Cloning,

Expression,

and

Gene

Hiroki

MATSUI1,

from

Koretsugu

Mutsumi

Characterization

Prevotella

OGATA1a,

NAKAMURA1,

a Cellulase

ruminicola

Kiyoshi

Rustem

of

TAJIMA1,

I. AMINOV2

Takafumi

and

NAGAMINE1,

Yoshimi

BENNO1,

1STAFF -Institute
, Tsukuba-shi 305-0854, Japan
2Department of Animal Sciences
, University of Illinois,
Urbana, IL 61801, USA
3Japan Collection of Microorganisms
, The Instituteof Physical and Chemical Research (RIKEN),
Wako-shi 351-0198, Japan
(Received May 28, 2001; Accepted June 29, 2001)
Abstract

Escherichia

cellulase

coli,

and

gene

its

from

Prevotella

enzymatic

4-methylumbelliferyl--D-cellobioside
from

12

positive

analyzed,

and

deduced
unidentified
from
was

anaerobic

Amino

amino

acid,

bacterium,

of

glycosyl

acid

The

carboxymethyl

sequence

and

6.8,

of

5,

product

sp.

strain

catalytic

however,

and

Heat

and

C.

the
in

lichenan.

treatment

fifth

of
amino

Escherichia
Temperature

at

70

for

the

10min

acid
coli
and

and

had

and
of

focusing

point
with

was

substituted

with

activity

the

and

nucleotides,

homology

optima

inactivated

selected

sequenced

showed

residue

pH

for

cellodextrinases

isoelectoric

celiulase

in

screened

endaglucanases

thermocellum,

Predicted

domain

967

with

expressed

was

was
of

homology

flavefaciens.

was

pPRC4

pPRC4

consisted

showed

F1,

in

and

library

designated

pairs)

ORF3

dalton)

cloned

genomic

clone

base

found.

37,000

expressed

(CMC),

respectively.

were

putative

was
A

(5,794

Ruminococcus

family

gene

cellulose

acttivity.

(ORF)
about

JCM8958T

characterized.

fragment

Clostridium
and

hydrolase

phenylalanine.

DNA

frames

succinogenes

6.09.

45

insert

reading

(322

Fibrobacter

motif

An

open

polypeptide

were

degrading

clones.
four

ruminicola

properties

toward
of

the

AvicelR,

celulase

were

cellulase.

Animal Scienee Journal 72 (5): 421-426, 2001

Key

The

words:

Cellulase,

rumen

microbial

host animals.
rumen
Ruminal

tote up to 60%

in starch

cellulose and
some

5, Prevotella

often

the

are able to
sources for

found

a variety

in the
of

predominant

contents22) and

diets.
species

may

prevotellas are considered

consti-

digestion

and

to be in-

utilization2), hemi-

pectin digestion3, 14)in the rumen.

strains of ruminal

ruminicola,

Rumen

actions with cellulolytic

bacteria were observed in
hemicellulose and pectin degradation in vitro3).
Therefore, ruminal prevotellasare believed to play
important roles for fiberdigestionin the rumen in
concert with other fibrolytic microorganisms.
Among ruminal prevotellas,
P. bryantiiB14 has been
intensivelystudied for production of endoglucanases,
xylanases, and glycosidases6,17).
8, These enzymes
were cloned, sequenced, and characterized7,
8, 16,
27).
Previously,we demonstrated that ruminal Prevotella
speciesproduced a range of polysaccharidedegrading
enzymes,
and
several carboxymethylcellulases

of

of total bacterial isolates on a silage

diet24). Ruminal
volved

are

are fed

are

the rumen

family

consists

microorganisms
into nutritional

that

prevotellas

isolated from

GH

such as bacteria, fungi, and

Prevotellas

of animals

digestion,

ecosystem

anaerobic microorganisms
protozoa.
These rumen
convert plant materials

Fiber

For

Prevotella, synergistic inter-

(CMCase)

in different sizes were

aPresent address: Analytical Instruments Division

, Genomic
Research Department, Shimadzu
Nakagyo-ku, Kyoto-shi 604-8511, Japan
Corresponding: Hiroki MATSUI
(fax:+81 (0) 298-38-2337,e-mail: matsui@gene.staff.or.jp)
Anim. Sci.J. 72 (5): 421-426, 2001

421

produced by
Corporation,

MATSUI,
P.rumnicola15).
xylanase

In

gene

has

rumnieola

so

rumnicola

for

this

gene

fare26).
the

the

P.

To

clarify
fiber
be

P.

bryantii,

only

and

sequenced

the

contribution

digestion

and

NAKAMURA,

one
for

P.

of P.

, the

details

of

sequenced

clone
the

harbouring

and

in

and

and

P.

strains

ruminicola
of
P.

at

E.

ml)

18h

was

added

digested
Zap

II

(Stratagene,

brary

was

in

in LB
at

described
f

was

of

Ogata

the

cellulase

gene

ability

DNA
A

was

set

of

pBAD-TOPO

vector

Carlsbad,

CA.,

Enzyme

carried

out

primers

cells

of

the

pelleted

clone

by

was

pellet

was

washed

buffer

resuspended

in

Model

250,
7

for

the

(pH

Branson,
30s,

5times.

as
used

as

renatured

phate

buffer

for

shaking.

of

activity

cloned

into

red.
with

The

et al.15).

the

cellulase

gene

6.8).
buffer

50mM

Washed
and

CT.,
crude

Anim. Soi.J. 72 (5): 421-426, 2001

Inc.

4).

with

Cupertino,
of

clearing
(ver.

reagents
(Kyoto,

were

sodium

pellet

sonicated

Danbury,
The

of

Inc.,
mass

images

were
Japan)

reagent

with

(SDS)

at

sodium
39

USA).

1.0.2)

for

(Apple

CA.
Com-

molecu-

estimated

using

Macintosh.
from

otherwise

digitized

Apparent
was

purchased

Congo

Hercules,

Macintosh

zone

enzyme

(wt/vol)
and

gels

gentle

to

(Bio-Rad,

elec-

phos-

with

the ncaptured

CA.

The

the

corresponding

a Power

an

load-

and

50mM

with 0.3%
were

mixed

CMC,

20h

MultiImager

equipped

All

were

at

gel

Fluor-STM

Multi-AnalystR

Briefly,

(9,000g20min,
with

lar

were

visualized

described

electrophoresis,
in

for

as

polyacrylamide

(wt/vol)

zone

sub39.

for 5min.

SDS-10%

incubated

used

of
at

sulfate
95

After

6.8)

were

mixture

were

3.0

6.0-8.0),-

incubated

dodecyl
at

pH

pH

performed

onto

clearing

was

(50mM,

denatured

(pH

The

at 30,
enyzme

8.2-8.8)

cell extracts

and

thermo-

exposed

(54mM,

was

was

at 150V.

analyzed

For

buffer

optimum.

sodium

was

was

pH

containing 0.15%

were

used0

mon-

Subsequently,

(50mM,

loaded

01Celex

celiulase

buffer

Crude

(Celex

analysis

Matsui

twice

same

reduc-

released

interval.

solution

of

trophoresed

Corporation,

zymogram

harboring

centrifugation

of

was

the

solution

analysis

and

the

10min.

pH

were

USA)

to

were

monitored

Glucose

Citrate

enzyme

buffer

gels

enzyme
for

buffer

volume

puter,

phosphate

output

product
(Invitrogen

according

substrates.

release

of

of

phosphate

determining

samples

as

(PCR)-amplification

and

buffer

substrate

was

degree

assayed.

for

of

USA).

activity

70

Tris-HCl

ing

assays

Cellulase

The

analyzed

was

PCR

crude

and

li-

Nu-

and

the

the

sodium

equal

The

(MUC).

reaction

and

The

determination

with 5

60, or

previouslyl5).

Lambda

USA).

2r5 '-AGGTTGGTACTTCTTGG-3')
chain

as

and

substrate

optimum

Zymogram

EcoRI

in

the

85

was

5.4),

prepared

An

degradation

et al.18).

50,

activity

Solid

5 '-ATTTTAATGCTGGCCGTA-3'

polymerase

et

medium

was

CA.,

insert

to

strate-crude

constructed

for

for

stability,
40,

Ampicillin (50g/

Jolla,

screened

in

Matsui

200rpm.

protocol21).

La

sequence

fluid-

broth

4-methylumbelliferyl--D-cellobioside
cleotide

rumen

of P. ruminicola

library

used

et al.23).

Germa-

phosphate

and

39.

Mo.,

concentrations.

Temperature

gene

standard

genomic

Louis, l
F. R.,

sodium

at

from

(CMC),
St.

cell extract

in Takenaka

from 5

required.

DNA
to

Japan
Wako,

described

agar.

endoglueanase

Chromosomal
according

from

in

shaking

1.8%
when

of

grown

grown

with

contained

Cloning

obtained

as

were

omer

(RIKEN,

was

strains

for

medium

was

medium

coli

37

conditions

Microorganisms

glucose-cellobiose
al.15).

growth

ruminicola

through-

Methods

JCM8958T

Collection
Japan).

and

crude

incubated

a standard
Bacterial

used

Darmstadt,

50mM

(wt/vol)
of

sugars

described
Materials

in

at 0.2%

mixed
ing

was

Co.,

(Merk,

dissolved

volumes

cellulase

Chemical

AvicerR

were

Equal

expressed

the

BENNO

Carbaxyrnethylcellulose

(SIGMA

(pH 6.8)

coli.

and

experiments.

USA),

of

characterized

cellulase

AMINOV

ichenan

a cellulase

and

the

the
out

ny)

JCM8958T,

properties

NAGAMINE,

studied.

cloned

ruminicola

enzymatic

Escheriehia

to

ruminal

we

TAJIMA,

cloned

should

study,

from

contrast

been

polysaccharidases
In

OGATA,

Nacalai

stated.

All

tesque,
reagents

grade.

was

Results

and

Discussion

library

derived

from

(Sonifier
USA)

cell

extract

with

genomic

screened

of

422

for

MUC

degrading

P.

ruminicola

activity.

was
clone

Cellulase

Fig.

1.

Amino

acid

Prevotella ruminicola
from

ruminal

Shadowed

amino

acid
P.r

F.s

Pi.e

2, O. joyonii

domain

with

family

of

cellulase

5 glycosyl

glycosyl

R.f
F.

cellulase

CdexA,

hydrolase

from

hydrolases

N.f
equi

B29; O.j

family

CelA,

bifunctional
Neocaltimastix

Cellulase 5A; O.j

CelA, O. joyonii

5.

F. s CdexA,

Ruminococcus

succinogenes

G;

Piromyces

cellulase
cellulase

of

ruminicola cellulase;

CMC/Xyn,

Cel 5A,

between
of

ruminicofa

catalytic

motif

Prevotella

F. sueeinogenes
A,

pinomyces joyonii

Motif

of

cellodextrinase;
A;

CeiG,

Numbers

Prevotella

its alignment

matches

Cel,

succinogenes

cellulase

sequence
and

cellodextrinase
F.s

from

microorganisms.

Abbreviations:
bacter

Gene

cellulase

Fibro-

flavefaciens
CMCase;
frontalis
CelB29, OrA; O.j

CelB

B2.

parentheses

family 5

are

glycosyl

GenBank

hydrolase

accession
is

numbers.

[LIV]-[LIVMFYWGA]

(2)-

[DNEQG]-[LIVMGST-x-N-E-PV[RHDNSTLIVFY](Henrissat,
1991.).

designated as pPRC4
clones. A DNA

was selected from

ria:celiulasefrom unidentifiedanaerobic bacterium

12 positive

fragment in the clone was sequenced

(U12011; 33%, identity),endoglucanase C307 precursorfrom Clostridiumsp.strainF1 (P23340, 30%),

endoglucanase C from C. thermocellum (1CEC,
30%), cellodextrinasefrom Fibrobactersuccinogenes

and analyzed for gene structure. The cloned DNA

fragment (GenBank

Accession Number;

AB022867,

5,794 base pairs) had 4 open reading frames (ORFs).

Amino

acid sequences deduced

ORF3,

and

ORF4

had

from ORF1, ORF2,

homology

(U07419, 28%),
and
cellodextrinase A
from
Ruminococcus fiavefaciens(P16169, 25%). Amino
acid sequence of the cellulasegene had motif of catalyticdomain of glycosylhydrolasefamiry510)(Fig.1).
The fifthamino acid residue of the cellulasewas
substitutedwith phenylalanine residue instead of

with D-alanine

permease, polynucleotide polymerase, cellulase,and

unknown polypeptide, respectively. The ORF3
was
further analyzed for nucleotide and amino

acid se-

quence structure. The ORF3

had 967 nucleotides.
Deduced amino acid sequence of the gene product had
322 amino
37,330.

acids with molecular

weight

(MW)

[LIVMGST].
Glycoside hydrolases belonging to
family 5 are widely spread among ruminal bacteria:
Butyrivibrio
fibrisolvens9)
(M3703), F. succinogenes1, 11)

of

Predicted isoelectricfocusing point (pI) was

6.09. Typical promoter

TATAAT)

was

Deduced

amino

region (-35TTGACA, -10

not found

upstream

acid sequence

(U94826, U33817), P. ruminicola26)(M83379), P.bryantii1

6) (M38216)
, R. albus19)(M30928), and R.
flavefaciens25)
(X51944), and ruminal anaerobicfungi:
eocallimastix frontalis5)
(U38843), N. patriciarum28)
N

of the ORF.

had homology

with

glycosyl hydrolases of the following anaerobic bacteAnim. Sci.J. 72 (5): 421-426, 2001

423

MATSUI,

OGATA,

TAJIMA,

NAGAMINE,

NAKAMURA,

(Z31364),
Orpinomyces joyonii 20) (AF015248),
Orpinomyces sp. PC-2 12) (U57818), Piromyces equi 4)
(AJ277483), and P.rhizinflata (U57818). This suggests that the gene was originated from common ancestor and was horizontally spread among ruminal
microorganisms.
The amino acid sequence of the
cellulase showed homology with cellodextrinase from

Fig.2.
Prevotella
M,

Zymogram
ruminicola

analysis
expressed

of

cellulase

in Escherichia

AMINOV

and

F. succinogenes,and R. flavefaciens.
Since P.
ruminicolaisa non-cellulolytic
bacterium,a possible
biologicalfunctionof the cellulase
is to degrade
cellodextrin
releasedfrom cellulose
degradationby
otherfiblolytic
microorganismsintosmallerfragments
which are availableto P. ruminicola.Thus, P.
ruminicola would be a consumer of cellooligosaccharides
in the rumen.
Sincecatalytic
domain of the cellulase
had homology with enodoglucanase,and the crude enzyme solution had activities
on CMC, we characterized
enzymatic propertiesusing CMC
as a substrate.
Substraterange,molecularweight,temperatureand
pH optima,and thermostability
of the cellulase
were
characterized
usingcrude extractof the clone. The
crude extractcould releasereducing sugar from
AvicelR and lichenan in addition to CMC.
Zymogram analysis
was carriedout to estimateMW
of the cellulase
expressedin E. coll. The estimated
MW was 44.2 kDa on zymogram analysis(Fig.2).
The MW includesleaderpeptideand His tag sequence
(about4.2kDa) derivedfrom the vector. Therefore,
the MW of the expressedcellulase
was a reasonable
sizewhen estimatedfrom amino acid sequence(37
kDa). Previously,
we found thatP. ruminicola
JCM
8958T produced a CMCase in the sizeof 40kDa 15).
The cellulase
obtainedhere would be identical
to this
protein. Temperatureoptimum, thermostability,
and
pH optimum of clonedcellulase
were characterized.

from
coli.

Marker.

Fig.3.
Temperature
and pH optima of a cellulase of
Prevotellaruminicola expressed iraEscherichia coli.
a)Temperature optimum.
b)pH optimum.
Buffers used were
citratebuffer (50mM, pH 3.0-5.4),sodium phosphate buffer (50
mM, pH 6.0-8.0), and Tris-HCl buffer(50mM, pH 8.2-8.8).
Anim. Sci.J.72(5):421-426,2001

BENNO

424

Cellulase Gene
The

celiulase

3-a).

was

The

The

active

highest

cellulase

between

activity

was

30

was

exposed

in

to

55

observed

various

from

Prevotella

Socity,

(Fig.

at

4)

45.

temperatures

ruminicola
50:
RY,

sequence

to

149-159.

Eberhardt

1991.

Gilbert

and

endoglucanases,

examine

thermostability

(data

not

shown).

activity

cellulase

was

was

to

over

The

at

less

ruminal

70

for

range

activity

bacteria

When
the

The

cellulase

was

(Fig.

3-b).

5.4-8.0

observed

family

and

the

15min,

of

was

of

50.

10%.

pH

properties

under

than

broad

highest

Enzyme

stable

heated

reduced

active

was

at

pH

5)

Cel5A

Fujino

Y,

from

6.8.

been

fungus

4,5, 11,13,20).

Although

the

pH

in

these

reports

optima

were

6)

to 5.5,

almost

the

pH

optimum

neutral.

(Fig.

1)

may

enzymatic
enzyme

of

of

ranged

this

properties

of

shift

of

optimized

the
in

pH

was

Wells

JE,

the

cellulase

were

and

that

this

rent

physico-chemical

enzymatic

characterized

8)

properties

in

this

amino

acid

sequence

of

the

cellulase

had

with

catalytic

zymatic

domain

of

were

examined

properties

substrate

in

this

zymological

study.

analysis

Furthermore,

Detailed

of the

regulation

polysaccharidases

of

should

significance

be

of Prevotella

the

cellulase

studied

to

ruminicola

in

en-

CMC

as
on

should

be

9)

en-

Gasparic

Ministry

of

was

Agriculture,

supported

Forestry,

other

understand

10)

the

KK,

Kim

Molecular

SC,

cloning

endoglucanase
S85

in Escherichia

nology,
2)

gene

27:

Cotta

MA.

from
gy,
3)

and

starch.
59:

Fishery.

BA.

digestion

in

JD,

Choi

of a novel

Fibrobacter

coli. Enzyme

and

YJ.

family

the

Gilbert

of

B14.

Wallace

genes

and
the

encoding

-L-arabino-

rumen

bacterium

FEMS

Microbiology

HJ.

and

A46.

B.
on

Journal

of

acid

cal

Journal,

280:

Iyo

AH,

Forsberg

Romaniee
of

the

celA

of

Butyrrvibrio

General

Microbiol-

glycosyl

hydrolases

1990.

classification

amino

JI,

sequencing

endogiucanase

2089-2097.

Henrissat

, Laurie

Cloning

strain

136:

1995.

DavidsonK

encoding

of

sequence

similarities.

309-316.

Biochemi-

1991.

CW.

Endoglucanase

succinogenes

enzymes

characterized

Canadian

Journal

succinogenes
Microbial

Li

X-L,

Chen

S85
by
of

ruminal
of

bacteria

Tech-

in

3)

the

Liu

belongs

a basic

to

from
class

C-terminal

Microbiology,

anaerobic

of

domain.

42:

14)

of microbial

synergism

Proceedings

Anim. Sci.J. 72 (5): 421-426, 2001

of

on

fibre

an

Nutritional

by

425

fungi

434-943.

of

970-974.
H,

of

BL,

Ushida

digested

index
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of

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63:

Tsai

C-F,

Neocallimastix

and

structurally
Applied

628-635.
Cheng

and
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K-J.

patriciarum

Canadian

1 Characxylanase

Journal

of

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1999.
K,

Miyazaki

xylan

fiber

Monocentric

xylanases.

Microbiology,

its product.

45:

Matsui

LG.

and

Selinger

and

ogy,

Microbiolo-

Ljungdahl

cellulases

J-H,

gene

maltooligasaccharides

Environmental

H,

Environmental

ratio

rumen.

Martin J,

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from

135-142.
GP,

related

1992.
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enCur-

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HJ.

DB,

hydrolases.

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of

and

Wilson
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274-277.

activities

palycentric

2000.

utilization

Applied

48-54.

Dehority

Bok

expression

Interaction

production

JH,

from

475-481.

the

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Woo

and

The
of

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12)

Cho

DB.

1995.

ruminicola

Flint

125:

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financially

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