Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus

Summary The proteolytic activity produced by a new species of Bacillus isolated in our laboratory was investigated. This enzyme was purified to homogeneity from cell-free culture liquids of B. thermoruber. The purification procedure included ion-exchange chromatography on DEAE-Sephadex A-50 and -casein agarose affinity chromatography. The protease consists of one polypeptide chain with a molecular weight of 39000±800. the isoelectric point was 5.3; the optimum pH and temperature for proteolytic activity (on casein) was found to be pH 9 and 45°C respectively. Enzyme activity was inhibited by PMSF and EDTA. The stability was considerably increased by addition of Ca²⁺, and the protease exhibited a relatively high thermal stability. The alkaline protease shows a preference for leucine in the carboxylic side of the peptide bond of the substrate. The K _m value for benzyloxycarbonyl-Ala-Ala-Leu-p-nitroanilide was 2.5 mM.     

β-Galactosidase in Alkalophilic Bacillus

The changes in ¹³C-NMR and ³¹P-NMR spectra and ¹H-NMR images in soybean cotyledons during germination were investigated. Using ¹³C-NMR, fatty acid signals in the form of triglycerides were observed in dry seeds, and those were observed approximately 18 days after the start of imbibition. Sucrose signals appeared at 16 hr and disappeared at 5 days. A signal was observed after 5 days, suggesting the activation of membrane metabolism in the cotyledons.

³¹P-NMR signals appeared 2 hr after imbibition before any apparent change in the ¹³C-NMR spectrum. The peaks identified as sugar phosphate, inorganic phosphate in the cytoplasm and in the vacuole, and an unassigned compound, were distinguishable after 5 days. The vacuole-associated inorganic phosphate peak became prominent 18 days after imbibition in ³¹P-NMR.

Distribution maps of free water indicated that the stored macro-molecular materials which bound water were consumed heterogeneously within the cotyledon. The relaxation time (T₁) increased suddenly between 18 days and 23 days after imbibition, which indicates the consumption of stored materials.

These findings suggest that cotyledons are the source of such compounds and the energy required for plant growth for approximately 18 days from germination until tri-foliolate leaves begin developing.     

Propeptide of Bacillus subtilis amylase enhances extracellular production of human interferon-α in Bacillus subtilis

The Gram-positive bacterium, Bacillus subtilis and related species are widely used industrially as hosts for producing enzymes. These species possess a high potential to produce secreted proteins into the culture medium. Nevertheless, the secretion of heterologous proteins by these species is frequently inefficient. In this study, the human interferon-α2b (hIFN-α2b) was used as a heterologous model protein, to investigate the effect of B. subtilis AmyE propeptide in enhancing the secretion of heterologous proteins in B. subtilis. We found that the secretion production and activity of hIFN-α2b with AmyE propeptide increased by more than threefold, compared to that without AmyE propeptide. The maximum amount of secreted hIFN-α2b with propeptide was 14.8 ± 0.6 μg ml⁻¹. In addition, the pro-hIFN-α2b bioactivity reached 5.4 ± 0.5 × 10⁷ U mg⁻¹, which is roughly the same level as that of the non-propeptide hIFN-α2b. These results indicated that AmyE propeptide enhanced the secretion of the hIFN-α2b protein from B. subtilis. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.     

A novel α-glucosidase produced by Bacillus amylolyticus

Bacillus amylolyticus produces -amylase, pullulanase and -glucosidase. By selection of carbon source in the growth medium, -glucosidase was produced preferentially and with exclusion of the other two activities. The -glucosidase was highly specific for maltose and to a lesser extent maltotriose but was inactive towards a range of other substrates including p-nitrophenyl -D-glucoside and isomaltose. Optima for activity were recorded at pH 7.0 and 40° C and the enzyme was insensitive to ethylenediaminetetraacetic acid.     

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 of α-amylase from Bacillus lentus cultures

Acetone fractionation of Bacillus lentus culture filtrate yielded the highest -amylase activity and the 66.6% fraction reached 13-fold that of the crude enzyme preparation. Gel filtration and ion exchange chromatography afforded a pure -amylase (relative molecular mass, 42 000). The pure enzyme was highly active on starch and dextrin. It produced a mixture of oligosaccharides as major products of starch hydrolysis. Maximal activity was reached at 70° C and pH 6.1. Ca²⁺, Na⁺, K⁺ and Sr²⁺ ions stabilized or slightly stimulated the enzyme whereas Ag⁺, Co²⁺, Hg²⁺, Zn²⁺, Cd²⁺ and Fe³⁺ ions strongly inhibited the activity. The enzyme contained 16 amino acids, of which aspartic and glutamic acids were present in the highest proportions. Correspondence to: S. H. Omar     

Control of ��-amylase production by Bacillus subtilis

This study proposes two adaptive control algorithms for the fed-batch production of α-amylase. The first one uses online information from hardware measuring glucose. Online information of both biomass and glucose concentrations measured with different frequency is used in the second algorithm. Hardware measuring variables are inputs for software sensors of glucose concentration and (specific) glucose consumption rate. Either of the algorithms do not require any kinetic coefficients. This is a benefit, because the kinetic coefficients can vary during cultivation and between cultivations, leading to low process reproducibility and the non-stationary state of the bioprocess. The results of simulation investigations show good performance of the proposed control schemes.     

Bacillus cytotoxicus—A potentially virulent food-associated microbe

Bacillus cytotoxicus is a member of the Bacillus cereus group with the ability to grow at high temperatures (up to 52℃) and to synthesize cytotoxin K-1, a diarrhoeagenic cytotoxin, which appears to be unique to this species and more cytotoxic than the cytotoxin K-2 produced by other members of this group. Only a few isolates of this species have been characterized with regard to their cytotoxic effects, and the role of cytotoxin K-1 as a causative agent of food poisoning remains largely unclear. Bacillus cytotoxicus was initially isolated from a food-borne outbreak, which led to three deaths, and the organism has since been linked to other outbreaks all involving plant-based food matrices. Other studies, as well as food-borne incidents reported to the UK Food Standards Agency, detected B. cytotoxicus in insect-related products and in dried food products. With insect-related food becoming increasingly popular, the association with this pathogen is concerning, requiring further investigation and evidence to protect public health. This review summarizes the current knowledge around B. cytotoxicus and highlights gaps in the literature from a food safety perspective.     

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     

Enzymatic characterization of Bacillus licheniformis γ-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was fused at its C-terminal end with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase. BlGGT and six fusion enzymes, BlGGT/SBD, BlGGT/AMYΔN476, BlGGT/AMYΔN443, BlGGT/AMYΔN376, BlGGT/AMYΔN195, and BlGGT/AMYΔN34, were over-expressed in Escherichia coli M15 cells and purified to apparent homogeneity by metal-affinity chromatography. The fusion constructions had no significant effect on the autocatalytic processing of BlGGT. Progressive decrease in the GGT activity of fusion proteins was associated with an increasing level of truncation, and only BlGGT/AMYΔN34 reserved the amylolytic activity. The protein fusions did not alter the optimal temperature and pH of BlGGT. However, as compared with the parental BlGGT, a significant change in circular dichorism and fluorescence spectra was observed in the fusion enzymes. Thermal unfolding of BlGGT, BlGGT/AMYΔN476, BlGGT/AMYΔN443, and BlGGT/AMYΔN376 followed the two-state unfolding process with a transition point (T(m)) of 61.3-63.1 °C, whereas BlGGT/AMYΔN195 and BlGGT/AMYΔN34 displayed two temperature transitions at 40.6 and 46.7 °C as well as at 62.8 and 62.9 °C, respectively. All of the fusion enzymes exhibited the raw-starch-binding ability, and the adsorbed proteins could be eluted from the adsorbent by 50mM Tris-HCl (pH 9.0) containing 2% soluble starch.     

Secretion of biologically-active human interferon-β by Bacillus subtilis

Human interferon-β (hIFN-β) was used as a heterologous model protein to investigate the effects of the Bacillus subtilis AmyE propeptide and co-expression of PrsA in enhancing the secretion of heterologous proteins in B. subtilis. Secretion and activity of hIFN-β with AmyE propeptide increased by more than four-fold compared to that without AmyE propeptide. Moreover, under conditions of co-expressed PrsA, the secretion production and activity of hIFN-β with AmyE propeptide increased by more than 1.5-fold. AmyE propeptide and co-expression of PrsA thus have an additive effect on enhancing the production of the hIFN-β in B. subtilis.     

枯草杆菌(Bacillus subtilis)稳定的基因表达系统

芽孢杆菌属(Bacillus)在应用微生物学方面有着悠久的应用历史,尤其是本世纪以来,该属在工业用酶生产中所起的作用愈来愈显重要。液化淀粉芽孢杆菌(B.amyloliquefaciens)曾占据α—淀粉酶工业生产的主导地位,而地衣芽孢杆菌(B.1icheniformis)则是当今世界工业用酶的主要来源之一。     

蛴螬病原菌(Bacillus sp.)的初步研究

蛴螬是农林生产上的重要地下害虫,由于长期栖居地下,防治较为困难。多年实践证明,采用综合防治措施可以有效地控制为害,利用病原微生物防治蛴螬是综合防治中的一个环节,是值得今后深入研究的课题。 1975年9月,我们在河北省任邱县白洋淀附近玉米地采到数头自然患病的阔胸犀金龟(Pento-don patruelis Frivaldszkz)三龄幼虫。1976年5     

Circular-dichroism studies on two β-lactamases from Bacillus cereus

The circular-dichroism (CD) spectra of beta-lactamases I and II from Bacillus cereus 569/H are reported, along with that of the beta-lactamase II free from carbohydrate. The results show that carbohydrate makes an appreciable contribution to the optical activity of beta-lactamase II in the far-ultraviolet, and that removal of carbohydrate greatly affects the optical activity of several aromatic side chains of the protein moiety. Both tyrosyl and tryptophanyl residues are affected, showing that some of these residues must be near to the surface of the protein moiety, close to the site of attachment of the carbohydrate. Although the far-ultraviolet CD spectrum of beta-lactamase II resembles that of a protein containing some beta-structure, it can be shown that this is a consequence of the optical activity of carbohydrate in this region of the spectrum, and that the protein is likely to contain alpha-helix rather than beta-pleated sheet structure. The overall structures of the protein components of beta-lactamases I and II are similar, but not identical, as shown by the dissimilarity of the CD spectra when calculated on a mean residue basis.