Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).     

Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli.

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli.

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.     

Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.     

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.     

Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product

N. OZCAN, C. CUNNINGHAM AND W.J. HARRIS. 1996. A gene encoding an enzyme which degrades cellulose ( end-1 ) was isolated from a library of Fibrobacter succinogenes SD35 DNA fragments and expressed in pUC18. The product of end-1 showed significant activity against carboxymethylcellulose but relatively minor activity against lichenan, xylan and avicel. The nucleotide sequence indicated a product of 388 amino acids with a molecular mass of 50.2 kDa. This was in agreement with the molecular size estimated by gel electrophoresis. No significant DNA sequence similarity was identified with any published endoglucanase.     

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene.

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan.     

Cloning and identification of cellulase genes from uncultured microorganisms in pulp sediments from paper mill effluent]

The metagenomic DNA of pulp sediments from paper mill effluent was extracted and purified. The 16S rDNA was amplified using the purified metagenomic DNA as template and a 16S rDNA library was prepared. Sequence analysis of 16S rDNA clones showed that diverse of uncultured bacteria inhabit in this environment, which can be classified into 4 clusters as Spirochaetes, Proteobacteria, Bacteroidetes and Firmicutes. A metagenomic library containing 10000 clones was constructed into cosmid vector, and the capacity of inserted DNA of which was 3.53 x 10(8) bp. Functional screening of the library resulted in isolation of two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressing beta-glucosidase activity. One clone expressing strongest enzyme activity from each activity category was chosen to be further analyzed. Three novel cellulase genes designated as umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expression. The umcel5L encodes an endoglucanase belonging to glycosyl hydrolase family 5, which is most related to an endoglucanase from Bradyrhizobium japonicum at 43% identity and 59% similarity. The umcel5M encodes a cellodextrinase belonging to glycosyl hydrolase family 5, which is most similar to a cellodextrinase from Fibrobacter succinogenes at 48% identity and 69% similarity. The umbgl3D encodes a putative beta-glucosidase belonging to glycosyl hydrolase family 3, which shares highest homology with a beta-glucosidase from Thermotoga maritima at 46% identity and 61% similarity. It is the first time to reveal the bacterial diversity of pulp sediments from paper mill effluent and clone novel cellulase genes from the bacteria by culture-independent method.     

Type II DNA restriction-modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.     

Molecular cloning and expression of a novel family A endoglucanase gene from Fibrobacter succinogenes S85 in Escherichia coli

A Fibrobacter succinogenes S85 gene that encodes endoglucanase hydrolysing CMC and xylan was cloned and expressed in Escherichia coli DH5 by using pUC19 vector. Recombinant plasmid DNA from a positive clone hydrolysing CMC and xylan was designated as pCMX1, harboring 2,043 bp insert. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The nucleotide sequence accession number of the cloned gene sequence in Genbank is U94826. The endoglucanase gene cloned in this study does not have amino sequence homology to the other endoglucanase genes from F. succinogenes S85, but does show sequence homology to family 5 (family A) of glycosyl hydrolases from several species. The ORF encodes a polypeptide of 654 amino acids with a measured molecular weight of 81.3 kDa on SDS-PAGE. Putative signal sequences, Shine-Dalgarno-type ribosomal binding site and promoter sequences (-10) related to the consensus promoter sequences were deduced. The recombinant endoglucanase by E. coli harboring pCMX1 was partially purified and characterized. N-terminal sequences of endoglucanase were Ala-Gln-Pro-Ala-Ala, matched with deduced amino sequences. The temperature range and pH for optimal activity of the purified enzyme were 55 approximately 65 degrees C and 5.5, respectively. The enzyme was most stable at pH 6 but unstable under pH 4 with a K(m) value of 0.49% CMC and a V(max) value of 152 U/mg.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1.

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.