Characterization, cloning and sequencing of a thermostable endo-(1, 3-1, 4) beta-glucanase-encoding gene from an alkalophilic Bacillus-brevis

(1)

@pried

.. Microbiology Biotechnology

Characterization, cloning and sequencing

of a thermostable endo-(1,3-1,4) ,fl-glucanase-encoding gene from an alkalophilic Bacillus b r e v i s

Maureen E. Louw 1, Sharon J. ReidWatson 1

1 Biotechnology Programme, Division of Food Science and Technology, CSIR, P. O. Box 395, Pretoria 0001, Republic of South Africa

2 Department of Microbiology, University of Cape Town, Private Bag, Rondebosch 7700, Cape Town, Republic of South Africa Received: 21 April 1992/Accepted: 3 August 1992

Abstract. A Bacillus brevis gene c o d i n g f o r a n e n d o - ( 1 , 3 - 1 , 4 ) - f l - g l u c a n a s e was c l o n e d in E s c h e r i c h i a coli a n d s e q u e n c e d . T h e o p e n r e a d i n g f r a m e c o n t a i n s a sequence o f 759 n u c l e o t i d e s e n c o d i n g a p o l y p e p t i d e o f 252 a m i n o a c i d r e s i d u e s . T h e a m i n o a c i d s e q u e n c e o f t h e / ~ - g l u c a - n a s e gene s h o w e d o n l y a 50°70 s i m i l a r i t y to p r e v i o u s l y p u b l i s h e d d a t a f o r Bacillus e n d o - ( 1 , 3 - 1 , 4 ) - f l - g l u c a n a s e s . T h e o p t i m u m t e m p e r a t u r e a n d p H for e n z y m e a c t i v i t y were 6 5 - 7 0 ° C a n d 8 - 1 0 , r e s p e c t i v e l y . W h e n h e l d at 7 5 ° C f o r 1 h, 7507o r e s i d u a l a c t i v i t y was m e a s u r e d . T h e m o l e c u l a r m a s s was e s t i m a t e d to b e a b o u t 29 k D a o n s o d i u m d o d e c y l s u l p h a t e ( S D S ) - p o l y a c r y l a m i d e gel elec- t r o p h o r e s i s a n d t h e e n z y m e was f o u n d to b e r e s i s t a n t to SDS.

Introduction

T h e e n z y m e e n d o - ( 1 , 3 - 1 , 4 ) - f l - g l u c a n a s e ( E C 3.2.1.73) is a b l e to h y d r o l y s e the m i x e d l i n k e d (1,3-1,4)-fl-glucans t h a t c o n s t i t u t e the m a j o r p a r t o f the e n d o s p e r m cell walls o f cereals such as o a t s a n d b a r l e y . S e v e r a l species o f t h e g e n u s Bacillus secrete f l - g l u c a n a s e s . M a n y o f the f l - g l u c a n a s e - e n c o d i n g genes h a v e b e e n c l o n e d a n d se- q u e n c e d : f r o m B. subtilis, ( M u r p h y et al. 1984, T e z u k a et al. 1989), B. a m y l o l i q u e f a c i e n s , ( H o f e m e i s t e r et al.

1986); B. macerans, (Borriss et al. 1990) a n d B. licheni- f o r m i s ( L l o b e r a s et al. 1991).

T h e m a j o r a p p l i c a t i o n o f e n d o - ( 1 , 3 - 1 , 4 ) - f l - g l u c a n a s e s a r e t o b e f o u n d w i t h i n t h e b r e w i n g i n d u s t r y , w h e r e t h e y a r e u s e d to s u b s t i t u t e a n d s u p p l e m e n t m a l t e n z y m e s ( C a n t w e l l a n d M c C o n n e l l 1983). T h e r m o s t a b l e fl-glucanases are p a r t i c u l a r l y s u i t e d f o r this, as t h e y a r e n o t i n a c t i v a t e d as r a p i d l y d u r i n g the k i l n i n g a n d m a s h i n g p r o c e s s e s .

I n this p a p e r we d e s c r i b e the i s o l a t i o n o f a n a l k a l o - p h i l i c B. brevis p r o d u c i n g a t h e r m o s t a b l e f l - g l u c a n a s e , the s u b s e q u e n t c l o n i n g a n d s e q u e n c i n g o f the gene a n d

the c h a r a c t e r i z a t i o n o f c e r t a i n n o v e l b i o c h e m i c a l p r o p - erties o f the e n z y m e .

Materials and methods

Isolation o f alkalophilic bacteria producing endo-(1,3-1,4)-fl-glu- canase. Approximately 3 g soil was pasteurized (Norris et al. 1981) before being used to inoculate 100 ml of alkalophilic enrichment broth, pH 10.0 (0.5% yeast extract, 0.5% polypeptone, 0.1% di- potassium hydrogen phosphate, 0.02% magnesium sulphate and 1% sodium carbonate as modified from Takeuchi et al. (1989) and incubated for 4-5 days at 37 ° C. Aliquots from the turbid broth cultures were then inoculated onto the same medium solidified with agar (15 g/l) and incubated at 37°C for 24-48 h.

The cultures were purified on nutrient agar plates, pH 9.0, at 37 ° C. They were then classified as Bacillus sp. after a Gram stain, a spore stain and a catalase test gave positive results. Colonies were then transferred to Luria agar plates (10 g tryptone, 5 g yeast extract, 10 g sodium chloride and 15 g agar per litre, adjusted to pH 9.0) and supplemented with 0.1% (w/v) lichenan from Cetrar- ia islandica (Sigma). After incubation at 37 ° C for 24-48 h, plates were flooded with 0.1% (w/v) Congo red (Cantwell and McCon- nell 1983). Colonies producing a clear zone were grown in Luria broth plus 0.1% lichenan for 2-3 days at 37°C and pH 9.0. The enzyme produced in the supernatant was then evaluated. Further strain identification was done by utilizing the API 50CH and 20E strips.

Endo-(1,3-1,4)-fl-glucanase determination. The determination of fl-glucanase activity was carried out using lichenan as a substrate (Tezuka et al. 1989). One unit was defined as the amount of enzyme that produced 1 ~tmol reducing sugar calculated as glucose per minute under the conditions of assay.

Bacterial strains, growth media and vectors. The Escherichia coli host strain for the original cloning experiment was JM101 (Mess- ing 1979), whereas for sequencing the E. col r e c - strain LKl12 was used (Zabeau and Stanley 1982). The cloning vector was the positive selection vector pEcoR251 (Zabeau and Stanley 1982).

Subcloning for sequencing was done using the high copy number Bluescript (SK) vector. E. coli L K l l 2 and JM101 were grown in Luria broth, pH 7.2, and the donor B. brevis strain was grown in Luria broth, pH 9.0, and maintained on Luria agar plates, pH 9.0. All strains were grown at 37 ° C.

Preparation o f DNA and recombinant DNA techniques. Chro- Correspondence to: T. G. Watson mosomal DNA was isolated from B. brevis according to the meth-

(2)

508

od of Lovett and Keggins (1979). Partial Sau3A restriction of the B. brevis DNA and isolation of size fradtionated fragments from low-melting-point agarose gels using agarase (CalBiochem) were according to the methods of Sambrook et al. (1989). DNA fragments ranging between 1.5 kb and 4 kb were isolated. Plasmid DNA was prepared by the method of Gryczan et al. (1978) and purified by cesium chloride-ethidium bromide equilibrium centri- fugation as described by Sambrook et al. (1989). A B. brevis ge- nomic library was constructed by ligating the partially digested Sau3A chromosomal DNA to Bg/II-endonuclease-digested pE- coR251. The E. coli JM101 and LKl12 ceils were made competent and transformed according to the method of Sambrook et al.

(1989). DNA fragments for subcloning were isolated from 0.8%

agarose TRIS/acetate gels using the Geneclean kit (Bio 101, Cal- if., USA). Positive transformants were selected for their ability to form clear halos on Luria agar plates containing 0.1 °70 (w/v) lichenan in addition to ampicillin (100 gg/ml) after flooding with Congo red.

Preparation of supernatant and cell lysate fractions. The recombinant E. coli strain was grown overnight in Luria broth plus 0.1%

lichenan and ampicillin (100 gg/ml). The supernatant as well as the periplasmic and sonicated fractions were assayed separately.

The recovery of extracellular, periplasmic and cytoplasmic enzymes was done as described by Cornelis et al. (1982).

Substrate specificity of the recombinant B-gluci~nase. In order to determine enzyme specificity, the activity of the sonicated fraction of the recombinant E. coli strain JM101 containing the cloned/~- glucanase gene was assayed according to Tezuka et al. (1989) at pH 9.0 using, in addition to lichenan, other high molecular mass substrates such as carboxymethylcellulose (CMC), avicel, laminarin and xylan at a final concentration of 0.5°70 (w/v). For calculat- ing specific activities, protein concentration of the sonicated fraction was measured using the biuret method (Herbert et al. 1971).

Sodium dodecyl sulphate-polyaerylamide gel electrophoresis (SDS-PAGE) protein assay. Proteins were analysed on 15% SDS- polyacrylamide gels (Laemmli 1970). To visualize enzyme activity, samples were pre-treated by incubating for 40 min at 60 ° C in sam- ple buffer and run on SDS-PAGE activity gels containing 0.1%

lichenan (Zverlov and Velikodvorskaya 1990). To remove the SDS the gels were washed in phosphate buffer, pH 6.3 (Beguin 1983).

The bands of enzyme activity were detected by staining the lichenan/PAGE gel with Congo red.

Restriction mapping and nucleotide sequencing. Restriction enzymes were from Boehringer Mannheim (FRG) and used according to the manufacturers specifications. The recombinant plasmids were characterized by restriction mapping using standard procedures (Sambrook et al. 1989). The Exo-III Mung bean nu- clease technique was used to create a deletion series for sequencing (Heinikoff 1987). The nucleotide sequence I of the fl-glucanase gene was determined by the dideoxynucleotide-chain-termination method (Sanger etal. 1977) using the Sequenase II Kit (USB) according to the manufacturers specifications. The nucleotide and deduced amino acid sequences were analysed using the Genetics Computer Group (GCG) software package (version 7.0). All the current databases accompanying the GCG package were screened for related nucleotide and amino acid sequences.

Results

Isolation o f B. brevis A L K 36

A p p r o x i m a t e l y 100 Bacillus s p p . were e v a l u a t e d in li- q u i d c u l t u r e a n d o f these, one s t r a i n , d e s i g n a t e d A L K 1 Nffcleotide sequence accession number. The nucleotide sequence reported has been assigned GenBank accession number M84339

36, was f o u n d to p r o d u c e a n a l k a l i n e e n d o - ( l , 3 - 1 , 4 ) - f l - g l u c a n a s e . This s t r a i n was i d e n t i f i e d as b e l o n g i n g to t h e species B. brevis. It was f o u n d t o be a b l e to g r o w at t e m - p e r a t u r e s r a n g i n g f r o m 3 7 ° C to 5 0 ° C a n d the p H g r o w t h r a n g e was b e t w e e n 7.0 a n d 11.0 w i t h o p t i m u m g r o w t h at p H 9.0.

E f f e c t o f p H on e n z y m e activity and stabifity

T h e a c t i v i t y o f the c r u d e e n z y m e e x t r a c t was d e t e r m i n e d at v a r i o u s p H v a l u e s (Fig. 1). T h e p H was a d j u s t e d using S o r e n s e n p h o s p h a t e b u f f e r at n e u t r a l t o a c i d p H values a n d T R I S b u f f e r at a l k a l i n e values. O t h e r c o n d i t i o n s were as for the s t a n d a r d a s s a y m e t h o d . T h e c r u d e en- z y m e e x h i b i t e d a c t i v i t y o v e r a b r o a d p H r a n g e w i t h o p t i - m u m a c t i v i t y at p H 9.0. A p p r o x i m a t e l y 8 0 - 1 0 0 % activi- t y o c c u r r e d b e t w e e n p H 7.0 a n d p H 10.0, a f t e r w h i c h the a c t i v i t y d e c l i n e d r a p i d l y .

E f f e c t o f temperature on activity and stability

T h e A L K 36 e n d o - ( 1 , 3 - 1 , 4 ) - f l - g l u c a n a s e e x h i b i t e d 9 5 - 100% a c t i v i t y b e t w e e n 65 ° C a n d 70 ° C (Fig. 2). A t 75 ° C at least 7 5 % o f m a x i m u m a c t i v i t y was r e t a i n e d . T h e r - m a l s t a b i l i t y o f the e n z y m e was m e a s u r e d at p H 7.0 b y i n c u b a t i n g t h e e n z y m e at d i f f e r e n t t e m p e r a t u r e s for t h e i n d i c a t e d t i m e p e r i o d . S a m p l e s were w i t h d r a w n a n d t h e r e s i d u a l a c t i v i t y m e a s u r e d a n d results s h o w n in Fig. 3.

A t 70 ° C, a p p r o x i m a t e l y 85% o f a c t i v i t y r e m a i n e d a f t e r 1 h w h e r e a s at 8 0 ° C t h e a c t i v i t y was f o u n d to d r o p o f f r a p i d l y .

Cloning o f the B. brevis A l k 36 fl-glucanase-encoding gene

E. coli JM101 was t r a n s f o r m e d w i t h t h e g e n o m i c l i b r a r y o f B. brevis. F r o m 1800 t r a n s f o r m a n t s f o u r s h o w e d hy- d r o l y s i s o f l i c h e n a n b y p l a t e assays. O n e o f the t r a n s f o r -

100 .

g

~ e0

N o

a -

0 I I I I I I I 1 I

3 4 5 6 7 8 9 10 11

p H "

Fig. 1. Infiuence of pH on endo-(1,3-1,4)-fl-glucanase activity.

The enzyme activities at various pH values were measured by the standard assay at 40°C

(3)

100

~ ~o

Q) .>_

-~

rr

o ,'o 5'0 o'o ;o ;o

Temperature (°C)

Fig. 2. Influence of temperature on endo-(1,3-1,4) fl-glucanase activity. The enzyme activities at various temperatures were measured by the standard assay at pH 7.0

Table 1. Substrate specificity of the/3-glucanase enzyme produced by Escherichia coli JM101 (pNA3)

Substrates U/ml U/rag protein

Lichenan 279.90 39.78

Laminarin 0.89 0.13

Carboxymethylcellulose 0.29 0.04

Xylan (birchwood) 0.43 0.06

Avicel 0.24 0.03

Table 2. Localization of the/3-glucanase enzyme produced by E.

coli JM101 (pNA3)

Cell fraction Distribution of total activity (%)

Extracellular 18

Periplasmic 36

Cytoplasmic 46

100

50

I I ~ 6[0

15 30 45

Incubation time (rain)

Fig. 3. Influence of temperature on the stability of endo-(1,3-1,4) /~-glucanase. The enzyme was assayed at pH 7.0

plasmic region with very little enzyme being secreted into the extracellular medium.

S D S - P A G E protein analysis

Zymograms that detected fl-glucanase activity of the renatured proteins separated by S D S - P A G E gave a molecular mass of approximately 29 kDa. When the gels

mants was selected and the residing plasmid was desig- nated pNA3. From preliminary mapping pNA3 was shown to harbour a 1.65-kb insert containing the fl-glucanase-encoding (bglBB) gene.

Substrate specificity o f the e n z y m e

The cloned B. brevis A L K 36 enzyme was characterized as an endo-(1,3-1,4)-fl-glucanase or lichenase on the basis o f its substrate specificity (Table 1).

Cellular localization o f fl-glucanase activity in E. coli JMIO1

In order to study the. distribution of the fl-glucanase syn- thesized in E. coli (pNA3) extracellular, periplasmic and cytoplasmic fractions were isolated and enzyme activity assayed (Table 2). Most o f the fl-glucanase enzyme activity was located in the cytoplasmic fraction and peri-

Fig. 4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis activity gel stained with Congo red: lane 1, sonicated extract of Escherichia coli JM101 (pEcoR251); lane 2, supernatant from Ba- cillus brevis Alk 36 grown for 24 h in Luria broth plus 0.1% lichenan; lane 3, sonicated extract of E. coli JM101 (pNA3)

(4)

510

TAT C ~ C J ~ T C ~ T ~ T A G G C ~ T T TAT TT T ~ , C A T T TA~TCJ~dkAT~T T ~ T ~ T ~ d k T ~ t F ~ i C ~ T T~T T T ~ T T ~ ~ T C ~ T T T ~ C ~ 1 0 0 t d ~ T T ~ . - , C A T J ~ T C ~ k T TC TCC~ATAT T T A ~ T A T T TC~I~T T ~ T T T T C ~ . _ - ~ A T T TT~CCC T C ~ ~ T ~ T T T T T T TC T ~ T ~ T ~ T ~ T 2 0 0 A A C C T G A C T T T T T G C A G T T A A T A G A A A A A G T A G T G A A A A A G A T T G A A T T T A T T T A A G A T T C A T T T A T A A T A A G A T T G A A A G C G C T T T C G A A A A T C C C G A A 3 0 0

- 3 5 - I 0

T T T T T A A A G T T G T T A C C G A A A A A G T A A C A A C A A A T T T T T T C G C T G T T T A C T A A C G C G C G T T A G T A A A C G G A T A G T A A C A A A A T G A T C C C T A T A A G G A G G A 400 S D

T G G A A G A T G G T A A A A A G T A A A T A T T T A G T T T T C A T T T C T G T T T T T T C T T T G T T G T T T G G A ~ T A T T T G T T G T T G G G T T T A G T C A T C A A G G G G T A A A A G C T G 5 0 0 M V K S K Y L V F I S V F S L L F G V F V V G F S H Q G V K A E

A A G A A ~ A G A G G C C A A T G G G A A C A G C G T T T T A T G A G T C G T T T G A T G C T T T T G A T G A C G A A C G T T G G T C T A A A G C A G G C G T C T G G A C A A A T G G C C A G A T G T T 600 E E R P M G T A F Y E S F D A F D D E R W S K A G V W T N G Q M F

C A A T G C A A C A T G G T A T C C A ~ A A C A G G T G A C G G C T G A C G G T C T A A T G A G A C T T A C T A T T G C A A A ~ A A ~ A C A A C A A G T G C T A G A A A C T A T A A A G C A G G A G A G 7 0 0 N A T W Y P E Q V T A D G L M R L T I A K K T T S A R N Y K A G E

C T T C G T A C C A A T G A T T T C T A T C A T T A T G G A C T C T T T G A A G T G A G T A T G A A G C C T G C G A A G G T A G A A G G G A C C G T G T C A T C C T T T T T T A C C T A C A C A G G G G 8 0 0

L R T N D F Y H Y G L F E V S M K P A K V E G T V S S F F T Y T G E

A A T G G G A T T G G G A T G G A G A T C C T T G G G A T G A A A T T G A T A T T G A G T T C T T A G G A A A G G A C A C G A C G A G A A T A C A A T T T A A T T A C T T T A C A A A T G G A G T A G G 9 0 0

W D W D G D P W D E I D I E F L G K D T T R I Q F N Y F T N G V G

z l d ~ G ~ T G ~ T T T TAC TATGATT TAGC~T T TGATC~2ATCJkGAGTCATT T ~ T A C GTATGCC T T TGI~T~AI~]xt'z-~-~AT TCCAT T ] I d 2 C T ~ T A T G T Ta~.T 1 0 0 0

G N E F Y Y D L G F D A S E S F N T Y A F E W R E D S I T W Y V N

G G A G A A G C G G T T C A T A C A G C G A C A G A A A A C A T T C C A C A G A C C C C C ~ C A G A A G A T C A T G A T G A A T T T A T G G C C G G G C G T G G G A G T A G A T G G A T G G A C A G G T G I I 0 0

G E A V H T A T E N I P Q T P Q K I M M N L W P G V G V D G W T G V

T A T T C G A T G G C G A T A A T A C G C C T G T A T A T T C A T A C T A T G A T T G G G T G A G G T A T A C A C C A C T T T A G A A T T A T C A G A T C C A C C A G T A A C A C C A G A A C C A C C A 1 2 0 0

F D G D N T P V Y S Y Y D W V R Y T P L

A T T G A A G A G C C G A C A G A A G A A C C A A T T G A A G A A C C A A T C G A A A A A C C A A T T G A A G A G C C T A T C G A G A A A A T C G A A A T A G A T G A G A A C G A C G A A A C G A A A G 1 3 0 0 A A A A C G T A A A C G A C T T T A A A G G A A T C T G G A G G T G A A A G A T T T A C C T T A A T A C A G G C G A C C T T C A A T G T A T T C A T T T A T T T T A C T A T T T T G G A C T T C T A G C 1 4 0 0 T T T T G C A A A T A G G A G T A A T G T T A C T T A A G G G C T A G A G T A A C A G A A A A G T A G G C T T T C A A G T A T T A A G A A A T T A C T G A T T A T C G T A A A A T A C A T T T T T T G T 1 5 0 0

Fig. 5. Nucleotide sequence of the insert in pNA3, containing the is shown in single-letter code below the coding sequence. Putative B. brevis bglBB gene. The nucleotide sequence is numbered promoter sequences (-35 and -10 regions) and the ribosome bind- throughout. The deduced amino acid sequence of the fl-glucanase ing site (SD) are underlined

were s t a i n e d , a z o n e o f a c t i v i t y was d e t e c t e d r u n n i n g t h e l e n g t h o f the P A G E gel (Fig. 4). T h i s i n d i c a t e d S D S - r e s i s t a n c e o f the e n z y m e , a n d it was f o u n d t h a t SDS-

Bm Bs Bs2 B1 Ba Bp Ct

P A G E gels c o u l d be s t a i n e d d i r e c t l y w i t h C o n g o r e d

w i t h o u t the r e m o v a l o f S D S . E n z y m e a c t i v i t y was as- Bb 53 51 50 51 49 54 46 s a y e d in t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f SDS Bm - - 69 69 68 70 88 49 ( r a n g i n g f r o m 0 . 1 % to 10%). It was f o u n d t h a t n o inhi- Bs - - - - 99 87 91 72 48 b i t i o n o f e n z y m e a c t i v i t y o c c u r r e d u p t o a c o n c e n t r a t i o n Bs2 - - - - - - 86 90 72 47

o f 10% S D S . B1 . . . . 84 73 54

Ba - - 71 46

Bp 46

N u c l e o t i d e sequence o f the b g l B B gene

T h e n u c l e o t i d e sequence o f the insert in p l a s m i d p N A 3 was d e t e r m i n e d (Fig. 5) a n d f o u n d to c o n t a i n a single o p e n r e a d i n g f r a m e ( O R F ) o f 759 nt, w h i c h b e g a n w i t h a n A T G at p o s i t i o n 407 a n d e n d e d w i t h a T A G at p o s i - t i o n 1165. T h e p r e d i c t e d size o f the p o l y p e p t i d e e n c o d e d b y this O R F was 252 a m i n o acids, w h i c h has a calcu- l a t e d r e l a t i v e m o l e c u l a r m a s s (Mr) o f 29000. A classical r i b o s o m e b i n d i n g site was l o c a t e d 6 - 1 4 nt u p s t r e a m o f t h e A T G i n i t i a t i o n c o d o n ( 5 ' - A A G G A G G A - 3 ' ) . A p u t a - tive p r o m o t e r s e q u e n c e , ( 5 ' - G A T T G A A T - N 1 6 - T A - T A A T - 3 ' ) was l o c a t e d u p s t r e a m f r o m the A T G s t a r t co- d o n f r o m p o s i t i o n 241 to 270 (Fig. 5). S e q u e n c e c o m p a r - isons at t h e n u c l e o t i d e a n d a m i n o a c i d levels i n d i c a t e d

Table 3. Similarity of nucleotides in different endo-(1,3-1,4)-fl- glucanase genes

Sequence data of the proteins were taken from Tezuka et al.

(1989) for Bacillus subtilis (Bs) and Murphy et al. (1984) for a se- cond B. subtilis strain (Bs2), Lloberas et al. (1991) for B. lichen#

formis (B1), Hofemeister et al. (1986) for B. amyloliquefaeiens (Ba), Borris et al. (1990) for B. macerans (Bm), Gosalbes et al.

(1991) for B. polymyxa (Bp), Zverlov et al. (1990) for Clostridium thermocellum (Ct) and B. brevis (Bb) (this paper)

t h a t this O R F was a f l - g l u c a n a s e - e n c o d i n g gene. T h e s i m i l a r i t y o f t h e d e d u c e d a m i n o a c i d sequence o f B. bre- vis f l - g l u c a n a s e t o the o t h e r r e p o r t e d f l - g l u c a n a s e s was d e t e r m i n e d o n the basis o f i d e n t i c a l a m i n o a c i d se- q u e n c e c o m p a r i s o n s ( T a b l e 3). T h e h i g h e s t s i m i l a r i t y was f o u n d to t h e p o l y p e p t i d e sequence f r o m B. macer-

(5)

6O B1 MS Y R V K R M L M L L V T G L F L S L S T F A A S A S A Q T . . . G G S F Y E P F - N N Y N T G L W Q K A D G Bs M P Y - L K R V L L L L V T G L F M S L F A V T S T A S A Q T . . . G G S F F D P F - N G Y N S G F W Q K A D G Ba M . . . . K R V L L I L V T G L F M S L C G I T S S V S A Q T . . . G G S F F E P F - N S Y N S G L W Q K A D G B s 2 M P Y- L K R V L L L L V T G L F M $ L F A V T A T A S A Q T . . . G G S F F D P F - N G Y N S G F W Q K A D G B m M - - K K K S C - F T L V T T F A F S L I - - - F S V S A L A . . . G S V F W E P L - S Y F N R S T W E K A D G Bp M - - M K K K S W F T L M I T G V I S L F - - - F S V S A F A . . . G N V F W E P L - S Y F N S S T W Q K A D G B b M V - K S K Y L V F I S V F S L L F G V F V V G F S H Q G V K A E E E R P M G T A F YE S F - D A F D D E R W S K A G V Ct M - - - K N R V I S L L M A S L L L V L S V I V A P F - - Y K A E A A T W N T P F V A V F R S N F D S V Q W K K - - -

* * ~ ~

• .

120 B1 Y S N G N M F N C T W R A N N V S M T S L G E M R L S LT S P - - S Y N K F D C G E N R S V Q T Y G Y G L Y E V N M K P Bs Y S N G N M F N C T W R A N N V S M T S L G E M R L A L T S P - - S Y N K F D C G E N R S V Q T Y G Y G L Y E V R M K P B a YS N G D M F N C T W R A N N V S M T S L G E M R L A L T S P - - S Y N K F D C G E N R S V Q T Y G Y G L Y E V E M K P B s 2 Y S N G N M F N C T W R A N N V S M T S L G E M R L A L T S P - - A Y N K F D C G E N R S V Q T Y G Y G L Y E V R M K P B m Y S N G G V F N C T W R A N N V N F T N D G K L K L G L T S S - - A Y N K F D C A E Y R S T N I Y G Y G L Y E V S M K P Bp Y S N G Q M F N C T W R A N N V N F T N D G K L K M S L T $ P - - A N N K F D C G E Y R S T N N Y G Y G L Y E V S M K P B b W T N G Q M F N A T W Y P E Q V T A D G L M R L T I A K K T T - - S A R N Y K A G E L R T N D F YH Y G L F E V S M K P Ct - - R W A K F V S T V L E A F T G D I S N G K M I L T L D R E Y G G S YP YKS G E Y R T K S F F G Y G Y Y E V R M K A

* W 9 * W W * * W W

180 B1 A K N V G I V S S F F T Y T G P T D - - G T P W D E ID I E F L G K D T T K V Q F N Y Y T N G V G N H E K I V N L G F D Bs A K N T G I V S S F F T Y T G P T D - - G T P W D E ID I E F L G K D T T K V Q F N Y Y T N G A G N H E K I V D L G F D B a A K N T G I V S S F F T Y T G P T E - - G T P W D E ID I E F L G K D T T K V Q F N Y Y T N G A G N H E K F A D L G F D Bs2 A K N T G I V S S F F T Y T G P T D - - G T P W D E ID I E F L G K D T T K V Q F N Y Y T N G A G N H E K I V D L G F D B m A K N T G I V S S F F T Y T G P A H - - G T Q W D E ID I E F L G K D Q T K V Q F N Y Y T N G V G G H E K V I S V G F D B p A K N T G I V S S F F T Y T G P S H - - G T Q W D E ID I E F L G K D T T K V Q F N Y Y T N G V G G H E K I I N L G F D B b A K V E G T V S S F F T Y T G E W D W D G D P W D E ID I E F L G K D T T R I Q F N Y F T N G V G G N E F Y Y D L G F D Ct A K N V G I V S S F F T Y T G P $ - - D N N P W D E ID I E F L G K D T T K V Q F N W Y K N G V G G N E Y L H N L G F D

W W W W W W * W W ~ W * * W W W W * * * * * W W * . . * * ~ * W W . ~ W W *

240 B 1 A A N S YH T Y A F D W Q P NS I K W Y V D G Q L K H T A T TQ I P Q T P G K I M M N L W N G A G V D E W L G S Y N G V Bs A A N A Y H T Y A F D W Q P N S I K W Y V D G Q L K H T A T N Q IP T T P G K I M M N L W N G T G V D E W L G S Y N G V B a A A N A Y H T Y A F D W Q P N S I K W Y V D G Q L K H T A T TQ I P A A P G K I M M N L W N G T G V D D W L G S Y N G V B s 2 A A N A Y H T Y A F D W Q P N S I K W Y V D G Q L K H T A T N Q IP TT L G K I M M N L W N G T G V D E W L G S Y N G V B m AS K G F H T Y A F D W Q P GY I K W Y V D G V L K H T A T A N IP STP G K I M M N L W N G T G V D D W L G S Y N G A B p AS TS F H T Y A F D W Q P GY I K W Y V D G V L K H T A T T N IP STP G K I M M N L W N G T G V D S W L G S Y N G A B b AS E S F N T Y A F E W R E D S I T W Y V N G E A V H T A T E N I P Q T P Q K I M M N L W P G V G V D G W T G V F D GD Ct AS QD F H T Y G F E W R P D Y I D F Y V D G K K V Y R G T R N I P V T P G K I M M N L W P GI G V D E W L G R Y D G -

* . • . W W . W . W . * W W . W . W . W W W W * W W W W W W * W W W • ,•

256 B I T - P L S R S L H W V R Y T K R Bs N - P L Y A H Y D W V R Y T K K B a N - P I Y A H Y D W M R Y R K K B s 2 N - P L Y A H Y D W V R Y T K K B m N - P L Y A E Y D W V K Y T S N B p N - P L Y A E Y D W V K Y T S N B b N T P V Y S Y Y D W V R Y T P L Ct R T P L Q A E Y G I C K I L S -

W .

Fig. 6. Alignment of the amino acid se- quence of the B. brevis (Bb) beta-gluca- nase with enzymes from different Bacil- lus strains. Identical and conserved ami- no acids among the eight proteins are marked with asterisks and dots, respec- tively

ans (53%) and to that of B. p o l y m y x a (54%), but all sequences showed significant sequence similarities. It is.

interesting to note that these two sequences from B. ma- cerans and B. p o l y m y x a are very closely related (88%

indentity), whereas the mesophilic fl-glucanases form a separate cluster based on h o m o l o g y as previously re- ported (Hofemeister et al. 1986). The B. brevis fl-gluca- nase gene and the Clostridium thermocellum laminari- nase show a greater divergence in sequence similarity.

Alignment of the predicted polypeptides from the differ- ent Bacillus fl-glucanases and the C. thermocellum lami- narinase (Fig. 6) revealed considerable h o m o l o g y in a number of highly conserved blocks, particularly in the central and the C-terminal parts of the proteins. The N- terminal region has the most pronounced variation be- tween all the different sequences.

Discussion

The endo-(1,3-1,4)-fl-glucanase isolated from B. brevis Alk 36 has some unique properties. It has a higher tem- perature optimum then previously reported for Bacillus lichenases, with the exception of B. macerans. The tem- perature profiles of the B. maeerans and B. brews li- chenases are similar, but the B. brevis lichenase exhibits a greater residual activity at a higher temperature than that of the B. macerans enzyme, viz 75% activity after 1 h at 75°C for B. brevis lichenase as opposed to ap- proximately 10% activity after 1 h at 70°C for B. ma- cerans enzyme.

The pH optimum of the enzyme is unusual as the pH optima for bacterial lichenases tend to fall between pH 6.0 and pH 8.0, whereas that of the B. brevis lichenase exhibits activities of greater then 80% from pH 7.0 to pH 10.0. This pH profile is, however, similar to that of the fl-l,3-glucanase isolated from an alkalophilic Bacil-

(6)

512

lus sp. by Nogi and Horikoshi (1990) and to the fl-l,3- glucanase or laminarinase isolated f r o m C. thermoceI- lum by Zverlov and Velikodvorskaya (1990). These alkalophilic laminarinase enzymes b o t h have similar ther- mostability profiles to that of the B. brevis lichenase.

The distinction between fl-l,3-glucanase and (1,3-1,4)-/?- glucanases can be rather tenuous as it is based on substrate specificity for laminarin as o p p o s e d to lichenan, respectively. Since the substrate specificity o f the B. bre- vis enzyme was found to be 300-fold m o r e for lichenan than laminarin, it was classified as a (1,3-1,4)-/?-glucanase. The laminarinase f r o m C. thermocelIum was, however, classified as such despite a 100-fold higher activity on lichenan (Zverlov and Velikodvorskaya 1990).

Another novel characteristic of the B. brevis (1,3- 1,4)-fl-glucanase enzyme is its resistance to SDS; this is the first report of an SDS-resistant fl-glucanase. A m o - lecular mass of a p p r o x i m a t e l y 29 k D a was derived by S D S - P A G E . This agrees with the molecular mass calculated f r o m the sequence of 29050 Da and is similar to the previously published endo-(1,3-1,4)-fl-glucanases, which are estimated to be between 25 and 30 kDa. An exception is the endo-(1,3-1,4)-fl-glucanase cloned f r o m B. circulans, which was reported to have a molecular mass of 40.5 k D a (Bueno et al. 1990). There is, however, some discrepancy as to whether this enzyme is a true lichenase due to the absence in the sequence of the conserved active centre found in all bacterial lichenases. In addition, its b r o a d enyzme substrate specificity suggests that it might no be a true lichenase (Gosalbes et al.

1991).

The N-terminal portion o f the/?-glucanase polypep- tide deduced f r o m the nucleotide sequence contains a typical signal sequence. A positively charged N-termin- us, with lysine residues at position 3 and 5, is followed by a highly h y d r o p h o b i c region f r o m residues 7 to 24.

There is a valine at position 29 followed by an alanine at 31 which conforms with the "-3, -1 rule" for cleavage sites p r o p o s e d by von Heijne (1988). This evidence points to a possible signal sequence of approximately 31 amino acids that m a y be cleaved f r o m the mature protein. However, the B. brevis/?-glucanase protein f r o m cell extracts of E. coli or supernatants of B. brevis had an Mr of 29 kDa, which agrees with the size calculated f r o m the nucleotide sequence. Localization experiments indicated that the protein was found mostly in the cytoplasmic and periplasmic fractions and to a lesser extent in the extracellular medium. It has been suggested that the production o f fl-glucanase alters the permeability of the outer m e m b r a n e of E. coli and so allows leakage of the periplasmic space proteins (Cantwell et al. 1988).

C o m p a r i s o n of the amino acid sequences of the different fl-glucanases showed that the genes are strongly conserved and that regions of identical amino acids are found t h r o u g h o u t the sequence. The most extensive of these is the block (amino acids 143 to 161) p r o p o s e d by Borris et al. (1990) to contain the active site of fl-glucanases. This was found to be conserved in all the fl-glucanase enzymes. The N-terminal region is least conserved between the all different sequences, in agreement with Hofemeister et al. (1986) who showed a great variation

in the signal sequences of the highly homologous B. sub- tilis and B. amyloliquefaciens fl-glucanase genes. C o m - parisons of the sequences of different/?-glucanase proteins exhibiting novel characteristics should facilitate the identification of the roles played by particular amino acid residues and, ultimately, the modification of these proteins to suit specific industrial processes.

Acknowledgements. The authors wish to thank Ilze Meyer for her expert technical assistance as well as Di James for her invaluable help in sequencing.

References

B6guin P (1983) Detection of cellulase activity in polyacrylamide gels using Congo Red-stained agar replicas. Anal Biochem 131 : 333-336

Borriss R, Buettner K, Maentsaelae P (1990) Structure of the beta- 1,3-1,4 glucanase gene of Bacillus macerans: homologies to other beta-glucanases. Mol Gen Genet 222:278-283

Bueno A, Vazquez de Aldana CR, Correa J, Villa TG, Rey F del (1990) Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-fl-glucanase in Escherichia coll. J Bacteriol 172: 2160- 2167

Cantwell BA, McConnell DJ (1983) Molecular cloning and expression of Bacillus subtilis fl-glucanase gene in Escherichia coli.

Gene 23:211-219

Cantwell BA, Sharp PM, Gormley E, McConnell DJ (1988) Mo- lecular cloning of Bacillus fl-glucanases. In: Aubert J-P, B6g- uin P, Millet J (eds) Biochemistry and genetics of cellulose de- gradation. Academic Press, London, pp 181-201

Cornelis P, Digneffe G, Willemot K (1982) Cloning and expression of a Bacillus coagulans gene in Escherichia coli. Mol Gen Genet 186: 50%511

Gosalbes M J, P6rez-Gonzfilez JA, Gonzfilez R, Navarro A (1991) Two fl-glycanase genes are clustered in BaciIluspolymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and a endo-fl-(1,3)-(1,4)-glucanase. J Bacte- riol 173:7705-7710

Gryczan TJ, Contente S, Dubnau D (1978) Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol 134: 318-329

Heijne G yon (1988) Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta 947 : 307-333

Heinikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28 : 351-359

Herbert D, Phipps P J, Strange RE (1971) Chemical analysis of microbial cells. Methods Microbiol 5B:209-344

Hofemeister J, Kurtz A, Borriss R, Knowles J (1986) The fl-glucanase gene from Bacillus amyloliquefaciens shows extensive ho- mology with that of Bacillus subtilis. Gene 49:177-187 Laemmli UK (1970) Cleavage of structural proteins during the as-

sembly of the head of bacteriophage T4. Nature 227 : 680-685 Lloberas J, Perez-Pons JA, Querol E (1991) Molecular cloning, expression and nucleotide sequence of the endo-fl-l,3-1,4-D- glucanase gene from Bacillus licheniformis. Eur J Biochem

197 : 337-343

Lovett PS, Keggins KM (1979) B. subtilis as a host for molecular cloning. Methods Enzymol 68:342-357

Messing J (1979) A multipurpose cloning system based on single stranded DNA bacteriophage M13. Recomb DNA Tech Bull 2:43

Murphy N, McConnell DJ, Cantwell BA (1984) The DNA sequence of the gene and genetic control sites for the excreted B.

subtilis enzyme fl-glucanase. Nucleic Acids Res 12:5355-5367

(7)

Nogi Y, Horikoshi K (1990) A thermostable alkaline fl-l,3-glucanase produced by alkalophilic Bacillus sp. AG-430. Appl Mi- crobiol Biotechnol 32: 704-707

Norris JR, Berkeley RC, Logan NA, O'Donnell AG (1981) The genera Bacillus and Sporolactobacillus. In: Starr MP, Stolp H, Trtiper HG, Barlows A, Schlegel HG (eds) The Prokaryotes, vol II: Springer, Berlin Heidelberg New York, pp 1724-1725 Sambrook J, Fritsch EF, Maniatis DF (1989) Molecular cloning: a

laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, N. Y.

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Nat Acad Sci USA 74 : 5463-5467

Takeuchi K, Nishino T, Oders M, Shiiuogaki H (1989) Alkaline

proteases and microorganisms producing same. US patent no.

4797362

Tezuka H, Yuuki T, Yabuuchi (1989) Construction of a/~-glucanase hyperproducing Bacillus subtilis using the cloned/3-glucanase gene and a multi-copy plasmid. Agric Biol Chem 53 : 2335-2339

Zabeau M, Stanley KK (1982) Enhanced expression of cro-/?-gal- actosidase fusion proteins studied under the control of the PR promoter of bacteriophage 2. EMBO J 1 : 1217-1224

Zverlov VV, Velikodvorskaya GA (1990) Cloning the Clostridum thermocellum thermostable laminarinase gene in Escherichia coli, the properties of the enzyme thus produced. Biotechnol Lett 12:811-816