Purification and properties of β-amylase from Bacillus circulans S31

A thermotolerant -amylase was purified from Bacillus circulans S31 isolated from soil in Hong Kong. The purified enzyme has an M _r of 64 kDa and was stable at 50°C and pH 7.0 for 30 min. Its K _m for starch was 0.9 mg/ml with a V _max of 0.3 mg/min. It was not activated by any metal ion although sulphydrys reagents were inhibitory.H.S. Kwan, K.H. So and K.Y. Chan are with the Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong S.C. Cheng is with the Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic, Hung Hom, Hong Kong.     

Causes of the Production of Multiple Forms of β-Galactosidase by Bacillus circulans

The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20–30 and 9–12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Continuous production of α-amylase byBacillus amyloliquefaciens in a two-stage fermentor

Summary The production of a secondary metabolite (-amylase) by a highly aerobic bacterium (Bacillus amyloliquefaciens) was examined in batch, single-stage chemostat, two-stage stirred tank, and two-stage stirred tank/tubular reactor configurations. The relative performance of these reactor systems as measured by product concentration and volumetric productivity was compared, and the effect of aeration rate on the extent of plug flow in the tubular reactor was examined.     

Production of trisaccharide from lactose using β-galactosidase from Bacillus circulans modified by glutaraldehyde

Summary Free amino groups of -galactosidase-1-from Bacillus circulans were partially modified using different glutaraldehyde concentrations to increase trisaccharide production from lactose. Glutaraldehyde of 0.01%–0.03% modified 15%–40% of the free amino groups of the enzyme. The maximum yield of trisaccharide increased from 6% to 12% depending upon the degree of modification with 25% conversion of 127 mM lactose. Modification of 50% of the free amino groups of the enzyme using 0.05% glutaraldehyde produced a considerable amount of tetrasaccharide along with trisaccharide even at the initial stage of the reaction.     

Solid state fermentation for production of α-amylase byBacillus megaterium 16M

Summary The ratio of buffer to wheat bran, incubation temperature and initial pH influence -amylase production byBacillus megaterium 16M under solid state fermentation. The enzyme, with pH and temperature optima at 6.0 and 70°C, is formed at a level of 30,000 units/g dry bacterial bran without coproduction of proteases and cellulases.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Purification and Properties of β-Galactosidases from Bacillus circulans

Six compounds, Z- and E-fadyenolide (3, 4), 1-ally1-2,3-(methylenedioxy)-4,5-dimethoxy-benzene (5), 4-methoxy-3,5-bis (3′-methyl-2′-butenyl)-benzoic acid (6), 2,6-dihydroxy-4-methoxy-dihydrochalcone (7), and 5-hydroxy-7-methoxyflavanone (8) were isolated from three species of Jamaican Piper, Piper fadyenii, C.D.C., Piper aduncum L. and Piper hispidum Sw. Three amides (9 ~ 11) of 3,5-dimethoxy-4-oxo-5-phenylpent-2-enoic acid using piperidine, pyrrolidine and morpholine, respectively, were synthesized from compounds 3 and 4, and tested for insecticidal activity against the tick Boophilus microplus (Canestrini) and the flour feetle, Tribolium confusum Duval. In our experiment, compounds 9 ~ 11 inhibited ovogenesis of B. microplus and were toxic to T. confusum. Compounds 3 ~ 8 were found to have no activity.     

Production of Maltohexaose by α-Amylase from Bacillus circulans G-6

An α-amylase which produces maltohexaose as the main product from strach was found in the culture filtrate of Bacillus circulans G-6 which was isolated from soil and identified by the author.

The enzyme was purified by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 column chromatography. The purified enzyme was homogeneous on disc electrophoresis. The optimum pH and temperature of the enzyme were around pH 8.0 and around 60°C, respectively. The enzyme was stable in the range of pH 5–10. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Fe²⁺ and Co²⁺ inhibited the enzyme activity. The molecular weight was about 76,000. The yield of maltohexaose from soluble starch of DE (dextrose equivalent*) 1.8-12.6 was about 30%, and the combined action of the enzyme and pullulanase or isoamylase increased the yield of maltohexaose.     

Cloning and Expression of a β-Galactosidase Gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta^®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Formation of amyloid fibrils from β-amylase

Fibril formation has been considered a significant feature of amyloid proteins. However, it has been proposed that fibril formation is a common property of many proteins under appropriate conditions. We studied the fibril formation of β-amylase, a non-amyloid protein rich in α-helical structure, because the secondary structure of β-amylase is similar to that of prions. With the conditions for the fibril formation of prions, β-amylase proteins were converted into amyloid fibrils. The features of β-amylase proteins and fibrils are compared to prion proteins and fibrils. Furthermore, the cause of neurotoxicity in amyloid diseases is discussed.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Production of β-glucosidase byCladosporium resinae

Summary Cladosporium resinae QM 7998 produced high activities of extracellular and constitutive -glucosidase when grown on a variety of sugars or cellulose. Starch and ribose induced enzyme synthesis several fold.Cladosporium resinae could utilize agricultural waste residues for growth and -glucosidase production. The initial pH of the medium had a marked effect on enzyme prowduction and optimum pH was between 4.0 and 5.0 depending on the assay method. Mixed culturing ofC. resinae with yeasts, viz.Saccharomyces cerevisiae andCandida utilis, increased the -glucosidase production while that with other fungi decreased the enzyme yield. The- glucosidase preparation fromC. resinae significantly increased the saccharification of rice and wheat straw (untreated or delignified) withTrichoderma reesei QM 9414 cellulase preparation.

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Résumé Cladosporium resinae QM 7998 produit des concentrations élevées de -glucosidase tant extracellulaire que constitutive lorsqu'elle croît sur une variété de sucres ou sur la cellulose. On a trouvé que l'amidon et le ribose augmentent de plusieurs fois la quantité d'enzyme synthétisée.Cladosporium resinae peut utiliser des résidus agricoles pour sa croissance et pour la production de -glucosidase. Le pH initial du milieu exerce un effet marqué sur la production d'enzyme et le pH optimum est compris entre 4.0 et 5.0 selon les conditions de l'essai. La croissance mixte deCladosporium resinae avec diverses levures, notammentSaccharomyces cerevisiae etCandida utilis, augmente la production de -glucosidase tandis que celle avec d'autres moisissures diminue le rendement en enzyme. La -glucosidase deCladosporium resinae augmente de manière significative la saccharification des pailles de riz et de froment (non-traitées ou délignifiées) traités par la cellulase deTrichoderma reesei QM 9414.

     

Purification and properties of recombinant β-galactosidase from Bacillus circulans

A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for the β1-3 linkage was expressed in Escherichia coli. The β-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal β1-3 linked galactose residues. However it did not hydrolyse β1-4 linked galactooligosaccharides. Moreover, Galβ1-3GlcNAc, Galβ1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10–46% yield by the transglycosylation reaction using this enzyme.     

Causes of the production of multiple forms of β-galactosidase by Bacillus circulans

The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D.     

Construction of an industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax β-amylase gene and secreting β-amylase

An industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax -amylase gene was developed by using two yeast integrating plasmids. One plasmid was constructed that contains the geneticin resistance gene (Gt^r) as the selection marker and the ribosomal DNA (rDNA) portion that comprises the 18S rDNA as the recombination site. The other plasmid contains the aureobasidin A resistance gene (AUR1-C) as the selection marker and the chromosomal Ty sequence as the recombination site. The -amylase activity of one clone of Saccharomyces cerevisiae transformed sequentially with these two plasmids was approx. 9 times higher than that of Saprolegnia ferax. This type of integration was mitotically stable even after 100 generations of cell multiplication under non-selective conditions.     

Characterization of cDNA clones for rye endosperm β-amylase and analysis of β-amylase deficiency in rye mutant lines

Summary In order to characterize a -amylase deficiency in the endosperm of mutant rye lines, homologous cDNA probes were prepared. A rye cDNA library was constructed from a normal line and screened with a barley -amylase probe. Three partial cDNA clones specific for endosperm -amylase in rye were isolated and characterized. The largest of these clones was used to investigate the expression of endosperm -amylase in mutant and normal lines by Northern hybridization. These experiments, as well as in vitro translation experiments, demonstrate the absence of endosperm -amylase mRNA in mutant lines. Sequencing of three different cDNA clones revealed a single nucleotide difference, which suggests that two genes encoding endosperm -amylase genes might exist in rye. From Southern blots we anticipate that these two genes are tightly linked. Results of these experiments and previous data indicating that the mutation was located within the -amylase locus on chromosome 5 are consistent with the hypothesis that the mutation results from a deletion simultaneously affecting the two genes. However, due to extensive polymorphism within normal lines used as control, additional experiments will be required to further substantiate this conclusion. The deduced amino acid sequence reveals the occurrence of three short glycine-rich repeats containing 11 or 12 residues close to the carboxyl terminus of the protein. A comparison of the nucleotide sequence between rye and previously described barley cDNA clones revealed ca. 90% homology at the amino acid level, except in this C-terminal repeated part, where it drops to 45%.     

Immunological studies on β-amylase of Bacillus cereus

Summary An antiserum against the -amylase from Bacillus cereus BQ10-S1 Spo II was prepared using rabbits. The antiserum obtained was confirmed to form a specific immunoprecipitate with the purified -amylase and showed a single band of protein with a molecular weight of 6.0x10⁴ on the nitrocellulose sheet by the Western-Blotting method. The antiserum showed a precipitin line with the -amylase from B. megaterium strain no. 32 by the Ouchterlony technique. However, the spur was formed on the Ouchterlony plate between the line of immunoprecipitin of the -amylase from B. cereus BQ10-S1 Spo II and that from B. megaterium strain no. 32. On the other hand, no immune reaction occurred with the -amylase from B. polymyxa no. 72 and those from higher plants such as soybean and barley. B. cereus BQ10-S1 Spo II was found to secret -amylase mainly from the mid to the late logarithnic phase of cell growth. With the use of antiserum, the amount of the -amylase secreted was estimated to be about 52 g/10⁹ cells, that of the parent strain (B. cereus BQ10-S1) about 14 g/10⁹ cells. These quantities of -amylase corresponded in each case with enzyme productivity of the two strains (about 1,100 U/ml and 270 U/ml).Abbreviations EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis