 to produce
the compounds listed in FIGS. 4-11 in order to degrade the
cellulose containing item as the 2-40 grows. An advantage of using
the 2-40 or systems of this invention is that the degradation of
the cellulose containing plant or item can be conducted in a marine
environment, for example under water.
[0098] It is one aspect of the present invention to provide a
nucleotide sequence that has a homology selected from 100%, 99%,
98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% to any of the sequences
of the compounds listed in FIGS. 4-11
[0099] The present invention also covers replacement of between 1
and 20 nucleotides of any of the sequences of the compounds listed
in FIGS. 4-11 with non-natural or non-standard nucleotides for
example phosphorothioate, deoxyinosine, deoxyuridine, isocytosine,
isoguanosine, ribonucleic acids including 2-O-methyl, and
replacement of the phosphodiester backbone with, for example, alkyl
chains, aryl groups, and protein nucleic acid (PNA).
[0100] It is another aspect of some embodiments of this invention
to provide a nucleotide sequence that hybridizes to any one of the
sequences of the compounds listed in FIGS. 4-11 under stringency
condition of 1.times.SSC, 2.times.SSC, 3.times.SSC 1, 4.times.SSC,
5.times.SSC, 6.times.SSC, 7.times.SSC, 8.times.SSC, 9.times.SSC, or
10.times.SSC.
[0101] The scope of this invention covers natural and non-natural
alleles of any one of the sequences of the compounds listed in
FIGS. 4-11. In some embodiments of this invention, alleles of any
one of any one of the sequences of the compounds listed in FIGS.
4-11 can comprise replacement of one, two, three, four, or five
naturally occurring amino acids with similarly charged, shaped,
sized, or situated amino acids (conservative substitutions). The
present invention also covers non-natural or nonstandard amino
acids for example selenocysteine, pyrrolysine, 4-hydroxyproline,
5-hydroxylysine, phosphoserine, phosphotyrosine, and the D-isomers
of the 20 standard amino acids.
[0102] It is to be understood that while the invention has been
described above using specific embodiments, the description and
examples are intended to illustrate the structural and functional
principles of the present invention and are not intended to limit
the scope of the invention. On the contrary, the present invention
is intended to encompass all modifications, alterations, and
substitutions within the spirit and scope of the appended
claims.
REFERENCES CITED
[0103] Andrykovitch, G. and I. Marx (1988). "Isolation of a new
polysaccharide-digesting bacterium from a salt marsh." Applied and
Environmental Microbiology 54: 3-4. [0104] Beguin, P. and J. P.
Aubert (1994). "The biological degradation of cellulose," FEMS
Microbiol Rev 13(1): 25-58. [0105] Chakravorty, D. (1998). Cell
Biology of Alginic Acid degradation by Marine Bacterium 240.
College Park, University of Maryland. [0106] Coutinho, P. M. and B.
Henrissat (1999). Carbohydrate-active enzyme server. Accessed Jan.
21, 2004 Coutinho, P. M. and B. Henrissat (1999). The modular
structure of cellulases and other carbohydrat-active enzymes; an
integrated database approach. Genetics, biochemistry and ecology of
cellulose degradation. T. Kimura. Tokyo, Uni Publishers Co:
15-23.
[0107] Distel. D. L., W. Morrill, et al. (2002). "Teredinibacter
turnerae gen. nov., sp. nov., a dinitrogen-fixing, cellulolytic,
endosymbiotic gamma-proteobacterium isolated from the gills of
wood-boring molluscs (Bivalvia: Teredinidae)." Int J Syst Evol
Microbiol 52(6): 2261-2269. [0108] Ensor, L., S. K. Stotz, et al.
(1999). "Expression of multiple insoluble complex polysaccharide
degrading enzyme systems by a marine bacterium." J Ind Microbiol
Biotechnol 23: 123-126. [0109] Gonzalez, J. and R. M. Weiner
(2000). "Phylogenetic characterization of marine bacterium strain
2-40, a degrader of complex polysaccharides." International journal
of systematic evolution microbiology 50: 831-834. [0110] Henrissat,
B. and A. Bairoch (1993). "New families in the classification of
glycosyl hydrolases based on amino acid sequence similarities."
Bichem J 293 (Pt 3): 781-8. [0111] Henrissat, B., T. T. Teeri, et
al. (1998). "A scheme for designating enzymes that hydrolyse the
polysaccharides in the cell walls of plants." FEBS Lett 425(2):
3524. [0112] Jonsson, A. P., Y. Aissouni, et al. (2001). "Recovery
of gel-separated proteins for in-solution digestion and mass
spectrometry." Anal Chem 73(22): 5370-7. [0113] Kelley. S. K., V.
Coyne, et al. (1990). "Identification of a tyrosinase from a
periphytic marine bacterium." FEMS Microbiol Lett 67: 275-280.
[0114] Kosugi. A., K. Murashima, et al. (2002). "Characterization
of two noncellulosomal subunits. ArfA and BgaA, from Clostridium
cellulovorans that cooperate with the cellulosome in plant cell
wall degradation." J Bacteriol 184(24): 6859-65. [0115] Laemmli, U.
K. (1970). "Cleavage of structural proteins during the assembly of
the head of the bacteriophage T4." Nature 277: 680-685. [0116]
Ljungdahl, L. G. and K. E. Eriksson (1985). Ecology of Microbial
Cellulose Degradation. Advances in Microbial Ecology. New York,
Plenum Press. 8: 237-299. [0117] Lou, J., K. Dawson, et al. (1996).
"Role of phosphorolytic cleavage in cellobiose and cellodextrin
metabolism by the ruminal bacterium Prevotella ruminicola." Apl.
Environ. Microbiol. 62(5): 1770-1773. [0118] Lynd, L. R., P. J.
Weimer, et al. (2002). "Microbial cellulose utilization:
fundamentals and biotechnology." Microbiol Mol Biol Rev 66(3):
506-77, table of contents. Shevchenko, A., [0119] M. Wilm, et al.
(1996). "Mass spectrometric sequencing of proteins silver-stained
polyacrylamide gels." Anal Chem 68(5): 850-8. [0120] Smith, R. D.,
J. A. Loo, et al. (1990). "New developments in biochemical mass
spectrometry: electrospray ionization." Anal Chem 62(9): 882-99.
[0121] Stotz, S. K. (1994). An agarase system from a periphytic
prokaryote. College Park. University of Maryland. [0122] Sumner, J.
B. and E. B. Sisler (1944). "A simple method for blood sugar."
Archives of Biochemistry 4: 333-336. [0123] Tomme, P., R. A.
Warren, et al. (1995). "Cellulose hydrolysis by bacteria and
fungi." Adv Microb Physiol 37: 1-81. [0124] Warren, R. A. (1996).
"Microbial hydrolysis of polysaccharides." Annu Rev Microbiol 50:
183-212.
[0125] Whitehead, L. (1997). Complex Polysaccharide Degrading
Enzyme Arrays Synthesized By a Marine Bacterium. College Park,
University of Maryland.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140248688A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140248688A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References