Appl Microbiol Biotechnol (1992) 38:243-247

Applied .. Microbiology Biotechnology © Springer-Verlag 1992

Cloning and expression of a thermostable exo-o -l,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli Yukio Takii, Katsuya Daimon, Yuzuru Suzuki Department of Agricultural Chemistry, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606, Japan Received 24 January 1992/Accepted 20 July 1992

Abstract. The gene coding for a thermostable exo-c~-l,4 glucosidase (a-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus A T C C 12016 was cloned within a 2.8-kb A v a I fragment of D N A using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exoa-l,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-AlaTyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.

Introduction Most bacterial a-glucosidases hitherto known fail to hydrolyze a-glucans such as soluble starch, amylose, amylopectin and glycogen (Kelly and Fogarty 1983; Fogarty and Kelly 1990). We found a novel p-nitrophenyl-a-Dglucopyranoside-hydrolyzing a-glucosidase in Bacillus stearothermophilus ATCC12016 (Suzuki et al. 1984). This enzyme was named exo-a-l,4-glucosidase (a-glucoside glucohydrolase: EC 3.2.1.20). The enzyme can release o~-glucose residues by cleaving a - l , 4 bonds successively from the non-reducing ends of maltosaccharides, a-limit dextrins, amylose, amylopectin, soluble starch and glycogen. However, the enzyme fails to hydrolyze the a - l , 6 bonds of isomaltose, panose, a-limit dextrins or the branched a-glucans mentioned above (Suzuki et al. 1984). Recently, Watanabe et al. (1990) found high sequence homology between Bacillus cereus ATCC7064 oligo-l,6-glucosidase and Saccharomyces carlsbergensis C B l l a-glucosidase. The same authors suggested that

Correspondence to: Y. Suzuki

these enzymes have the same folded conformation. This strongly indicates the presence of c o m m o n structural motifs between a-glucosidases acting on c~-l,4-glucosidic bonds and those acting on a-l,6-glucosidic bonds. In the present study, as a first step towards fulfiling this proposal, a gene coding for Bacillus stearothermophilus exo-a-l,4-glucosidase was cloned and expressed in Escherichia coli.

Materials and methods Escherichia coli strains and cultivation. E. coli strains JM109 and C600 (Brent and Irwin 1987) were used. Aerobic cultivation was carried out at 37° C. L-Broth (pH 7.2) consisted of 1% (w/v) peptone, 0.5% yeast extract, 0.5% NaC1 and 0.1% glucose. Medium I (pH 8.0) contained 0.3% KHzPO4, 0.6% NazHPO4, 4% NH4C1, 0.05% NaC1, 0.2% glucose, 0.2% casamino acid, 2 mM MgSO4, 0.1 mM CaC12 and 50gg ampicillin/ml. Liquid cultivation was carried out by rotatory shaking at 190 cycles/rain (3.4 cm amplitude) of 2-1 Erlenmeyer flasks each containing 200 ml culture medium, or by reciprocal shaking at 120 oscillations/min (3.9 cm amplitude) of test tubes (1.8 x 18 cm) each containing 5 ml culture medium. Cell concentrations were expressed as dry cell weight per millilitre of culture (Suzuki et al. 1976). DNA manipulations. All DNA manipulations were as described by Maniatis et al. (1982). Restriction endonucleases and T4 DNA ligase were used as specified by the suppliers (Toyobo, Osaka, Japan; Takara Shuzo, Kyoto, Japan). Cloning of exo-c~:l,4-glucosidase gene. Chromosomal DNA from B. stearothermophilus ATCC12016 ceils in the exponential growth phase (Suzuki et al. 1984) was purified as described by Miura (1967). The B. stearothermophilus DNA was partially digested with restriction endonuclease AvaI. Vector plasmid pUC19 was cleft to completion with A vaI and treated with calf intestine alkaline phosphatase (Boehringer Mannheim Yamanouchi, Tokyo, Japan). Both digests (1 ~tg of strain ATCC12016 DNA and 0.3 ~tg of vector DNA) were ligated for 16 h at 4°C with T4 DNA ligase. The mixture was used to transform E. coli JM109. Th transformants (about 200 per plate) were grown on 1.5°70 agar plate of L-broth containing 50 ~xgampicillin/ml, 0.1 mM isopropyl-thiogalactoside (IPTG) and 20 ~tg/ml 5-bromo-4-chloro-3-indolyl-fl-Dgalactoside. Colourless clones achieved were screened for exo-c~1,4-glucosidase activity at 60° C by using p-nitrophenyl-c~-D-glucopyranoside (pNPG) (Paoni and Arroyo 1984). One positive clone

244 was obtained among 830 colonies. A plasmid (named pBST) was isolated from the clone. E. coli JM109 was transformed again with pBST, which yielded 100070 ampicillin-resistant colonies presenting exo-a-l,4-glucosidase activity. Endonuclease analysis of pBST on agarose gel electrophoresis (Maniatis et al. 1982) revealed the insertion of 2.8-kb Aval fragment of DNA within pUC19. This fragment was isolated from the gel. The fragment was ligated with pBR322 previously treated with AvaI and with alkaline phosphatase. The ligation mixture was used to transform E. coli C600. An ampicillin- and tetracycline-resistant transformant presenting exo-c~-l,4-glucosidase activity was isolated; it contained a plasmid, named pBST(322). This plasmid comprised a 2.8-kb AvaI fragment inserted within pBR322. Transformation of E. coli C600 with pBST(322) gave the host exo-a-l,4-glucosidase activity.

Purification o f cloned exo-c~-l,4-glucosidase. All steps of enzyme purification were carried out at 4°C unless otherwise stated. E. coli JM109 bearing the hybrid plasmid pBST was cultivated on medium I in Erlenmeyer flasks with shaking at 37°C for 16 h. Cells (wet weight, 3.4 g from a 1.2-1 culture) collected by centrifugation at 5000 g for 30 min was disrupted in 50 mM potassium phosphate/5 mM ethylenediaminetetraacetate (EDTA), pH 7.0, by sonication (Suzuki et al. 1976). The suspension was centrifuged (8000 g, 30 min). The supernatant (67 ml) was heated at 60° C for 15 min, and then it was centrifuged. The supernatant was dialyzed against 5 m s potassium phosphate/25 ~M EDTA (pH 6.8, buffer A). The dialyzate was applied to a hydroxylapatite Bio-Gel HTP (Bio-Rad Laboratories, Richmond, Calif., USA) column (4.5 x 24 cm) equilibrated with buffer A. The column was washed with 900 ml buffer A and then with 1000 ml of a linear 5-200 mM phosphate gradient in buffer A. The flow rate was 16 ml/h. Active fractions (15 ml each) that appeared between 140 and 180mM phosphate were combined. The solution was dialysed against buffer A. The diaiyzate (275 ml) was applied to a diethylaminoethyl (DEAE)-Sephacel (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) column (1.5 x 20 cm) equilibrated with buffer A. The column was washed with a linear 0-100 mM NaC1 gradient in buffer A (240 ml). The flow rate was 20 ml/h. Active fractions (each 5 ml) eluting between 50 and 90 mM NaC1 were pooled. The solution (122mi) was concentrated by ultafiltration with Amicon (Danvers, Mass., USA) PM-10 membrane. The concentrate (3.1ml) was applied to a Bio-Gel P-150 column (2.0x95cm) equilibrated with buffer A/0.1 M NaC1/0.02% NAN3. The column was washed with the same buffer at a rate of 15 ml/h. Active fractions (62-79, 2 ml each) were combined (Fig. 1). The solution (40 ml) was stored at - 2 0 ° C. B. stearothermophilus ATCC12016 exo-a-l,4-glucosidase was purified as described previously (Suzuki et al. 1984). Enzyme assay. Cellular, extracellular and periplasmic exo-c~-l,4glucosidase activities were assayed with pNPG (Suzuki et al. 1976; Watanabe et al. 1989). One unit (U) of exo-a-l,4-glucosidase was defined as the amount of enzyme hydrolysing 1 ~tmol pNPG for 1 min at 60° C and pH 6.8. Protein concentration was determined by the method of Lowry et al. (1951).

Results and discussion Figure 2 shows the restriction m a p of the 2.8-kb A v a I D N A f r a g m e n t f r o m the B. s t e a r o t h e r m o p h i l u s A T C C 1 2 0 1 6 c h r o m o s o m a l D N A , the f r a g m e n t that was within the h y b r i d p l a s m i d pBST (see Materials a n d methods). O n d o u b l e i m m u n o d i f f u s i o n , the a n t i s e r u m against B. s t e a r o t h e r m o p h i l u s A T C C 12016 exo-c~- 1,4glucosidase p r o d u c e d single precipitin lines with this enzyme a n d with the cell extract of E. coli JM109 b e a r i n g pBST (Fig. 3). Both lines fused completely w i t h o u t a

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Fig. 2. Restriction maps of the cloned fragment of Bacillus stearothermophilus ATCC12016 DNA associated with the exo-~-l,4glucosidase activity. Three digests were prepared from the 2.8-kb AvaI fragment in pBST with PvuII, SacI and XhoI. Each fragment was ligated with pUC19 digested by two of the corresponding nucleases, except that SalI was used instead of XhoI. The resultant plasmids, pBST(PX), pBST(SaA) and pBST(ASa), shown in the figure were used to transform E. coli JM109. Transformants were assayed for exo-c~-l,4-glucosidase as in Materials and methods. The box in the figure indicates the fragment of B. stearothermophilus ATCC12016 DNA cloned in the hybrid plasmid. The thin horizontal lines attached to the box represent the part of the vector pUC19 in the hybrid plasmid. Vertical broken lines crossing the boxes indicate endonuclease restriction sites; +, positive and - , negative enzyme activities of transformants tested; E, EcoR1; A, AvaI; B, BanIII; H, HincII; H', HindIII; N, NruI; P, PvuII; Sa, SacI; Sc, ScaI; X, XhoI

spur. These data suggested that the gene coding for the exo-o~-l,4-glucosidase is located within the 2.8-kb A v a I fragment. E. coli JM109 b e a r i n g pBST a n d E. coli C600 b e a r i n g pBST(322) were cultivated at 37 ° C in tubes with shaking for 16 h in L-broth c o n t a i n i n g 50 ~tg a m p i c i l l i n / m l ( W a t a n a b e et al. 1989). N o significant difference was

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]Fig. 3. Double immunodiffusion tests. Rabbit antiserum (6 gl) (Suzuki et al. 1984) against B. stearothermophilus ATCC12016 exo-ce-l,4-glucosidase was added to the centre well (3 mm diameter) in an agarose plate; wells B and E contained the exo-a-l,4glucosidase (3.1 ~tg) purified from strain ATCC 12016; wells A and D contained the exo-a-l,4-glucosidase (1.2 gg) purified from E. coil JM109 bearing pBST; wells C and F contained 10 ~tl cell extract (0.35 U) from E. coli JM109 bearing pBST. The same precipitin pattern was observed when wells C and F each contained extracellular, cytoplasmic or periplasmic fraction from this strain, or when the wells contained cell extract of strain ATCC12016. The above subcellular fractions used were prepared as in Fig. 4 and concentrated by using an Amicon Centricon-10 microconcentratoe Immunodiffusion was at 4°C for 24h (Suzuki et al. 1984). The plate was soaked at 22°C overnight in 0.85% NaC1/0.1% NaN3 and was photographed

found between the level of exo-c~-l,4-glucosidase production (0.81-0.92 U / m l of culture) for cells bearing pBST and that for cells bearing pBST(322). A l s o , the level of enzyme production was not affected by the addition of I P T G ( 0 . 1 - 1 0 m s ) to the culture of E. coli JM109 bearing pBST. F r o m these findings, the gene is most probably transcribed under the control o f its own p r o m o t e r present in the 2.8-kb A v a I fragment. Figure 2 shows clearly that the gene is situated within the 2.0-kb P v u l I - X h o I region of the 2.8-kb A v a I fragment. This is consistent with evidence that the relative molecular mass (M,.) of the ATCC12016 enzyme is about 62000 (see Fig. 5). E. coil JM109 bearing pBST was cultivated at 37°C in tubes with shaking (Fig. 4). E. coli produced cellular

Table 1. Purification of exo-c~-l,4-glucosidase from Escherichia coti JM109 bearing pBST

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(hours)

Fig. 4. Change in exo-~-l,4-glucosidase activity in the extracellular fraction (©), the cellular periplasmic fraction (~7) and the cellular cytoplasmic fraction (0) during cultivation of E. coil JM109 bearing pBST. Cells were grown on medium I in test tubes with shaking at 37° C. The cell concentration of the culture (D) and its pH (A) were measured at defined intervals. The subcellular fractionation of culture was carried out as described before (Suzuki et al. 1976; Watanabe et al. 1989). The enzyme activity was expressed as the value found in 1 ml of culture

exo-ot-l,4-glucosidase in parallel with growth. The production reached nearly a m a x i m u m after 16 h o f cultivation, corresponding to the early stage of the stationary phase. The level of enzyme production (3.3 U / m l culture, 2.8 U / m g dry cells) was about sevenfold higher than that observed for B. stearothermophilus ATCC12016 (Suzuki et al. 1984). The enzyme appeared in the culture broth after 12 h of cultivation, corresponding to the middle stage of the logarithmic phase, and it accumuled slowly. However, the level o f extracellular enzyme increased abruptly after 20 h of cultivation. This was consistent with a decrease in cell concentration. Only 2% and 4% o f the total enzyme activity was found in the periplasm in cells after 16 h and 24 h of cultivation, respectively. The extracellular, cellular and periplasmic forms of exo-ot-l,4-glucosidase showed identical serological patterns on double immunodiffusion with the antiserum against B. stearothermophilus ATCC12016 enzyme (Fig. 3). These observations, along with the kinetics of enzyme production (Fig. 4), suggest that exo-c~-l,4-glucosidase is produced in E. coil JM109 bearing pBST as a cytoplasmic protein, and it is later released outside the cells only as a result of cell lysis, as in B. stearothermophilus ATCC12016 (Suzuki et al. 1984).

Purification steps

Total protein (mg)

Total activity (U)

Specific activity (U/rag protein)

Yield (%)

Purity

Crude extract Heat treatment fraction Hydroxylapatite eluate DEAE-Sephacel eluate Bio-Gel eluate

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Fig. 5A-E. Electrophoresis of purified exo-a-l,4-glucosidases from strains of B. stearotherrnophilus ATCC12016 and E. coli JM109 bearing pBST. Disc gel electrophoresis was performed at 4° C for 3 h at 2 mA/tube, using 5 ~tg each of exo-a-l,4-glucosidase of E. coli JM109 bearing pBST (A) or of B. stearothermophilus ATCC12016 (B) (Davies 1964). Sodium dodecyl sulphate (SDS)-gel electrophoresis was carried out for 8 h at 25° C, using 3 ~tg of exo-a-l,4-glucosidase from E. coli JM109 bearing pBST (C) or 7 ~tg of B. stearothermophilus ATCC12016 exo-a-l,4-glucosidase (D) or 2 Ixgeach of the relative molecular mass (Mr) markers (Weber and Osborn 1969). The disc gels (A,B) and the SDS gels (C, D) (each gel diameter=0.5 cm) were stained with Amidoblack and with Coomassie brilliant blue, respectively (Davis 1964; Weber and Osborn 1969). In the SDS gel (E), the following Mr markers were used: 1, bovine serum albumin (Mr, 67000); 2, ovalbumin (M, 45000), 3, bovine erythrocyte carbonic anhydrase (Mr, 29000) and 4, soybean trypsin inhibitor (Mr, 20100)

Table 1 summarizes the purification of cloned exo~-l,4-glucosidase (see Materials and methods). The final preparation gave a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) (Fig. 5). The purified enzyme absolutely agreed with B. stearothermophilus ATCC12016 exo-a-l,4-glucosidase in its Mr (62000) determined by SDS-PAGE (Fig. 5), isoelectric point (5.0) (Wrigley 1968), electrophoretic behaviour on native and denaturing polyacrylamide gels (Fig. 5), the N-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-AlaTyr-Gln-Ile-Tyr,) (Watanabe et al. 1990), its antigenicity judged by double immunodiffusion (Fig. 3), the temperature dependence of its activity (most active at 70 ° C) and of its stability (50°70 inactivation at 69 ° C in 10 min) (Fig. 6), the p H dependency of its activity (optimum at p H 6.1) (Suzuki et al. 1984), its activity for maltose, maltotriose and soluble starch (Table 2), and in the lack of its activity for isomaltose (Table 2).

o

~

,~

Fig. 6. Effect of temperature on the activity (A) and stability (O) of the exo-a-l,4-glucosidase purified from E. coli JM109 bearing pBST, and on the activity (I,) and stability (O) of the exo-c~-l,4glucosidase purified from B. stearothermophilus ATCC12016. The activity was measured at temperatures from 4 to 90° C (Suzuki et al. 1984), and the activity observed at 70° C was expressed as 100%. The enzyme (1.1 gg/ml in buffer A) was treated for 10 min at various temperatures, and then the activity recovered was determined (Suzuki et al. 1984). The activity observed at 4°C was defined as 100°70

Table 2. Hydrolysis of various sugars by exo-a-l,4-glucosidase from E. coli JM109 bearing pBST

Sugars

Final concentration

Maltose Maltotriose Isomaltose Soluble starch

10 mM 10 mM 10 mM 1°7o

Glucose formation (~tmol/min/mg . protein) a 530 663 0 311

~ Determined at 60°C and at pH 6.8 by the method of Trinder (1969) as quoted by (Suzuki et al. 1984) The cloned exo-~-l,4-glucosidase appeared to be non-glycosylated like the B. stearothermophilus ATCC12016 enzyme (Dubois et al. 1956; Suzuki et al. 1984). On double immunodiffusion, the antiserum against B. stearothermophilus A T C C 12016 exo-a- 1,4glucosidase produced single precipitin lines with the cell extract from E. coli JM109 bearing pBST, the exo-a1,4-glucosidase purified f r o m this E. coli strain, the cell extract f r o m strain ATCC12016, and the exo-~-l,4-glucosidase purified from this microbe (Fig. 3). These lines fused completely with each other without a spur. All the observations presented above suggest that exo-a-l,4-glucosidase is produced in E. coli without any processing or modification, as in B. stearothermophilus ATCC12016.

247

References Brent R, Irwin N (1987) Vectors derived from plasmids. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Seidman JG, Struhl K (eds) Current protocols in molecular biology. Wiley, New York, pp 1.5.1-1.5.8 Davis BT (1964) Disc electrophoresis II. Method and application to human serum proteins. Ann NY Acad Sci 121:404-427 Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350-356 Fogarty WM, Kelly CT (1990) Recent advances in microbial amylases. In: Fogarty WM, Kelly CT (eds) Microbila enzymes and biotechnology, 2nd edn. Elsevier Applied Science, London pp 71-132 Kelly CT, Fogarty WM (1983) Mirobial c~-glucosidases. Process Biochem 18:6-12 Lowry OH, Rosebrough N J, Farr AI, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193 : 265-275 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. Miura K (1967) Preparation of bacterial DNA by the phenol-pH 9-RNases method. Methods Enzymol 12."543-545 Paoni NF, Arroyo RL (1984) Improved method for detection of

glycosidases in bacterial colonies. Appl Environ Microbiol 47 : 208-209 Suzuki Y, Kishigami T, Abe S (1976) Production of extracellular c~-glucosidase by a thermophilic Bacillus species. Appl Environ Microbiol 31 : 807-812 Suzuki Y, Shinji M, Eto N (1984) Assignment of a p-nitrophenyle~-D-glucopyranoside-hydrolyzing a-glucosidase of Bacillus stearothermophilus ATCC12016 to a novel exo-c~-l,4-glucosidase active for oligomaltosaccharides and c~-glucans. Biochem Biophys Acta 787:281-289 Trinder P (1969) Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 6: 24-27 Watanabe K, Iha H, Ohashi A, Suzuki Y (1989) Cloning and expression in Escherichia coli of an extremely thermostable oligo-l,6-glucosidase gene from Bacillus thermoglucosidasius. J Bacteriol 171 : 1219-1222 Watanabe K, Kitamura K, Iha K, Suzuki Y (1990) Primary structure of the oligo-l,6-glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene. Eur J Biochem 192: 609-620 Weber K, Osborn M (1969) The reliability of molecular weight determination by dodecyl sulfates-polyacrylamide gel electrophoresis. J Biol Chem 244: 4406-4412 Wrigley CW (1968) Analytical fractionation of plant and animal proteins by gel electrophoresis. J Chromatogr 36:362-365