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.     

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 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.     

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.     

Multiple β-1,3 Glucanases in the Lytic Enzyme Complex of Bacillus circulans WL 12

The lytic enzyme complex produced by Bacillus circulans WL 12 upon induction with the myceiia of Piricularia oryzae P₂ was analyzed by large scale polyacrylamide gel electrophoresis, Six β-1,3 glucanases were separated. Activity profiles were obtained of these multiple β-1,3 glucanases against P. oryzae P₂ cell walls, Saccharomyces cerevisiae walls, laminarin, oat glucan, Phytophthora glucan and pachyman.     

Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced -amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55C. The -amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of -amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.     

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%.     

Effect of glutaraldehyde on oligosaccharide production by β-galactosidase from Bacillus circulans

Summary The oligosaccharide-producing activity of -galactosidase-1, one of the isomers of -galactosidase (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was changed after immobilization onto porous silica gel (Merckogel) by crosslinkage with glutaraldehyde. The reason for this modification was studied by treating the free enzyme with glutaraldehyde. Glutaraldehyde of 0.025% to 3% modified 40% to 90% of the free amino groups with or without intermolecular crosslinking. The maximum yield of oligosaccharides increased from 12% to 40% depending upon degree of modification, while native enzyme gave only 6% trisaccharides during hydrolysis of 127 mM lactose. The K _m value for the enzyme treated with glutaraldehyde was also increased.     

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 β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

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.     

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.     

Multiple Forms of Rice Seeds β-Amylases on Zymogram

Hen lysozyme modified with histamine (HML) and Japanese quail lysozyme (JQL) were treated with immobilized metal ion affinity chromatography to analyze the states of their imidazole groups. When Ni(II) was used as the metal ion immobilized, JQL was strongly retained in a Ni(II)-chelating Sepharose column, while hen lysozyme and HML were hardly retained in the same column. All of these lysozymes have a histidine imidazole group at the 15th position, while JQL has an additional histidine imidazole group at the 103rd position and HML has an additional imidazole group covalently attached to Asp101. Thus, I concluded that the imidazole group at the 103rd position of JQL is exposed to the solvent and recognized by the metal ion, but that the imidazole group attached to Asp101 in HML is localized to a hydrophobic region and not recognized by the metal ion.     

Immobilization of β-Galactosidase by Polyacrylamide in the Presence of Protective Agents

β-Galactosidase from Aspergillus oryzae was immobilized in crosslinked polyacrylamide gel beads. The presence of the enzyme inhibitor, such as glucono-δ-lactone or galactono-γ-lactone, during polymerization procedure enhanced the residual enzymatic activity in the polymer beads, and activity yield attained up to 45%. Such enhancement effect was also observed when bovine serum albumin, dithiothreitol or glutathione was added during polymerization. Temperature and pH optima were not affected by the immobilization. The Michaelis constants for free and immobilized β-galactosidase were comparable. Lyophilized beads exhibited good stability without loss of enzymatic activity when stored at 4°C for 47 days.     

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.     

Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9 mg/g and 36.4 h of contact, resulting in a biocatalyst with 595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7 g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.     

Immobilization of β-Galactosidase by physical adsorption on Chromosorb-W

Summary -Galactosidase was immobilized by physical adsorption on Chromosorb-W. 94.8 % of the initial protein was adsorbed at 4^ºC, pH=5.5 and ionic strength=0.31. The specific activity of the immobilized derivative was 105.3 U/mg and K_M for o-nitrophenyl- -D-galactopyranoside was 0.12 mM.     

Stimulation of Lactic Streptococci in Milk by β-Galactosidase

Acid production in milk by lactic streptococci was stimulated by added beta-galactosidase. Both glucose and galactose accumulated rapidly in the presence of this enzyme. Glucose accumulation ceased as the culture entered the most rapid period of acid production, whereas galactose accumulation continued. In cultures without added beta-galactosidase, a low concentration of galactose accumulated in the milk, whereas glucose was not detected after 2 hr of incubation. Cultures grew and produced acid faster in broth containing glucose rather than galactose or lactose. These observations suggest that the lactic streptococci do not metabolize the lactose in milk efficiently enough to permit optimum acid production and that a phenomenon such as catabolite repression functions to allow for a preferential use of glucose over either galactose or lactose. In addition to providing the culture with a more readily available energy source, it is possible that the culture produced more acidic metabolites as a result of preferentially utilizing the glucose released by the action of the beta-galactosidase.     

Production of β-glucosidase by Aspergillus terreus

The production of -glucosidase by Aspergillus terreus was investigated in liquid shake cultures. Enzyme production was maximum on the 7th day of growth (2.18 U/ml) with the initial pH of the medium in the range of 4.0–5.5. Cellulose (Sigmacell Type 100) at 1.0% (wt/vol) gave maximum -glucosidase activity among the various soluble and insoluble carbon sources tested. Potassium nitrate was a suitable nitrogen source for enzyme production. Triton X-100 at 0.15% (vol/vol) increased the enzyme levels of A. terreus. The test fungal strain showed an ability to ferment glucose to ethanol.