Vol.

169,

No.

June

29,

1990

3, 1990

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

CLONING CODING

AND EXPRESSION

OF CLOSTRIDIUM

FOR THERMOSTABLE IN

K.Tuka,

of Molecular Kurchatov

May 2,

G.A.Velikodvorskaya

and

Genetics, USSR Academy of 46, 123182 Moscow, USSR

Sciences.

1990

screening approach two independent genes directing the synthesis of thermostable glucanases with an exo-mode of action have been isolated from pUClS-based gene bank in E.coli TGl. The genes are located on 3,4 and 11,3 kb DNA fragments showing no homology. E.coli-derived exoglucanases, presumably. cellobiohydrolases, are able to cleave lichenan, carboxymethyl cellulose, xylan and p-nitrophenyl derivatives of cellobioside and lactoside. Cellobiose is the main degradation product of carboxymethyl cellulose, treated with the identified exoglucanases. With p-nitrophenilB-D-cellobioside as substrate the enzymes had a pH optimum around 6.5 and a temperature optimum at 650C. The identified and expressed enzymes differ from all other Cl.thermocellum proteins known to date. 0 1990 *cademlc Press, klc. Summary Cl.thermocellum

By

sq.

(CELLOBI~HYDROLASES) CELLS

COLI

B.K.Bumazkin, A.Ya.Strongin*

1055-1060

GENES

THERMOCELLUM

EXOGLUCANASES

ESCHERICHIA

V.V.Zverlov,

Institute

Received

AND

INTRODUCTION

special

bacterium

produces

cellulose.

to

glucose

characterization

of

have

the

facilitated

whom

anaerobic thermophilic complex able to

a cellulose-cleaving

The individual 4). studied comparatively of Cl.thermocellum

*To

an

Cl.thermocellum

correspondence

and

consisting

enzymes

of

poor cellulase

up

the

of

the

to now. genes in

corresponding of

be

20

The E.coli

enzymes

(1,

complex cloning and

and the

E.coli-derived

understanding

should

about

multicellulase

convert

mechanisms

2,

have

3, been

expression proteins

of

cellulose

addressed.

CMC, carboxymethyl cellulose: EG, Abbreviations used: CBH, cellobiohydrolas$; BG, beta-glucosidase; XL, endoglucanase; 50 mM phosphate-citrate buffer, pH 6,3; xylanase: PC-buffer, 4-methyl-umbelliferril@-D-cellobioside; MU-G, rl-methylMU-GP, pNPC, p-nitrophenylB-Dumbelliferril@-D-glucopyranoside; cellobioside; pNPLac, p-nitrophenyl-fl-D-lactoside; pNPG, pnitrophenyl-B-D-glucopyranoside; pNPGa1, p-nitrophenyl-B-Dp-nitrophenylfi-D-xylopyranoside: galactopyranoside; pNPXy1, ampicillin. AP, OOO6-291XM 1055

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

169,

No.

conversion cloning 2 XLs

of

have

knowledge

3,

been no

this

AND

catalized by the 19 Cl.thermocellum reported

attempts

CBH genes paper

have

we present

cloning and expression coding for the cells, specificity, distinct

the

of two enzymes from

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

coding

complex. for 15

Recently, EGs, 2 BGs

multicellulase genes,

(5-12).

successive

Cl.thermocellum In

BIOCHEMICAL

1990

all

On the

other

describing been

the

to

our

selection

of

reported.

specific

selection

Cl.thermocellum with strict other

hand,

the and

known

procedure,

genes exoglucanase

in

E.coli or CBH

Cl.thermocellum

cellulases. MATERIALS --

AND METHODS

Cl.thermocellum F7 was obtained from Institute of Microbial Biochemistry and Physiology, USSR Academy of Sciences, Puschino, Moscow region. The E.coli strain TGl was employed for transformation with plasmid pUC19. Preparation of Cl.thermocellum total DNA and E.coli plasmid DNA, endonuclease treatment, ligation and transformation, agarosegel electrophoresis and Southern blot-hybridization were carried out by conventional techniques (12, 13). For the construction of Cl.thermocellum pUC19-based gene bank in E.coli TGl lo-20 kb long Sau3A-fragments were isolated by agarose-gel electrophoresis and ligated with BamHI-treated pUC19 (14). Transformed cells were plated on L-agar, containing 0,l mg/ml Ap and after 12-14 h at 370C each plate was overlayed with 5 ml of 0,7% agar, containing 1 mg/ml MU-G or MU-G2 or 0,5% (w/v) CMC. After heating at 60°C for 3-4 h transformants producing WC-cleaving activity were detected by staining with Congo red (15). In case of fluorogenic substrates MU-G and MU-G2 the clones surrounded with a fluorescent halo were selected for the further analysis after 20-30 min heating at 60°C. For the activity measurment the cells were grown at 370C in L-broth, containing 0,l mg/ml Ap, collected by centrifugation and desintegrated in PC-buffer. The cell homogenate was heated for 5060 min at 60°C, the pellet was removed by centrifugation (25.000 x g; 30 min), the supernatant was then treated with solid ammonium sulphate and the fraction precipitated between 0.3 and 0,9 saturation was collected by centrifugation and dissolved in PC-buffer. The enzyme activity was assayed at 60°C in PC-buffer with pNPC, pNPLac, pNPG, pNPGa1 and pNPXy1. One unit of enzyme corresponds to the release of 1 pmole of p-nitrophenol per rain Th e activity per mg of protein. was also measured with highmolecular weight substrates CMC, lichenan and xylan. Reducing sugars released from the substrates were determined with the 3,5dinitrosalicylic acid reagent (16), assuming that one unit of enzyme corresponds to the release of 1 pmole of glucose equivalent per min per mg of protein. The activity of the enzymes was also determined by a viscosimetric assay using CMC as substrate (17). Reaction mixtures containing 2% CMC (w/v) in PC-buffer and an enzyme sample were incubated at 60°C for 5-90 min. The reaction was stopped by heating for 3 min in boiling water bath. The viscosity was measured at 60°C with an Ostvald's viscosimeter. The reciprocal of the specific viscosity was calculated using the equation l/q.,=to/t-to, where t, and t correspond to the flow time of PC-buffer and sample, respectively. 1056

BIOCHEMICAL

Vol. 169, No. 3, 1990

The protein (18). Analysis chromatography

RESULTS

AND A

of was

the

like

activity.

approach

It

well

known

size

less

therefore

EG-expressing CBHs

with

only

the

last

clones

with

the

phenotype

applying

5000

independent

this

have

been

Assuming

for

They

pCU309. EcoRI

ligated.

that

inserted

fragment

two

the of

delete

resulted

Further 7,3

in 3,4

and

kb

the

long

gene

to

with

the

plasmid

same

select bank.

bank

of

desirable

kb,

pCU304 the

CBHs

gene

about

phenotype the

inserted

designated pCU303

EcoRI-fragment

plasmid

showing

kb),

the

11,3

be

BGs

tried

the

cleaving not

MU-G2,

from

plasmids

lo,?

respectively. to

with

from

of

have

the

screening

carried

fragments

we

dye.

will

with and

MU-G2'

clones

red

capable

MU-G

and

residues

clones contrast

MU-G-

(20).

the

not

that,

CMC-

approach

DNA and

with

in

yielding

Congo

off

substrates

one.

selected.

CMC

with

and

clones CBH-

residues

CBH-expressing

clones.

C.thermocellum

degrade

stained

Moreover,

TGl

following

glucose

chains

red.

cle,ave

pCU303

Blue

expressing

the

splitting

fluorogenic

E.coli

bank,

on EGs

5 are

Therefore,

Congo

When

that than

oligosaccharide

both

the

gene

exo-cellulases

moiety.

hydrolysing

cut

with Coomassie of CMC by thin-layer (19).

select

based

clones

are

of

to

is

is

Contrary, termini

approach

the

with

cellobiose

used

pUC19-based

This

oligomers

stained

measured products according to

was

Cl.thermocellum

assumptions.

the

was

DlSCUSSIPN

special

from

concentration the degradation performed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was

and (the

CMC-

size

of

MU-G-

the

MU-G2+

PST1 HIMI

HIND111 PST1

P!

HIND111

.HINDIII

FSTI Lens

th

13100

1

I

RI PST1 PST1

ECORI

Fig 1. Physical maps of indicates Cl.thermocellum plesmid pUC19 DNA. EcoRI are indicated by arrows.

the

plasmids DNA fragment. sites used

1057

pCU303 and pCU304. Thin Boxed line indicates for the construction of

line the pCu304

CIP