Vol. 58, No. 11

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1992, p. 3455-3465

0099-2240/92/113455-11$02.00/0 Copyright X 1992, American Society for Microbiology

Cloning, Characterization, and Nucleotide Sequence of a Gene Encoding Microbispora bispora BglB, a Thermostable ,-Glucosidase Expressed in Escherichia coli RICHARD M. WRIGHT,* MICHAEL D. YABLONSKY, ZAMIR P. SHALITA, ANIL K. GOYAL, AND DOUGLAS E. EVELEIGH Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick; New Jersey 08903-0231 Received 21 April 1992/Accepted 10 August 1992

Genomic DNA frgments encoding 1-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing e-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbeliiferyl-o-D-glucoside. Two genes encoding 13-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a 13-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60°C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-f-D-glucosides and was thermostable, retaining about 70%o of its activity after 48 h at 60°C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bgiB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with 13-glucosidases from glycosal hydrolase family 1.

Cellulose, an abundant but recalcitrant biopolymer, is composed of repeating glucose units linked by P-1,4-glycosidic bonds. Cellulases, produced by a wide variety of microorganisms, degrade such polymers and play a major role in the recycling of biomass. Cellulases are multicomponent complexes which are often composed of endoglucanases (1,4-0-D-glucan glucano-hydrolase, EC 3.2.1.4), cellobiohydrolases (1,4-0-D-glucan cellobiohydrolase, EC 3.2. 1.91), and cellobiases (1,4-0-D-glucoside glucohydrolases, EC 3.2.1.21). Cellobiases, while specific for cellobiose, belong to a very diverse family of enzymes (,-glucosidases) capable of hydrolyzing a broad spectrum of ,-glucosides. Cellulase components are thought to act in a stepwise process and can act synergistically to achieve more efficient degradation (10, 53, 54, 56). The major end product of concerted endoglucanase and cellobiohydrolase activity is cellobiose. Cellobiose is then hydrolyzed to glucose by 3-glucosidases. The removal of cellobiose from the reaction by ,B-glucosidases circumvents a major rate-limiting step during enzymatic cellulose hydrolysis, as both endoglucanase and cellobiohydrolase activities are frequently inhibited by cellobiose (10, 55). However, many P-glucosidases are also end product inhibited by glucose. Therefore, cellulases which include ,-glucosidases that are resistant to end product inhibition can more rapidly degrade cellulose and produce concentrated (20 to 30%) glucose syrups (49). Thermophilic bacteria produce cellulases (including 1-glucosidases) that are thermostable and able to withstand the rigors of reuse. Accordingly, these enzymes are of potential industrial value. Several thermophilic ,-glucosidases from various bacterial genera have been characterized (1, 3, 8, 22, *

34), and certain of the genes have been cloned and characterized in heterologous hosts (15, 28, 37). To obtain microorganisms that produce 3-glucosidases that are both thermostable and resistant to end product inhibition, we isolated thermophilic cellulolytic actinomycetes from thermal ecological niches and evaluated their enzymes by assaying for ,B-glucosidase activity (against para-nitrophenyl-1-glucoside) in the presence of up to 30% glucose at 60°C (49). Of several isolates, the most attractive was Microbispora bispora (31, 49). In order to study the enzyme components independently and to characterize the genes encoding them, we have cloned several M. bispora cellulase components (56). These include two genes (bgl4 and bglB) which encode distinct 3-glucosidases. This report describes the cloning of the thermostable 3-glucosidase gene (bglB) from M. bispora, characterization of the nucleotide sequence encoding bglB, and properties of the enzyme expressed in Escherichia coli.

MATERIALS AND METHODS

Bacterial strains and plasmids. M. bispora NRRL 15568 (49) was the source of DNA for cloning the ,-glucosidase genes. The cloning vectors were plasmids pBR322 (5) and pUC119 (46). E. coli strains DH5a {F- endAI hsdRl7(rK MK+) supE44 X- thi-1 recA4 gyrA reLAI A(lacZYAargF)U169 [(80d1acA(1acZ)M15]} and HB101 [F- hsdS20 (rB- mB-) recA13 leuB6 ara-14 proA2 lacYl galK2 rpsL20(Strr) xyl-S mtl-l supE44 X-] (6) were used as host strains. Cloning procedure. Total DNA from M. bispora was prepared by the procedure of Collmer and Wilson (9). A partial digest of M. bispora DNA was prepared with restric-

Corresponding author. 3455

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WRIGHT ET AL.

tion endonuclease PstI. A portion of the digest was mixed with plasmid pBR322, previously digested with PstI and dephosphorylated with calf alkaline phosphatase (final DNA concentration, 40 ,ug/ml). This mixture was incubated at 16°C overnight with T4 DNA ligase and then used to transform CaCl2-treated competent E. coli HB101 cells (18). A total of 11,000 tetracycline-resistant transformants containing M. bispora genomic inserts were transferred to 96-well culture plates containing Luria broth with 0.2% (wt/vol) agar supplemented with 15 p,g of tetracycline per ml and grown overnight at 37°C, glycerol was added to 20% (vol/vol), and the cultures were frozen at -80°C. These clones served as master cultures during screening for 0-glucosidase activity. In situ activity screen for 1K-glucosidase. Recombinant clones from the genomic library were replica plated onto M9 minimal medium (supplemented with 5 g of glycerol per liter, 2.5 g of sorbitol per liter, 5 g of cellobiose per liter, and 15 ,ug of tetracycline per ml) and incubated at 37°C for 48 to 72 h. After growth, the colonies were overlaid with 0.6% (wt/vol) top agar containing 1 mM 4-methylumbelliferyl-13-D-glucoside (MUG) (Sigma Chemical Co., St. Louis, Mo.) in 50 mM phosphate-citrate (PC) buffer, pH 6.5, with 0.1% sodium dodecyl sulfate (SDS) and 0.02% Triton X-100. Detection of clones that expressed ,-glucosidase activity was accomplished by heating the plates at 60°C for up to 16 h and periodically viewing them on a UV transilluminator (365 nm) to detect production of the fluorescent methylumbelliferone. Restriction enzyme analysis. Restriction analysis of the cloned fragments was carried out as described by Sambrook et al. (38). Southern hybridization. M. bispora genomic DNA and recombinant plasmids were digested with restriction endonucleases, fractionated by gel electrophoresis, and then vacuum blotted onto a ZetaBind-charged nylon membrane (Cuno Inc., Meriden, Conn.) by the method of Sambrook et al. (38). Hybridizations were carried out by the method of Southern (42). The probe was labeled by sulfonating the cytosine residues of single-stranded DNA, and hybridization was detected by a sandwich immunoenzymatic reaction as described in the ChemiProbe kit (FMC BioProducts, Rockland, Maine). Enzyme localization. E. coli cells were osmotically shocked by the method of Neu and Heppel (30) to release any product of the cloned periplasmic ,-glucosidase gene (bglB). The cells were then completely disrupted by sonication (Vibracell 50 model; Sonics and Materials, Danbury, Conn.) to obtain the intracellular fraction. Periplasmic, intracellular, and cellular debris fractions, as well as culture broth, were individually assayed for BglB activity against MUG. In addition, all fractions were assayed for E. coli alkaline phosphatase, a periplasmic marker (50). Preparation of cell-free extract. Cells were harvested at early stationary phase by centrifugation at 4,000 x g for 10 min, and the supernatant was decanted. The cells were washed by resuspension in 0.5 volume of 50 mM phosphatecitrate buffer, pH 6.5, and recentrifuged, and again the supematant was discarded. The cells were then resuspended in PC buffer (1/100 the original culture volume) and disrupted by sonication on ice until cells were broken, as determined by microscopic observation. Cellular debris was pelleted by centrifugation at 15,000 x g for 20 min, and both supernatant and debris were recovered for analysis. Enzyme activity and protein concentration assays. ,B-Glucosidase activity in cell-free extracts was determined by measuring the release of 4-methylumbelliferone from MUG;

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p-nitrophenol from p-nitrophenyl-j3-D-glucoside (PNPG), p-nitrophenyl-o-D-cellobioside (PNPC), p-nitrophenyl-P-Dxyloside (PNPX), p-nitrophenyl-3-D-lactoside (PNPL), and p-nitrophenyl-a-D-glucoside (PNPaG); or glucose from cellobiose, esculin, arbutin, and salicin. Substrate concentrations were 1 mM for MUG; 5 mM for PNPG, PNPC, PNPX, PNPL, and PNPaG; and 30 mM for cellobiose, esculin, arbutin, and salicin in PC buffer. The assays were run at 60°C for 1 h. The release of 4-methylumbelliferone was measured fluorometrically at 450 nm (26), while that of p-nitrophenol was measured spectrophotometrically at 405 nm (11). Glucose production was measured with a hexokinase-glucose6-phosphate dehydrogenase kit (Sigma). For p-nitrophenyl substrates, 1 U of enzyme activity corresponds to the release of 1 ,umol ofp-nitrophenol per min. With MUG as substrate, 1 U corresponds to the release of 1 ,umol of aglycone per min. With esculin, arbutin, and salicin as substrates, 1 U corresponds to the release of 1 ,umol of glucose per min. With cellobiose as the substrate, 1 U is defined as the release of 2 p,mol of glucose, since a single cleavage yields two molecules of glucose. Protein concentrations were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, Calif.) by measuringA595 and comparing the results to bovine serum albumin standards. Specific activity is defined in units per milligram of protein. Characterization of enzyme products by HPLC. Reaction mixtures (1 ml) containing 50 mM cellobiose in PC buffer and crude extract (50 jig of protein) from M. bispora or recombinant E. coli were incubated at 60°C for 12 h. Samples (20 ,ul) were injected into a high-pressure liquid chromatography (HPLC) unit fitted with a Sugar-Pak 1 column (Waters Chromatography, Milford, Mass.) at 90°C and eluted with H20 at a flow rate of 0.5 ml min-'. Reaction products were detected with a refractometer (Waters Chromatography, Milford, Mass.) and plotted versus elution time. Electrophoresis and zymograms. Proteins were separated by electrophoresis through 10% (wt/vol) polyacrylamide gels in the presence of 0.1% (wt/vol) SDS as described by Laemmli (24) by using a Hoefer SE 600 vertical slab unit (Hoefer Scientific Instruments, San Francisco, Calif.). The gels were silver stained by the procedure of Rabilloud et al. (36). For native zymograms, the SDS was omitted from samples and buffers. Following electrophoresis, gels were washed twice for 20 min in PC buffer and then heated at 60°C for 10 min in a minimal volume of PC buffer containing 1 mM MUG. The gels were then viewed on a UV transilluminator (365 nm) and photographed immediately. To confirm the apparent molecular mass, the cloned BglB enzyme was also subject to nondenaturing gradient (8 to 25%) polyacrylamide gel electrophoresis (PAGE) (PhastSystem; Pharmacia, Piscataway, N.J.). BglB was identified by zymogram staining as described above and marked on the gel. The gel was then stained with Coomassie brilliant blue and migration of BglB was compared with that of standards. Staining for glycoproteins. Crude protein extracts from M. bispora and recombinant E. coli expressing bglB were tested for glycosylation. Following nondenaturing PAGE as outlined above, the gels were Schiff stained for glycoproteins by the method of Glossmann and Neville (12). Determination of pH optimum. Optimum pH for 3-glucosidase was determined by assaying for MUG activity in PC buffer with pH ranging from 4.0 to 8.0. All reaction mixtures were then placed on ice and adjusted to pH 10 with 0.5 M glycine buffer (pH 10.8), and fluorescence was measured. Temperature optimum and thermostability. The temperature optimum for ,-glucosidase was determined by assaying

VOL. 58, 1992

M. BISPORA 3-GLUCOSIDASE B

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for MUG activity over a range of temperatures between 37 and 90°C. Thermostability was tested by preincubating enzyme samples at 60°C for up to 48 h, pelleting any precipitated proteins by centrifugation at 10,000 x g for 15 min, and then assaying the supernatant for residual activity against MUG at 60°C. End product inhibition. The effect of glucose concentration on BglB activity was tested by assaying for activity against MUG at 60°C in PC buffer, pH 6.5, containing increasing concentrations (0 to 40% [wt/vol]) of glucose. DNA sequencing. Unidirectional nested deletion subclones were constructed from plasmids containing the bglB insert in both orientations by using exonuclease III and Si nuclease as outlined in the Erase-A-Base kit (Promega Corp., Madison, Wis.). Both DNA strands were sequenced by the dideoxynucleotide chain termination method (39) by using Taq DNA polymerase (Promega) with supercoiled plasmid templates. Reactions were run in parallel at 70°C with standard and deaza nucleotide mixes to resolve GC compressions during electrophoresis. Computer analysis of DNA and protein sequences. DNA sequences were analyzed by using the GCG software package (Genetics Computer Group, University of Wisconsin, Madison) to determine open reading frames (ORFs), GC content, codon bias, deduced amino acid sequence, and multiple sequence alignments. Nucleotide sequence accession number. The GenBank nucleotide sequence accession number for the M. bispora bglB gene is M97265.

RESULTS

Cloning and gene characterization. E. coli transformants containing M. bispora genomic DNA were screened for P-glucosidase activity as described in Materials and Methods. Of the 11,000 clones screened, 11 were positive. Plasmid DNA was isolated from these clones and used to retransform E. coli HB101 in order to confirm that the ,B-glucosidase activity was plasmid associated and not the activation of a cryptic E. coli P-glucosidase (33, 40). Analysis of the genomic inserts by restriction endonuclease mapping revealed that two types of inserts coded for the expression of 3-glucosidase. One, an 8-kb PstI fragment termed MUG1, was isolated from 7 of the 11 positive clones. The other insert, a 9.5-kb PstI fragment termed MUG2, was isolated from four positive clones. The restriction maps of MUG1 and MUG2 fragments were different (Fig. 1). In addition, the MUG2 clones showed greater MUGase activity than the MUG1 clones, as indicated by the amount of fluorescence observed from in situ zymograms and in quantitative assays against 3-D-glucoside substrates. The two putative genes were designated bgLA (MUG1) and bglB (MUG2). The fragment containing the bglB gene was studied further. In order to establish a more precise location for the gene within the insert, the 9.5-kb fragment encoding Bg1B and several smaller internal restriction fragments were subcloned into pUC119, and the clones were assayed for activity against MUG (Fig. 1). A clone harboring plasmid pX29,

3458

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WRIGHT ET AL.

2 3

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TABLE 1. BglB activity in crude protein extracts prepared from E. coli harboring pX92 against glucoside substrates Substratea Sp act (mU)

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FIG. 2. Southern hybridization analysis of M. bispora genomic DNA and cloned fragments which express P-glucosidase activity in E. coli. DNA fragments which encode bglA (4.0 kb) and bglB (2.2 kb) were excised with XhoI from plasmids pX49 and pX29, respectively, and purified. The purified fragments, along with M. bispora genomic DNA previously digested with XhoI, were separated on a 0.8% agarose gel and blotted onto a ZetaBind membrane (shown). The bglB fragment was labeled antigenically and used as the hybridization probe. Lane 1, 2.2-kb XhoI fragment encoding bglB; lane 2, 4.0-kb XhoI fragment encoding bgL4; lane 3, sheared calf thymus DNA (nonspecific control); lane 4, M. bispora genomic DNA digested with XhoI.

PNPaG, p-nitrophenyl-a-D-glucoside.

concentration (peak 2) accompanied with an increase in glucose concentration (peak 3). No broadening of the buffer peak (peak 1), corresponding to glucose-i-phosphate or glucose-6-phosphate, was detected. Identification of 13-glucosidase by PAGE and in zymograms. ,-Glucosidase activity in gels following nondenaturing PAGE was assessed by staining with MUG. The zymogram 12

3

AS which contained an internal 2.2-kb XhoI fragment ligated into the unique SalI site of pUC119, showed about five times more activity toward MUG than the clone carrying the larger 9.5-kb fragment (specific activities, 3 and 0.6 mU, respectively). The expressed activity was uniform, irrespective of induction of the lac promoter with isopropylthiogalactoside. Southern hybridization. A labeled DNA probe prepared from the 2.2-kb XhoI fragment containing bglB was tested for hybridization against both M. bispora genomic DNA which had been digested with XhoI and a 4-kb fragment which contained bglA (Fig. 2). The bglB probe showed hybridization to a single 2.2-kb band in the M. bispora genomic digest and none, even under low-stringency conditions, to the bglA fragment. This confirmed that bglB originated from M. bispora and supports the idea that at least two different genes encode 0-glucosidases in M. bispora. Localization of BgIB in E. coli. E. coli cells containing the plasmid pX29 were osmotically shocked, and the cellular fractions were assayed for 3-glucosidase with MUG as a substrate. When expressed in E. coli, the ,B-glucosidase encoded by bglB was largely confined to the intracellular fraction (90% of activity). Substrate specificity of BglB. Crude protein extract prepared from an E. coli culture harboring plasmid pX29 was assayed for activity against several ,B-D-glucoside substrates. The BglB enzyme was more than 10 times more active toward cellobiose than toward the aryl-p-D-glucoside substrates (Table 1). Characterization of products by HPLC. The products of enzyme action against cellobiose were analyzed by HPLC. The chromatograms (Fig. 3) showed a decrease in cellobiose

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FIG. 3. Characterization of cellobiose hydrolysis products by HPLC. Crude extracts from M. bispora and recombinant E. coli were incubated with 50 mM cellobiose for 12 h at 60°C, and the products were analyzed by HPLC. (A) Standards (elution profile), PC buffer (peak 1), 10 mM cellobiose (peak 2), and 10 mM glucose (peak 3); (B) control experiment, 50 mM cellobiose in PC buffer incubated for 12 h at 60°C; (C) recombinant E. coli crude extract incubated with 50 mM cellobiose in PC buffer for 12 h at 60°C; (D) M. bispora crude extract incubated with 50 mM cellobiose in PC buffer for 12 h at 60°C.

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M. BISPORA 1-GLUCOSIDASE B

kDa 1 97

3459

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FIG. 5. Determination of molecular mass by SDS-PAGE. The

cloned BgIB activity band was excised from the zymogram (Fig. 4), FIG. 4. Zymogram stained for 13-glucosidase activity with MUG. Cell-free extracts were separated by nondenaturing PAGE, washed, stained, and photographed as outlined in Materials and Methods. Lane 1, crude extract (100 pg of protein) from E. coli cariying plasmid pX29 which contains bglB insert; lane 2, crude extract (100 pg of protein) from E. coli carrying plasmid pUC119 with no insert; lane 3, crude extract (5 p.g of protein) prepared from M. birpora.

(Fig. 4) showed that crude extract from E. coli expressing BglB (lane 1) gave an activity band that corresponded to that of one of the 13-glucosidases from M. bispora (lane 3). In M. bispora, a second band with less activity toward MUG migrated below Bg1B. At this time, it is not clear whether or not this is the enzyme encoded by bglA. In addition, there was a minor activity band directly below the major Bg1B band in the M. bispora preparation that was difficult to distinguish. Occasionally, this band also appeared in the clone preparations. The control, E. coli carrying only pUC119, showed no activity (lane 2). Determination of molecular mass by SDS-PAGE and nondenaturing gradient PAGE. The activity band (cloned BglB) from the PAGE zymogram was excised, extracted from the gel by maceration, subjected to SDS-PAGE, and silver stained. A doublet with bands that migrated at approxiimately 52,000 and 51,400 Da was clear (Fig. 5). The propor-

tions of the upper and lower band in the doublet differed in some BglB preparations, suggesting that the lower band may be a proteolytic cleavage product. This interpretation is supported by the fact that the smaller protein occasionally exhibited some activity in zymograms. In addition, nondenaturing gradient PAGE (not shown) was also used to elicit the molecular mass of cloned Bg1B. Following electrophoresis, zymogram staining, and protein staining, BglB activity migrated with a molecular mass of approximately 52,280 Da. T'he molecular mass values exhibited by nondenaturing gradient PAGE and SDS-PAGE are in good agreement. Glycoprotein analysis. Crude protein extracts, prepared from M. bispora and recombinant E. coli expressing BglB, were analyzed for glycoproteins by Schiff staining after nondenaturing PAGE. No band corresponding to BglB was positive in either extract, indicating that BglB is nonglycosylated in both organisms (not shown). Determination of pH optimum. Crude protein extract, prepared from recombinant E. coli expressing Bg1B, was

extracted from the gel, subjected to SDS-PAGE, and silver stained. Lane 1, low-molecular-weight markers; lane 2, cloned BglB band excised from zymogram.

assayed against MUG over a range of pH values to determine the effect of pH on BglB activity. BglB expressed in E. coli exhibited a pH optimum of about 6.2. Determination of temperature optimum and thermostability. Crude protein extracts, prepared from recombinant E. coli expressing Bg1B, were assayed at various temperatures against MUG to determine the optimal temperature for Bg1B activity. The optimal temperature for cloned M. bispora BglB was 60°C, and this enzyme was thermostable, retaining about 70% of its activity after 48 h at 60°C (Fig. 6). The initial drop in BglB activity that occurred within the first hour of preincubation was not due to denaturation of the enzyme. Some BglB coprecipitated with the large amount of E. coli proteins denatured during this time, and it was active upon resuspension. Glucose concentration versus BgIB activity. The effect of glucose concentration on BglB activity against MUG is shown in Fig. 7. Surprisingly, BglB activity increased 2.5fold at glucose concentrations of 2 to 5% and did not become inhibited until the glucose concentration reached about 40%. Nucleotide sequence of bgLB. The entire nucleotide sequence of the 2.2-kb insert originating from M. bispora was determined for both strands with no gaps (Fig. 8). A single long ORF extending from nucleotides 323 to 1744 that encoded a protein of 473 amino acids with a calculated molecular weight of 52,227 was found. This ORF is preceded by A-T rich regions which resemble actinomycete transcriptional promoter regions (21) and a potential ribosome binding site (AGGAGC) that is 7 bp upstream from the putative translation start site. In addition, the ORF is followed by a pair of 19-bp inverted repeats 5 bp downstream from the stop codon which may serve as a transcription termination site. The presence of potential promoter and terminator sequences suggests that the bglB transcript is monocistronic. GC content of bglB. The overall G+C content of the cloned DNA insert was calculated at 72%. The GC content within the bglB ORF was slightly higher (73%) than outside it (69%). These values agree very well with those published for Microbispora spp. (total GC content of 71.3 to 73%) (31). Homology between BgIB and other 1-glucosidases. The TFASTA algorithm was used to identify deduced amino acid sequences in the GenBank and EMBL data bases and

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WRIGHT ET AL.

3460

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Pre-incubation Time at 60 C (h) FIG. 6. f-Glucosidase activity as a function of temperature. (A) Optimum temperature. Crude extract (500 ILg of protein) prepared from recombinant E. coli carrying pX29 was assayed at different temperatures against 1 mM MUG in 50 mM PC buffer, pH 6.5, for 10 min. (B) Thermostability. Crude extract (500 pg of protein) was preincubated at 60°C in 50 mM PC buffer, pH 6.5. Residual activity was assayed in the same buffer containing 1 mM MUG for 10 min at 600C.

them with BglB. Significant homology was found between Bg1B and members of family 1 glycosyl hydrolases (Fig. 9) (20). Family 1 includes 3-glucosidases from Agrobacterium faecalis, Bacillus polymyxa, Caldocellum saccharolyticum, and Clostridium thermocellum (14, 16, 28, 47). Other enzymes grouped in this family include phospho-3glucosidase from E. coli (40); phospho-3-galactosidases from compare

Lactobacillus casei, Staphylococcus aureus, and Streptococcus lactis (4, 7, 35); and a ,B-galactosidase from Sulfolobus solfataricus (27) and human lactase phlorizin hydrolase which has multiple homologous regions (29). DISCUSSION

3-Glucosidases are produced by plants and animals as well microbes and often show broad substrate specificities (55). Microbial p-glucosidases, however, can usually be categorized into two large groups: those specific for cellobiose and those with a more relaxed specificity, also hydrolyzing aryl-glucosides. At least one Thermomonospora and two Bacillus species produce both enzyme types, i.e., one enzyme with greater affinity for cellobiose and another acting preferentially against aryl-glucoside substrates (3, 13, as

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43). In contrast, C. thermocellum produces a ,-glucosidase which is active against both cellobiose and aryl-P-D-glucosides, though with a different temperature optimum for each substrate (2). M. bispora possesses two different genes, bgLA and bglB, which encode 0-glucosidases. Restriction mapping revealed that the two cloned fragments encoding ,3-glucosidase activity had different patterns, and Southern analysis showed no cross-hybridization between the two genes, even under low-stringency conditions. On the basis of this information, bglA and bglB appear distinct. This is not uncommon, as several bacteria have been shown to carry multiple genes encoding ,B-glucosidases (14, 17, 48). In fact, genes encoding as many as 15 endoglucanases, two xylanases, and two ,B-glucosidases have been reported for C. thermocellum (19, 25). Multiple genes encoding different cellulolytic enzymes may offer a selective advantage to an organism by enabling it to degrade cellulose, a substrate that not only varies greatly in terms of degree of crystallinity and polymerization but also changes during the course of degradation, more efficiently. BglB is one of two enzymes from M. bispora that showed activity toward ,-glucoside substrates. The natural role of each enzyme (BglA and BglB) in M. bispora is not certain. However, cloned BglB showed greater activity toward cellobiose than aryl-13-D-glucosides (Table 1), suggesting that it functions as a cellobiase in vivo. Preliminary evidence (not shown) suggests that BglA is more active toward aryl-3-Dglucosides than cellobiose and therefore the M. bispora system may be similar, in terms of substrate specificities, to the Thermomonospora and Bacillus enzymes. The bglB-encoded enzyme exhibited an approximate molecular mass of 52,000 Da by SDS-PAGE and nondenaturing gradient PAGE; this mass was predicted from the deduced amino acid sequence. BglB is similar in size to many bacterial ,B-glucosidases, including those from A. faecalis [47], B. polymyxa (13), Bacillus subtilis (43), C. saccharolyticum (28), C. thermocellum (2), and an extremely thermophilic anaerobic bacterium, WaiW.2 (34). Larger bacterial P-glucosidases, including an 85-kDa enzyme from Clostridium stercorarium, have been reported (8). The cloned bglB gene may be regulated by an actinomycete promoter within the insert rather than by the plasmid-encoded lac promoter, since it was expressed in E. coli at the same level, irrespective of the presence or absence of

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P-GLUCOSIDASE

M. BISPORA

B

3461

1 101 201

GAGCTCGGTAGCCCTGGTGTAGACGGGCCGCATCGACGGCTGCGGAAGCTGGCGGCTGCTGCGGTACATCGTCTTCCGGTGGCCGGCCGGCCGCGCGTGC

301

GATCATTGCAGGAGCGGTCAGGATGACCGAATCGGCCATGACATCACGGGCGGGCCGAGGACGCGGGGCCGACCTCGTGGCCGCGGTCGTCCAGGGACAC

TCGGATGCTCACTCATGACGCCTGAACGACTACTTCTGGCCGCTGGTGGTGCTCACCCCGGACAACCCCACGGTGCAGGTGGCGCTCTCCCAGCTCGCGA GCGGCTACGTCCAGGACTTCGCGCTCGGCCTGACGGCACGCGGTGGCGACCCTGCGCTCGTGCTCATCTTCATCCTTCTCGGCAAGCAGATCATCGGAGG M A

S

A

A

D

A

A

E S

T

G

L

D

A

S

M

F

T

P

R

S

D

A

F

G

G

I

R G

W

G

G

R

A

A

T

A

D

A

L

A

A A V V Q G

V

Y

Q

H

E G A W R E

I

D

401

GCGGCGGCGAGCGACGCCGCCGGGGACCTGTCTTTCCCTGACGGCTTCATCTGGGGGGCCGCCACCGCCGCCTACCAGATCGAGGGCGCCTGGCGAGAGG

501

ACGGTCGCGGGCTCTGGGACGTCTTCTCCCACACCCCGGGGAAGGTGGCGAGCGGCCACACCGGGGACATCGCCTGCGACCACTACCACCGGTACGCGGA

G

R

G

D

L

R

V

W

L

D

V

A 'G

M

S

F

L

H

T

D

G

P

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G

V

Y

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A

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S

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V

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A

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T

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I

I

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A

V

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C

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D

G

H R Y

G

S

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A D

N

V

P

A

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G

F

D

Y

D

R

D

L V

E

L

L

G

G

H

I

T

P

Y

P

T

L

Y

H

W

D

L

P

Q

L E

T

D

701

GGCCTGGACTTCTACGACCGGCTCGTGGACGAGCTGCTCGGGCACGGCATCACGCCGTACCCCACCCTCTACCACTGGGACCTGCCGCAGACCCTGGAGG

801

ACCGGGGCGGCTGGGCGGCGCGCGACACCGCGTACCGGTTCGCCGAGTACGCGCTCGCCGTCCACCGGCGGCTCGGCGACCGGGTCCGGTGCTGGATCAC

901

GCTGAACGAGCCGTGGGTGGCGGCCTTCCTCGCTACGCACCGGGGAGCACCCGGGGCGGCCGACGTGCCGCGCTTCCGTGCACCACCTGCTGC

R

L 1001 1101 1201

G

G

N

W A A

P

E

T

R D

W V A A

A

L

F

Y

A

R

F

T

A

E

A L A V H R R

Y

R G A P G A A D

H

V

L G

R F

P

R V R C W

D

T

I

R A V H H L L L

G H G L G L R L R S A G A G Q L G L T L S L S P V I E A R P G V R G GG TCACGGCCTCGG CCTCCG CCTGCG CTCGGCGGG CG CCGG CCAGCTTGGCCT CACG CTCAG CCTCTCCCCGGTGATCGAGGCCCGCCCCG&GGGTCCG&GG G G R R V D A L A N R Q F L D P A L R G R Y P E E V L X I M A G H G AGG CGG CCG CCGGGTGGACGCG CTCGCCAACCGGCAGTTCC TCGA CCCGG CG CTG CG CGG CCGC TACCCGGAGGAGG TCCTCAAGATCATGGC CGGGCA A R L G H P G R D L E T I H Q P V D L L G V N Y Y S H V R L A A E

CGCGCGGCTCGGCCATCCGGGACGGGACCTGGAGACGATCCACCAGCCGGTGGACCTGCTCGGGGTGAACTACTACTCCCACGTACGTCTCGCGGCCGAG G

E

A

P

R

N

L

P

S

G

E

G

I

R

E

F

R

P

T

A

V

T A

W

P

G

D

R P

D

G L

R T

1301

GGCGAGCCGGCGAACCGGCTTCCCGGGAGCGAGGGCATCCGGTTCGAGCGTCCGACGGCGGTGACGGCATGGCCTGGCGATCGACCCGACGGGCTGCGCA

1401

CCCTGCTGCTGCGGCTCTCCCGCGACTACCCGGGAGTCGGCCTGATCATCACCGAGAACGGGGCCGCGTTCGACGACCGGGCGGACGGCGACCGGGTGCA

L

L

L

R

L

S

R

D

Y

P

G

V

G

L

I

I

T

E

N

G

A A

F

D

D

R A D G D

R V H

1601

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

1701

-----------------R D V V R R N G L R N G E * -4---------ATCGGGACGTCGTGCGGCGCAATGGTCTGCGGAACGGCGAGTGAGCGCCCACCCCTGAGACGGTCGCGGCCCGGGCCGGGGTGTCGCGGGCGACCGTCTC

1501

1801 1901 2001 2101 2201

GCGGGTGGTGAACGGCCGGCAGACCGTGACCCGGAGATCCGGAGTCGTGCTCCGCGCGATCAGGAGCTGGGGTACGTCCACTCGGCGGCGCGCAGCTGGT CACCCAGCGCACGACTCGGTCGCCTCGTGGTCTCCGAGCGCGACCAGTCTCTCCGACGACCGATGTCTCACGTGATCGCTCGCGAGCCTGAGCTGAGAAG CGACGGCAGTGTGCTGATCGTCGCGAGCTCGCCGGAGCACGCCGATCGAGCGCTTCATCGCGGCGGCCACGTCGACGGCGTCATGCTGATCTCGATGCAC GGTGCCGACCCGCTGCCCGCCGCCCTGCCGCATGGCGTCCCCGTGGTCTCGTAGGCCGGCCCGCGTCCCGGTGCCGCTGCCGTACGTGGAGCAACGACAA

CGTGGCGGCGG FIG. 8. Nucleotide and deduced amino acid sequences of bglB. The deduced amino acid sequence appears above the first letter of the respective codons. The putative Shine-Dalgarno ribosome binding site is overlined. A pair of inverted repeats which may serve as a transcription terminator are overlined with facing arrows. Numbers on the left refer to the first nucleotide of each line.

the lac inducer isopropylthiogalactoside. Regions of the nucleotide sequence (A-T rich), upstream from the putative translation start site for bglB, resemble some actinomycete transcription promoters (21). Low transcription levels caused by the inability of E. coli RNA polymerase to recognize the M. bispora bglB promoter could account for the relatively low specific activity of cloned Bg1B in the crude extracts compared with that in enzyme extracts from M. bispora (0.13 and 5.0, respectively). Nevertheless, BglB expression (in a crude protein extract) was comparable to that of products of other cloned bacterial ,B-glucosidase genes which exhibit specific activities for cellobiose that range from 0.08 to 1.83 (2, 8, 13, 17, 23, 34, 37, 43, 44). The increase in BglB activity exhibited by subclones carrying the smaller (2.2-kb) genomic fragment might have resulted from deletion of a negative regulator element within the larger fragment (9.5 kb). Cell-associated 0-glucosidases are often not as thermostable as the secreted endoglucanases or cellobiohydrolases (55), although exceptions exist (28, 34). Even though M. bispora BglB is a cell-associated enzyme (intracellular in E. coli), it is one of the more thermally stable 3-glucosidases reported thus far. BglB showed optimal activity at 60°C and retention of roughly 70% of its activity after 48 h at 60°C. In comparison, Trichoderma reesei P-glucosidase (in Cellulase 150L; Genencor, South San Francisco, Calif.) was completely inactivated after only 10 min at 60°C (not shown).

The effect of glucose on BglB activity revealed that not only was the enzyme resistant to end product inhibition, but it was actually activated at glucose concentrations from 2 to 5% (0.1 to 0.3 M). Although rare, there is precedence for enhancement of 3-glucosidase activity by glucose. For example, a twofold activation of ,B-glucosidase activity (at 0.1 M glucose) has been reported for a Streptomyces sp. (32). Comparisons between the deduced amino acid sequence of M. bispora BglB and sequences within the data bases revealed that BglB belongs to glucosyl hydrolase family 1 (20). This family includes ,-glycosidases from organisms representing all three domains (Archaea, Bacteria, and Eucarya) (52). Even certain enzyme domains of human and rabbit lactase phlorizin hydrolases (intestinal brush border enzymes) showed significant similarity to this group. Perhaps, not surprisingly, these domains have been shown to exhibit some cellulolytic activity (29). BglB contains regions, particularly in the N-terminal and C-terminal portions, that showed significant similarity to the rest of the family, suggesting that these conserved regions may be involved in binding or catalysis. Several residues (Asp, Glu, and His) which are believed to act as catalytic sites during the hydrolysis of ,B-glycosidic bonds via an acid catalysis mechanism (41) are conserved in BglB and throughout family 1. Inhibitors, such as 2',4'-dinitrophenyl-2-deoxy2-fluoro-j3-D-glucopyranoside, have been used to trap covalent intermediates and identify the active-site nucleophile of

3462

WRIGHT ET AL.

APPL. ENvIRON. MICROBIOL.

Mbbglb MT E SAM T S RA Csbgls Ctbgla Bpbgla Asbgls Bpbglb Hulph3 Hulph4 Ecbglb Lcpbgal Sapbgal Slpbgal Sabgal .

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D X .Tu S H T .L R9B N T LQ PH G VN GKRM BP R I:ISGK L Di L D K QMR. A .... Tt LB BNY A .... Kg LEBDNY DHVWVH D R. .EBN IV SQEVS

Mbbglb D RV V Csbgls L K A ::@@ Ctbgla I K S Bpbgla I R T _ Asbgls V E A RLE2 Bpbglb Hulph3 V K A Hulph4 VS H S Ecbglb F T C I a Lcpbgal Sapbgal Slpbgal VNGI'I Ssbgal LNAV IN1

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Mbbglb Cabgls Ctbgla

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DAAGDLS

D PNTLAAR ......MT ...... *

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Mbbglb Cabgls Ctbgla Bpbgla Asbgls Bpbglb Hulph3 Hulph4 Ecbglb Lcpbgal Sapbgal Slpbgal Ssbgal

AVVQ GHAAA S

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Mbbglb Csbgls Ctbgla Bpbgla Asbgls Bpbglb

Hulph3

A

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SEIAK

171 140 141 141 146 142 157 144 147 136 136 145 184 221 193 194 194 199 195 211

199 199 189 189 198 239

P V. IEARPGVRGG GRRVD 266 PVYLQTERLG YKVSEI REM VSLSS 248 ASEKAE IEE SYHYP *ELSF 243 . . . . .S.... W A V P YSTSEE KAE CARTI 243 . S ... AIP ASDGEA LKE EERAF 248 HV D ... A ASERPE VAE EIRRD 244 S L S THWAEPK SPGVPR VEE EDRML 264 TI SSDWAEPR DPSNQE VEE ERRYV 252 LVYP L TCQP Q DMLQAM EN 248 TVYP Y ..... .SDSAVB HB ELQD 238 T K Y P F . . . . . DPSNPE VRE EELED 239 T K Y P Y . . . . . DPENPA VRE ELED 248 SYYP L ..... RPQ ENE VEIAE 285

M. BISPORA ,3-GLUCOSIDASE B

VOL. 58, 1992 Mbbglb

Csbgls Ctbgla Bpbgla Asbgls Bpbglb Hulph3 Hulph4

ALAB A L AEl' QLD G S LAG

ALR. RR I S VLK. L V LK R S I Y Q E V F. K .

S L H SI

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QF SuL QFH G QFMG Ecbglb . . RR' Lcpbgal A LEI Sapbgal IRIH Slpbgal I IH Ssbgal R L N R I

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I F RN I FKN VQAR TL A TY L TY L I IKGE .

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Mbbglb Csbgls Ctbgla Bpbgla Asbgls Bpbglb Hulph3 Hulph4

S T S S T T r T

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M F M M T

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DT EV GY HQE S RE S D K

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TV T G

RL . AA VR . LY KY FI. VN . RF RV. AD II . R. IVQHKTPRLN LAYNLNYATA CVS HDE SINK

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LLDYLVQKDL ALKLYKKKG I LVDWFAE QGA MMEALGDRMP MVEWY. . . GT MKWKVGNRSE MKTRIRDRSL MQRFFRDHNI T LALVKE I LD T MEGVQH I LS T MEGVNH I LA .

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FIG. 9. Amino acid sequence alignment of Bg1B and the members of ,B-glycosidase family 1. Alignment was maximized by introducing gaps denoted by dots. Identical residues are shaded black, conservative substitutions are shaded dark gray, and similar substitutions are shaded light gray. Proposed catalytic residues are denoted with a circle. Numbers to the right represent the last residue in each line. Abbreviations: Mbbglb, M. bispora P-glucosidase B; Csbgls, C. saccharolyticum 3-glucosidase S; Ctbgla, C. thermocellum ,-glucosidase A; Bpbgla, B. polymyxa 3-glucosidase A; Asbgls,Agrobacterium sp. ,B-glucosidase S; Bpbglb, B. polymyxa ,B-glucosidase B; Hulph3, human lactase phlorizin hydrolase domain 3; Hulph4, human lactase phlorizin hydrolase domain 4; Ecbglb, E. coli f-glucosidase B; Lcpbgal, L. casei phospho-13-galactosidase; Sapbgal, S. aureus phospho-13-galactosidase; Slpbgal, S. lactis phospho-13-galactosidase; Ssbgal, S. solfataricus P-galactosidase.

an A. faecalis 1-glucosidase as Glu-358 (51). Most recently, Trimbur et al. (45) used region-directed mutagenesis to confirm that Glu-358 is indeed the nucleophile. This residue corresponds exactly to Glu-378 of M. bispora BglB. Other

conserved residues implicated as possibly involved in positioning the nucleophile (51) correspond to Asn-379 and Gly-380 of BglB. In addition, Asp-394 may play a role as an acid-base catalyst, protonating the leaving group and subse-

3464

WRIGHT ET AL.

quently deprotonating the water molecule as it attacks (45). Such a mechanism would be analogous to that of hen and T4 lysozymes and has been postulated for many cellulases. ACKNOWLEDGMENTS This work was supported by the U.S. Department of Energy (DE-F605-87ER13794) and the New Jersey Agricultural Experiment Station (no. D-01111-02-92). REFERENCES 1. Ait, N., N. Creuzet, and J. Cattaneo. 1979. Characterization and purification of a thermostable p-glucosidase from Clostridium thennocellum. Biochem. Biophys. Res. Commun. 90:537-546. 2. Ait, N., N. Creuzet, and J. Cattaneo. 1982. Properties of P-glucosidase purified from Clostridium thermocellum. J. Gen. Microbiol. 128:569-577. 3. Bernier, R., and F. Stutzenberger. 1988. Extracellular and cell-associated forms of beta-glucosidase in Thermomonospora curvata. Lett. Appl. Microbiol. 7:103-107. 4. Boizet, B., D. Villeval, P. Slos, M. Novel, G. Novel, and A. Mercenier. 1988. Cloning and expression of the phospho-13galactosidase gene from Streptococcus lactis into Escherichia coli. Gene 62:249-261. 5. Bolivar, F. 1978. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant molecules. Gene 4:121-136. 6. Boyer, H. W., and D. Rouland-Dussoix. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-472. 7. Breidt, F., Jr., and G. C. Stewart. 1987. Nucleotide and deduced amino acid sequences of the Staphylococcus aureus phospho3-galactosidase gene. Appl. Environ. Microbiol. 53:969-973. 8. Bronnenmeier, K., and L. Staudenbauer. 1988. Purification and properties of an extracellular 3-glucosidase from the cellulolytic thermophile Clostnidium stercorarium. Appl. Microbiol. Biotechnol. 28:380-386. 9. Collmer, A., and D. B. Wilson. 1983. Cloning and expression of a Thermomonospora fusca YX endocellulase gene in E. coli.

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