Analysis of the active center of Bacillus stearothermophilus neopullulanase.

The active center of the neopullulanase from Bacillus stearothermophilus was analyzed by means of site-directed mutagenesis. The amino acid residues located in the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanse, Taka-amylase A, and other amylolytic enzymes. When amino acid residues Glu and Asp, corresponding to the putative catalytic sites, were replaced by the oppositely charged (His) or noncharged (Gln or Asn) amino acid residue, neopullulanase activities toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages disappeared. When the amino acids corresponding to the putative substrate-binding sites were replaced, the specificities of the mutated neopullulanases toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of neopullulanase participated in the dual activity toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. Pullulan is a linear glucan of maltotriosyl units linked through alpha-(1----6)-glucosidic linkages. The production ratio of panose from pullulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the alpha-(1----4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanse which exhibited higher specificity toward the alpha-(1----6)-glucosidic linkage.     

Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages.

The transglycosylation reaction catalyzed by neopullulanase was analyzed. Radioactive oligosaccharides were produced when the enzyme acted on maltotriose in the presence of [U-14C]glucose. Some of the radioactive oligosaccharides had only alpha-(1----4)-glucosidic linkages, but others were suggested to have alpha-(1----6)-glucosidic linkages. The existence of alpha-(1----6)-glucosidic linkages in the products from maltotriose with neopullulanase was proven by proton NMR spectroscopy and methylation analysis. We previously reported that the one active center of neopullulanase catalyzes the hydrolysis of alpha-(1----4)- and alpha-(1----6)-glucosidic linkages (Kuriki, T., Takata, H., Okada, S., and Imanaka, T. (1991) J. Bacteriol. 173,6147-6152). These facts proved that neopullulanase catalyzed all four types of reactions: hydrolysis of alpha-(1----4)-glucosidic linkage, hydrolysis of alpha-(1----6)-glucosidic linkage, transglycosylation to form alpha-(1----4)-glucosidic linkage, and transglycosylation to form alpha-(1----6)-glucosidic linkage. The four reactions are typically catalyzed by alpha-amylase, pullulanase, cyclomaltodextrin glucanotransferase, and 1,4-alpha-D-glucan branching enzyme, respectively. These four enzymes have some structural similarities to one other, but reactions catalyzed by the enzymes are considered to be distinctive: the four reactions are individually catalyzed by each of the enzymes. The experimental results obtained from the analysis of the reaction of the neopullulanase exhibited that the four reactions can be catalyzed in the same mechanism.     

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.     

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.     

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.     

Continuous production of panose by immobilized neopullulanase

Panose is a mildly sweet trisaccharide composed of three glucose units which has the nature of an anti-cariogenic sugar in foods. Continuous production of panose from pullulan was investigated by using a column of immobilized neopullulanase. Although enzyme immobilization by ionic bond is one of the most general methods for the production of non-ionic materials from non-ionic substrates, the activity of the neopullulanase immobilized on DEAE-cellulose significantly decreased. When the neopullulanase was immobilized on a carrier with spacer arms by covalent bond through the formation of the schiff base, the activity was fully expressed. Optimum temperature and pH for the reaction of the immobilized neopullulanase were 55–60°C and 6.0, which were almost the same as those of the free enzyme. The immobilized neopullulanase column was employed for the continuous production of panose from pullulan. More than 92% of pullulan was converted by this system into the final products; panose, maltose, and glucose. The ratio of panose in the mixture of products was 84%, which was significantly higher than that (around 70%) of a batch system using the free enzyme. The immobilized neopullulanase was very stable, and more than 90% of the initial activity was retained after 150-h continuous reaction at 55°C.     

Purification and characterization of branching specificity of a novel extracellular amylolytic enzyme from marine hyperthermophilic Rhodothermus marinus

An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.     

Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.     

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product.

Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is unique among those of alpha-amylase family enzymes due to a loop (residues 193-218) that is located at the end of the cleft around the nonreducing region and forms a 'dam'-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis.     

Characterization of ApuB, an extracellular type II amylopullulanase from Bifidobacterium breve UCC2003

The apuB gene of Bifidobacterium breve UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located alpha-amylase-containing domain which hydrolyzes alpha-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes alpha-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the apuB gene in B. breve UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.     

Pullulan 4-glucanohydrolase from Aspergillus niger

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6³-α-glucosylmaltotriose, 6²-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.     

Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.     

Complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with alpha-(1,6) glycosidic linkages

Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of alpha-(1,4), alpha-(1,4), and alpha-(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes alpha-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes alpha-(1,6) glycosidic linkages with a lesser efficiency. X-ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]2 or P5; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaealpha-amylase showed that the (-) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the alpha-(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites -1 and -2 at the nonreducing end of TVAI are effective in the hydrolysis of alpha-(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch-binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan-binding domain.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library

It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42 degrees C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making alpha-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, alpha-amylase, and 4-alpha-glucanotransferase.     

Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.     

alpha-L-fucosidase from aspergillus niger: demonstration of a novel alpha-L-(1----6)-fucosidase acting on glycopeptides

An alpha-L-fucosidase which hydrolyzes fucose from alpha-(1----6)-linkage to N-acetylglucosamine was found in Aspergillus niger. The enzyme was purified by affinity chromatography with bovine IgG glycopeptide-Sepharose 4B. The enzyme preparation released fucose from bovine IgG glycopeptide and fucosylated asialoagalactofetuin, but failed to cleave 1----2, 1----3 or 1----4 linkages of alpha-L-fucosides.     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Catalytic properties of the cloned amylase from Bacillus licheniformis.

A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage. The enzyme also hydrolyzed pullulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage. The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.