The complete hydrolysis of cellulose requires a number of different enzymes ... of cellulase genes and specifically discusses (i) strategies for the isolation of ...
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ISOLATION, CHARACTERIZATION AND MANIPULATION OF CELLULASE GENES BERNARD R. GLICK and J. J. PASTERNAK Department
of Biology,
University of Waterloo, Canada NZL 3G1
Waterloo,
Ontario,
ABSTRACT The
complete
endoglucanase,
hydrolysis
of cellulose
exoglucanase
‘cellulase’complex
requires
and B-glucosidase.
called a cellulosome. of the cellulase
a number
of different
enzymes
including
These enzymes function in concert as part of a
In order (i) to develop a better understanding
biochemical
nature
components
and (ii) to utilize cellulases either as purified enzymes or as part of an engineered
organism for a variety of purposes, technology
exoglucanase
researchers
on the current status of the isolation, characterization
genes and specifically
discusses (i) strategies
DNA
This review
and manipulation of endoglucanase,
of the cellulase genes
regulatory elements; (iii) the expression of cellulase genes in heterologous
host organisms and (iv) some of the proposed Key words:
for the isolation
and B-glucosidase genes; (ii) DNA sequence characterization
and their accompanying
of its integral
have, as a first step, used recombinant
to isolate the genes for these enzymes from a variety of organisms.
provides some perspective of cellulase
complex as well as the genetic regulation
of the
uses for isolated cellulase genes.
cellulose, cellulase, endoglucanase, recombinant
DNA technology
361
exoglucanase, cellobiohydrolase,
O-glucosidase,
362
B. R. GLICK and J. J. PASTERNAK
INTRODUCTION Cellulose,
which is the most abundant
underutilized processes
resource
polymer
and is frequently
in the biosphere,
a wasted
byproduct
is at the present
of agricultural
(64). The possibility of using cellulose as an inexpensive feedstock
and industrial
for the production
of bulk chemicals such as ethanol and acetone is an attractive; but, to date, unrealized significant
portion
enzymatically
of the recent
research
effort to meet this objective
hydrolyzing cellulose to glucose prior to converting
Initially, it was thought that mass production would be sufficient mediated
for breaking
by a single enzyme.
has been
However,
cellulase
Rather, it is a complex of several different
goal. A aimed at
it to other materials
of cellulase from microbial and/or
down cellulose.
time an
(64).
fungal sources
activity in vivo is not enzymes which act in
concert (15, 48, 49). The cellulase complex forms a unique structure which has been called a cellulosome cellulosome
and appears
to be ubiquitous
includes (i) endoglucanase
glucose molecules
their non-reducing
cellulolytic
microorganisms
(49).
The
activity which hydrolyzes b-1,4 linkages between adjacent
within the amorphous
chain in the middle; (ii) exoglucanase
among
regions of the cellulose polymer thereby breaking the
activity which degrades
ends producing glucose, cellobiose and/or
the nicked cellulose chains from cellotriose;
(iii) cellobiohydrolase,
another type of exoglucanase activity which removes larger polysaccharides
from the non-reducing
end of the cellulose molecule;
and (iv) ILglucosidase, or cellobiase, which converts cellobiose to
glucose (12, 15). The cellulase complex of different microorganisms
comprises a variable number
of different isozymes of each of the major types of cellulase enzymes. Cellulomonas and Pseudomonas each encode several different
In recent years recombinant and manipulating of recombinant understanding
For example, Clostndium,
species-specific
endoglucanases.
DNA technology has provided a means for isolating, characterizing
the genes for a large number of different proteins. DNA technology
applied
of the catalytic functioning
to the cellulase and regulation
It is expected that the use
system will facilitate
(i) a better
of these enzymes, (ii) insight into the
CELLULASE
nature
of the cooperative
complete
interactions
between
GENES
different
hydrolysis of cellulose, (iii) the development
native cellulose; viz. the conversion novel cellulolytic microorganisms other properties
363
enzymes
that are involved
of practical systems for the utilization of
of waste biomass into usable products
by the introduction
in the
and (iv) developing
of exogenous genes and thereby enabling
of the host organism to be exploited more fully.
This review deals with (i) strategies for the isolation of cellulase genes; (ii) the distinctive features of those cellulase heterologous
genes which have been isolated;
(iii) the expression
of cellulase
genes in
host organisms; and (iv) some of the uses to which the isolated genes might be put.
GENE ISOLATION STRATEGIES Since the first reported
isolation of a B-glucosidase gene from Escherichia adecarbox&ta
(1) and a Cellulomonas jimi exoglucanase
in 1981
gene in 1982 (103) there has been an explosion of
activity in the area of cellulase gene cloning such that by July, 1989 more than thirty different cellulase
genes
endoglucanase
have
been
isolated.
of the
recent
genes, without question, can be attributed
Congo Red-carboxymethylcellulose that
Much
express
endoglucanase
overlay technique
activity
(95).
With
for selecting recombinant this selection
to find the very small fraction of recombinant
endoglucanase
activity in a clone bank. E. coli are screened
carboxymethylcellulose
endoglucanase-producing
sodium
chloride.
surrounded
different
technique,
E. coli clones it is relatively
clones which express and secrete or derivatives of it, clone banks of
the bacterial
colonies
with agar containing
and then incubating the plates, usually at 37°C for several hours.
this time, the carboxymethylcellulose
carboxymethylcellulose
With this technique,
by overlaying
in isolating
to the use of the simple yet powerful
straightforward
recombinant
success
colony
molecules that are present in the immediate are
partially
digested.
To
visualize
the
During
vicinity of an digestion
of
the petri plate is flooded with a solution of Congo Red and then with If a bacterial
by a yellow
colony produces
halo; whereas,
endoglucanase,
the background
then
will be red.
the colony will be The Congo
Red-
364
B. R. GLICK and J. J. PASTERNAK
carboxymethylcellulose
procedure
has permitted
researchers
to isolate endoglucanase
genes that
are expressed in E. cob from clone banks of Streptomyces (11, 87), Clostridium (5, 17, 38, 62, 78), Thermoanaerobacter (35), Thermomonospora (22, 37), Erwinia (2, 6, 7), Pseudomonas (23, 52, 80, NM), Cellvibrio (108), Ruminococcus (3, 43, 70), Cellulomonas (67), Bacteroides (13) and Bacillus (47, 61, 75, 88, 109) species.
However,
for this strategy
synthesized
to be effective
in a heterologous
endoglucanase
gene must be both
host cell such as E. coli and capable of functioning
must be secreted
the substrate,
(i) the cloned endoglucanase
and (ii) the
either to the growth medium or to the host cell periplasm since
because of its size, cannot enter the cell.
When endoglucanase
activity remains
localized within the cytoplasm of the host, it is necessary to either partially or fully lyse the host cells to detect enzyme activity.
In these cases, replica plates are used to ensure that viable cells
are available for further use. The isolation of endoglucanase Badus
genes from some Clostrzdium and
species was facilitated by exposing the host cells to chloroform
lysozyme
(38,
109) prior
carboxymethylcellulose incompatability functional
to assaying
procedure.
in the secretion
secretion
The requirement
activity
with
the
Congo
Red-
for host cell lysis may reflect either an
signals used by the donor and host organisms
or the lack of a
signal sequence.
In addition to the Congo Red-overlay select for the expression fluorescence
for endoglucanase
vapour (17, 109) and/or
technique, other methods have been employed that directly
of cloned endoglucanase
of methylumbelliferon
genes in host bacteria.
For example, the
under UV light at 254 run which results from the hydrolysis
of either 4-methylumbelliferyl-B-D-glucoside
or cellobioside
(36, 45, 84) can be detected
the production
of either ‘shallow craters’ (18) or ‘halos’ (45, 66) surrounding
solid medium
containing
endoglucanase
aktivity.
carboxymethylcellulose
have been used to denote
and
colonies grown on the presence
of
CELLULASE
GENES
365
While most of the published work on the cloning of cellulase genes has been devoted specifically to the isolation of endoglucanase available for the identification Brown (1) isolated transforming
of cloned B-glucosidase
a gene which encodes
and
their clone bank into E. coli and then selecting for transformants
which could grow
medium
with cellobiose
activity
may
MacConkey-cellobiose
also
be
as the sole carbon selected
using in
the
a
source.
Clones
chromogenic plating
substrate
medium
solid medium in which l3-glucosidase-positive
which express such
(59)
or
as with
colonies turn red (Glick and
unpublished).
When only a portion that is synthesized protein.
For example, Armentrout
adecarboxyfata by
5-bromo-4-chloro-3-indolyl-B-D-glucopyranoside
Pastemak,
genes.
l3-glucosidase from Escherichia
on a minimal R-glucosidase
genes, there are some simple yet powerful selection schemes
of the gene for a particular
in the host microorganism
target protein has been cloned, the peptide
may not be able to express the activity of the intact
In such instances it is often possible to screen transformed
host cells for the presence
of the protein of interest using immunological
specificity as an indicator of the presence
of the target protein.
and endoglucanase
In this way exoglucanase
(103, 106), an endoglucanase
gene from Batik
of part
genes from Cellulomonasfimi
subtiZk (81) and a g-glucosidase
gene from
Schizophyllum commune (65) have been isolated.
Immunological eukaryotic
genes
transcriptional presence
detection
is an effective approach when working with eukaryotic DNA because
are unlikely
and translational
to be expressed signals between
in E. coli as a result
the donor and host organisms
of introns within eukaryotic DNA which cannot be processed
these drawbacks mRNA from the filamentous programme
the synthesis of intron-free
of (i) differences
in
and (ii) the
by E. coli. To overcome
fungus S. commune was isolated and was used to
copy DNA (cDNA) which was subsequently
cloned into
the E. coli expression vector lambda gtll (65). The S. commune B-glucosidase was produced in vivo as a fusion protein which included a portion of the vector-encoded
g-galactosidase
protein
B. R. GLICK
366
(65).
In this way it is possible
bacterial
and
J. J. PASTERNAK
to isolate and express the genes for eukaryotic
cellulases
in
hosts.
In eukaryotic systems that have cellulase activity the specific mRNAs that encode these enzymes constitute
only a small fraction of the total mRNA population.
It is often necessary to enrich for
the target mRNA and to distinguish those cDNA clones that do not carry the target sequences. To meet these ends, “differential hybridization” of different eukaryotic cellulase genes (10,76,83,
has been employed in the isolation of a number 89,98,99).
Briefly, with this approach, mRNA
is isolated from cells with normal levels of cellulase activity (Le., non-induced have been grown in the presence cellulase (Le., induced cells). of cDNA.
of cellulose or cellulose derivatives
Each mBNA fraction is used separately
The cDNA from the induced
bacteriophage
cell population
cells) and those that
to enhance
to program the synthesis
is cloned into either
vector, replica plated and then separately screened using radiolabelled
the induced and non-induced
fractions as hybridization
probes.
the levels of
a plasmid
or
cDNA from
Putative positive clones are those
that yield a signal when they are probed with cDNA from induced cells and give no signal with cDNA from non-induced
cells. Not all clones that are selected in this way will contain cellulase
cDNA, thus, the positive clones must be further characterized.
The method of hybrid mRNA
selection, ie., using the DNA from the positive clones to select mRNAs from the induced cell population,
is combined
with in vitro translation
of the selected mRNA in a rabbit reticulocyte
lysate or wheat germ extract cell-free system to identify the bona fide cellulase gene-containing clones.
The desired clones are those that select mRNA that encodes cellulase as detected
antibodies that were raised against the purified cellulase. Alternatively, libraries may be constructed
by
as indicated above, cDNA
in a vector, such as lambda gtll, which facilitates the expression of
the cDNA as part of a fusion protein (65).
Another widely employed strategy for the isolation of genes with similar functions is the use of “heterologous”
hybridization
probes.
Such probes may share some sequence
similarity with the
CELLULASE
target DNA although they are derived from a different cellulase genes show little inter-species cellulase genes from different DNA sequences
organism.
similarity and, therefore,
bacterial genera.
among plant cellulase genes.
of a 595 nucleotide
367
GENES
Unfortunately,
prokaryotic
are of little use for identifying
However, there may be more commonality
of
Recently, Tucker et al. (97) reported the isolation
long cDNA clone encoding a portion of bean abscission cellulase which was
selected by hybridization
using a full-length avocado cellulase cDNA as the probe.
The isolated
bean partial cDNA was found to be 55% identical at the amino acid level to the corresponding region from avocado.
The occurence of similar cellulase genes in two diverse plants may denote
a shared ancestral gene as well as a gene that plays an important plants.
On the other
hand, bacterial
interspecies
sequence
In addition
to preparing
and fungal cellulase
role in the life cycle of these
genes which have little or no
similarity probably have evolved independently
purified
enzymes
to elicit antibodies
of each, other.
which can be used to select
expressed genes, the amino acid sequence of a purified cellulase enzyme can be used to deduce the sequence chemically unexpressed
of the cellulase gene.
synthesized
With this information
and then used as a hybridization
an oligodeoxyribonucleotide
can be
probe to indicate the presence
of an
gene. A B-glucosidase gene from Agrobacterium has been isolated with this approach
(101).
CHARACTERIZATION
OF CELLULASE
GENES
Many of the cellulase genes that have been cloned have been characterized analysis (Table I). generalizations
On the basis of these studies and other work, at the protein
In a
the lack of sequence similarity, at
and DNA levels, among cellulases and their genes, there are certain general
features that these cellulases share. N-terminal
level, some
regarding the structure and function of cellulases are beginning to emerge.
recent review, Knowles et cd (46) noted that nothwithstanding both the protein
by DNA sequence
Cellulases appear to contain three separate
hydrolytic domain, (ii) a serine and threonine,
domains (i) an
and to a lesser extent proline, rich
366
B. R. CLICK and J. J. PASTERNAK
‘hinge’ region and (iii) a C-terminal to the substrate.
domain which is responsible
When there is sequence similarity at the protein level among cellulases within
the same genus it is generally confined to the C-terminal the lack of sequence similarity may be the consequence within
an organism
mutagenesis (‘ancestral’)
for the binding of the enzyme
selection
to create sequence
pressure
and maintain
region of the enzyme. of independent
may be sufficient intraspecific
gene
gene evolution; whereas,
to enable
families.
As noted above,
gene
duplication
The evolutionary
for cellulase genes may have been a gene(s) that encodes
and source
a pre-existing
glycolytic enzyme(s).
In addition to characterizing the promoters
that regulate
the structural portion of cellulase genes, several groups have studied cellulase
gene expression
mechanism by which these genes are regulated. AMP may be important
transcription
genes
to understand
the
For example, there are several reports that cyclic
in regulating cellulase gene expression (32, 53, 94, 105). In this regard,
a putative cyclic AMP binding site has been identified endoglucanase
and have sought
(32, 105).
A comparison
upstream
of two separate P. jluorescens
of the DNA regions
upstream
from the
initiation sites of several Cellulomonasjimi cellulase genes suggested that a common
regulatory element which overlaps the -35 regions of the promoters
for these genes might exist
(29). In T. fusca, an A-T rich region which includes part of the ceE promoter
and some upstream
DNA as well as a putative positive regulatory protein which binds adjacent to this region, has been implicated in the transcriptional understanding elaborated.
of the mechanisms
control of this gene (56,57).
of transcriptional
regulation
At the present time a detailed
of cellulase genes remains to be
CELLULASE
GENES
369
Table I. Cellulase genes that have been sequenced.
Organism
Activity
Bacillus subtilir
endoglucanase
Gene
82
endoglucanase
69
endoglucanase
86
endoglucanase
61
Bacillus sp. 1139
endoglucanase
Bacillus sp. N-4
endoglucanase
Caldocellum saccharolyticum
endoglucanase exoglucanase
18 ceL4 celB
B-glucosidase Cellulomonar jimi
uda
&
19 19 84
celB
exoglucanase endoglucanase
Cellulomon~
References
60 71
ted
80, 106
cenB
73
endoglucanase
68
Clostridium acetobutylicum
endoglucanase
111
Clostridium thermocellum
endoglucanase
celA
4
celB
30
celC
8.5
celLI
34
celE
33
celZ
31
Erwinia chlysanthemi
endoglucanase
Persea americana
endoglucanase
96
Pseudomonas jluorescens
endoglucanase
32
endoglucanase
105
Schizophyllum commune
!3-glucosidase
65
Streptomyces sp. KSM-9
endoglucanase
CUSA
66
Trichodenna reesei
cellobiohydrolase
CBH I
16, 90
CBH II
8, 98
EG I
76
EG III
83
endoglucanase
370
B. R. GLICK and J. J. PASTERNAK
EXPRESSION Although
a number
IN DIFFERENT
of studies using expression
vectors have found that the overproduction
cellulase genes occurs in E. coli, in many instances in the cytoplasm which makes its purification product.
To overcome
this problem,
facilitate the secretion
(24, 42, 58).
carrying a plasrnid which encoded
of
either all or a part of the enzyme is found
difficult thereby increasing
several groups have developed
of the overproduced
or to the cell growth medium
HOST ORGANISMS
the cost of the final
cloning protocols
enzyme either to the E. coli periplasmic Gilkes et al. (24) mutagenized
cellulase activity and selected
which
space (72)
a strain of E. coli
for a mutant that leaked the
cellulase activity from the periplasm into the growth medium.
However, this mutant leaked other
periplasmic
greater than one half of the total
enzymes into the growth medium and moreover
enzyme activity was still retained within the cells. Consequently,
this mutant is unlikely to provide
a practical means of obtaining large amounts of cellulase secreted into the growth medium by E. cob.
Another
method
for obtaining
extracellular
of cellulase by E. coli was devised by
production
a plasmid vector which carries the kil gene.
Kato et al. (42) who constructed responsible
for colicin El release and when it is expressed the outer membrane
permeable
and the enzymes
including plasmid-encoded
which would otherwise
be localized
cellulase, are found in the extracellular
provide a useful means of getting E. coli to secrete plasrnid-encoded
This gene is
E. cob becomes
in the periplasmic
medium.
space,
This approach may
proteins to the extracellular
medium.
Fortuitously,
Lo et al. (58) found that when some cloned gene products which are expressed in
E. coli contain
extracellular periplasm.
the appropriate
medium
despite
signals at the protein
the fact that in E. coli secretion
When an endoglucanase
enzyme activity (>90%)
level they can be secreted
is normally exclusively to the
gene from B. subtih was expressed
was found in the extracellular
into the
in E. coli, most of the
medium without any evidence of lysis of
CELLULASE
GENES
371
the host cells (58). A more complete understanding
of the amino acid sequences which form the
part of the foreign
for secretion
development
protein
which is responsible
in E. coli may lead to the
of vectors that contain the signal(s) for selective secretion
of the protein products
of cloned genes.
E. coli is generally used as the initial host organism for the isolation and expression of bacterial genes.
However,
Consequently, cellulase
other
microorganisms
also
have
a number of different microorganisms
genes
including
Sacchnromyces
potential
utility
have been transformed
cerevikze
as host
organisms.
with vectors carrying
(14, 77, 91, 99, 107, IlO), Pseudomonas
jluorescens (52), Bacillus subti1i.s (SO, 51, 59, 81, 93), Bacillus megaterium (44, 50, 51, 92), Bacillus stearothemtophilus combination
(93), Zymomonas mobilis (54, 63), Azotobacter vinelandii (27, 79), a fused
ofEnterococcus faecium and Furobacterium varium (9), Brevibacterium lactofennentum
(74), Rhodobacter capszdatus (40) and Streptomyces lividans (21, 87). It is important these experiments
are preliminary
and should be viewed as only a first step in the development
of a range of different cellulolytic microorganisms. some of these microorganisms
to note that
are elaborated
Some of the reasons for specifically selecting
below.
S. cerevisiae and Z. mobilis are organisms that efficiently convert simple sugars such as glucose into ethanol.
They have been used as hosts for the expression of cellulase genes.
of active cellulase genes might enable these organisms obtainable
from lignocellulosic
materials,
to alcohol.
to directly convert cellulose, which is These studies have shown that both S.
cerevisiae (14, 77, 91, 99, 107, 110) and Z. mobilis (54,63) can express cell&se the demonstration
The presence
activity. However,
that an individual cellulase gene can be expressed in either S. cerevisiae or Z.
mobilis is a necessary cellulose into alcohol.
but not sufficient step in developing A considerable
amount of additional
conversion of cellulose into glucose within a fermentative
a strain that can efficiently convert work is required
microorganism
to the scientific hurdles, it should be noted that the conversion
before efficient
is achieved.
of lignocellulosics
In addition into alcohol
B. R. GLICK
372
requires
a substantial
feedstock
before
and J. J.
PASTERNAK
input of energy for either the chemical or physical pretreatment
the cellulose
can be hydrolyzed
enzymatically.
cellulose directly to alcohol may not be economically
The ability of diazotrophic
microorganisms
limited by the supply of metabolizable diazotrophs
Thus, a process
to grow and proliferate
in the environment
carbon that is available to them.
is often
One way to provide
with a carbon supply that is sufficent to power both nitrogen fixation and cell growth
possibly, combine
should have an advantage over other soil microorganisms
broad-host-range
A cellulolytic
in its ability to proliferate
with isolated
Two different groups have recently transformed
cellulase
genes.
diazotrophic
When A. vinelumfii was transformed
plasmids carrying an isolated endoglucanase
despite the fact that the gene was stably maintained
observations).
(27, 55).
(100) or,
and thereby it should be better suited to provide some of the fixed nitrogen
which is required for plant growth. microorganisms
microorganisms
both of these activities in a single microorganism
in the environment
detected
to convert
viable.
is to either establish a close association of cellulolytic and diazotrophic
diazotroph
of the
with
gene no enzyme activity could be (Glick and Pasternak, unpublished
The basis for this lack of activity may be the metabolic load which the plasmid and
the proteins
which it encodes
imposes
experiments,
when an endoglucanase
on the transformed
cells (26, 28).
set of
gene from P. jluorescens that had been cloned into pBR322
was used to transform A. vinekmfii, the plasmid DNA became integrated although the endoglucanase
In another
into the host genome
gene sequence was not (79). Thus, the addition of cellulase genes
to A. vinelandii is, by itself, not sufficient for creating a cellulolytic diazotroph.
Transformation
of R capsulatzuwith a broad-host-range
plasmid carrying cellulase genes resulted
in expression in this host, provided that a DNA fragment containing a R capsulam promoter was appropriately
positioned
on the plasmid with respect
enzymes were not efficiently carboxymethylcel~ulose
secreted
to the cellulase
and the transformed
as a sole carbon source.
genes.
However,
the
organism was unable to grow on
In sum, engineered
cellulolytic diazotrophs
that
CELLULASE GENES
373
might be suitable for field testing as plant growth promoting developed
rhizobacteria
are far from being
and the efficacy of this strategy remains to be demonstrated.
Cellulomonasjimi
cellulase genes were introduced
into Brevibacterium lactoferentum
a better means of obtaining both higher levels of expression Since B. lactofermentum is used for the industrial
and secretion
production
of glutamic
to provide
than in E. coli (74). and lysine (74), a
cellulolytic B. lactofetmentum might provide an economic means of producing glutamic acid and lysine from cellulosic wastes.
Finally, it was found that when S. Zividans was transformed
cellulase gene from T. fusca, a very high level of cellulase activity was produced of this activity was found in the extracellular
with a
and, as well, all
medium (21) so that this system seems to have
potential.
In a unique experiment,
Chen et aL (9) created a stable cell fusion product between Enterococcus
faecium and Fusobacterium varium and then transformed fusion with an E. coli spheroplast a stable fusant with cellulase approach properties
this fusant by polyvinyl alcohol-mediated c-
which carried a plasmid-encoded activity could be isolated
may be useful in creating
by this procedure
novel ‘microorganisms’
of the starting microorganisms
cellulase gene. The fact that suggests that this
with unique combinations
of the
including the ability to hydrolyze cellulose.
To obtain cellulases that both are produced in high yield and secreted into the medium several Bacil1u.s species have been used as host cells. Additional a host microorganism from other gram-positive fermentations. stearothermophih
include (i) it may facilitate
attributes
the expression
bacteria and (ii) there is considerable
While B. subtih are attractive
for using Bacillus species as of exogenous
industrial experience with Bacillus
may suffice for many applications, alternative
B. megaterium and B.
systems which offer the advantages
plasmid stability (SO) and growth at a higher temperature
genes derived
(93), respectively.
of increased
B. R. GLICK and J. J. PASTERNAK
374
FUTURE PROSPECTS The binding and catalytic domains of the cellulase molecule are physically distinct regions (20, 25, 39). Either the binding or the catalytic domains can be isolated separately, free from the rest of the enzyme molecule either by manipulating protein
(20, 25, 39) with each domain
separate
protein
domains,
retaining
each with a portion
catalysis, is the first step towards developing functions. contained
For example,
Warren
both endoglucanase
to crystalline constructs
its characteristic
synthetic protein engineered
and exoglucanase
The isolation
of
constructed
cellulases for specific a fusion protein
which
activities although it lacked the ability to bind
cellulose because of the absence of an intact binding domain.
One can envision
in which the cellulose binding domain from an isolated cellulase gene is genetically value such as insulin or interferon.
could be purified in one step by adsorption
purification
activity.
digesting the
of the enzymic activity, i.e., either binding or
et al. (102) genetically
fused to the gene for a protein of commercial protein
a cloned gene or by enzymatically
of the target protein.
Such a fusion
to cellulose thereby lowering the cost of
In such a case, after the fusion protein is isolated, the cellulose
binding domain could then be removed by enzymatic digestion yielding the protein of interest in an intact form (25).
It is worth noting that although the term “cellulase” is widely used, it meaning is often inprecise and ambiguous.
To avoid confusion,
we suggest the terms endoglucanase,
S-glucosidase whenever possible to describe the individual components be reserved
for discussions of the multi-component
The advent of recombinant
exoglucanase
or
and that the term cellulase
complex.
DNA technology has dramatically
altered and accelerated
research
in the cellulase field. In the past three to four years a large number of cellulase genes have been isolated, characterized the development the enormous
and expressed in a variety of hosts. With this work forming the foundation,
of new cellulolytic organisms which can be used to exploit, in a myriad of ways, amount of waste cellulose which is produced
as a byproduct
of industrial
and
CELLULASE GENES
agricultural
375
practice may be feasible.
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