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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


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



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.


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


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


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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