Raw starch digesting amylase from Thermoactinomyces thalpophilus F13

Thermoactinomyces thalpophilus produced a raw-starch digesting amylase (RSDA) when grown on both cereal and tuber starches. Glucose, maltose and glycerol repressed enzyme production. Highest activity was recorded on rice starch (39 U ml^-1). Considerable variability existed in the effectiveness of nitrogenous nutrients to stimulate expression of RSDA. Multiple pH optima for RSDA production and activity suggests enzyme heterogeneity.     

Cloning and expression of a pathway for benzene and toluene fromBacillus stearothermophilus

Bacillus stearothermophilus strain BR325 demonstrating broad aromatic substrate capability was isolated from petroleum-contaminated soil. The chromosomally-located aromatic pathway from this isolate was cloned intoEscherichia coli as a 32 kb insert in cosmid pHC79, conferring growth on benzene, phenol, and toluene as sole carbon sources.     

Intraspecific protoplast fusion of amylase-producing strains of Candida fennica

The production of -amylase was increased by protoplast fusion of auxotrophic mutants of Candida fennica FTPT-8903. One prototrophic fusant was 90% and 32% more efficient in producing -amylase in semi-solid and liquid fermentation, respectively, than the parental strains. Protoplast fusion did not significantly stimulate the synthesis of glucoamylase in the fusants.     

Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.     

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl₂.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

Optimization of a Thermostable Lipase from Bacillus stearothermophilus P1: Overexpression, Purification, and Characterization

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55°C. It was highly stable in the temperature range of 30–65°C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37°C in 0.1% Chaps and Triton X-100.     

Delignification of wood pulp by a thermostable xylanase from Bacillus stearothermophilus strain T-6

During the bleaching of wood pulp for the paper industry, large amounts of chlorinated aromatic compounds are produced and released into the environment. These compounds are extremely toxic and are a major source of pollution. The paper and pulp industry is seeking for alternative methods for bleaching pulp. One such method involves the use of hemicellulases to release the colored lignohemicellulose. We have isolated and characterized several thermophilic bacteria which produce xylanases. One such strain, T-6, produced high levels of extracellular xylanase, free of cellulase and proteinase activities. Strain T-6 was classified as a strain of Bacillus stearothermophilus and was able to grow on defined medium containing xylose, methionine and asparagine at 65 °C. Xylanase activity was induced by either xylose or xylan; no activity was detected with other carbon sources, such as glycerol, acetate, lactose, glucose, maltose, fructose, mannose, galactose or sucrose. Xylanase constitutive mutants were obtained following mutagenesis and detection on p-nitrophenol -d-xylopyranoside containing agar plates. Xylanase T-6 was produced on large scale, and was purified and concentrated by a single adsorption-desorption step from a cation exchanger. The overall purification yield of a 1000 liter fermentation was 45%, resulting in a 98% pure enzyme. Xylanase T-6 was shown to partially remove lignin from unbleached pulp at 65 °C and pH 9.0, without loss in pulp viscosity. The enzyme-treated pulp was used to make handsheets that had higher brightness than untreated pulp.     

Resistance of bacteria from cooling waters to bactericides

Summary Bacteria from water cooling systems developed resistance to three different bactericides i.e. quarternary ammonium compound (QAC), isothiazolone and thiocarbamate. Resistance was induced by exposing isolates to increasing sublethal concentrations for a period of 10 weeks.Bacillus subtilis became resistant to 1000 mg l^–1 QAC. Cross-resistance was also detected, e.g. isothiazolone induced resistance to QAC and thiocarbamate.     

Fusion of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> leucine aminopeptidase II with the raw-starch-binding domain of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 -amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70°C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k _cat value.     

Differential expression of thermophilic phosphatases in the wild type and auxotrophic mutant strains of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

In the wild type strain (stock no. 1227) of Thermoactinomyces vulgaris, as reported earlier [Sinha and Singh (1980) Biochem. J. 190, 457–460], all phosphatase isoenzymes (three alkaline — AlpI, AlpII and AlpIII, and one acidic — Acp) are present. However, the auxotrophic mutants, the strains 1286 (thi ⁻), 1279 (nic ⁻, ura ⁻) and 1278 (thi ⁻, ura ⁻) exhibited two alkaline phosphatase isoenzymes (AlpII and AlpIII), but AlpI was lacking. In the strain 1261 (nic ⁻, thi ⁻), only AlpIII was expressed, and AlpI and AlpII isoenzymes were missing. The results suggest that the strains, which require either thiamine (1286 and 1278) or nicotinamide (1279) for their growth, were AlpI ⁻ mutants; and the strain (1261), which requires both thiamine and nicotinamide for its growth, was AlpI ⁻/AlpII ⁻ double mutant. There was no direct correlation between uracil auxotrophy and the expression of phosphatases. The uniform expression of AlpIII and Acp in all the strains, irrespective of their nutrient requirements, suggest that these constitutive phosphatases are species-specific. The specific activities of the thermophilic acid and alkaline phosphatases were maximum in the wild type strain (1227) of T. vulgaris. The next in phosphatase activity was the strain 1279 (an AlpI ⁻ mutant), followed by their decrease, in order, in the strains 1286 and 1278 (which were also AlpI ⁻ mutants); while least activity of these enzymes was observed in the obligate thermophile strain 1261 (AlpI ⁻/AlpII ⁻ double mutant).     

Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids

Seven thermophilic anaerobic bacteria which ferment xylan were isolated from natural geothermal features in the western United States. Typically, these strains were Gram-negative non-sporeforming rods with an unusual double-layered cell wall which resembled that observed in Thermobacteroides acetoethylicus. The strains differed from known thermophilic anaerobes in their ability to utilize a very wide variety of carbohydrates and in their ability to grow in a chemically-defined medium and/or at pH 3.5. Four of the strains contained cryptic plasmids of 1.2 or 1.5×10⁶ daltons. The taxonomic characteristics of the strains are discussed in terms of their relatedness to those of Thermoanaerobium, Thermoanaerobacter, and Thermobacteroides species.Contribution No. 3222 of the Central Research and Development Department     

Directed Mutagenesis of the Conserved Asparagine Residues of Bacillus Stearothermophilus Leucine Aminopeptidase II

Summary To understand the structure–function relationships of Bacillus stearothermophilus leucine aminopeptidase II (LAPII), each of the four conserved asparagine residues was replaced with leucine, aspartate, and lysine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes with an apparent molecular mass of approximately 44.5 kDa were purified to homogeneity by nickel-chelate chromatography. Substitution of Asn-245, Asn-335, and Asn-341 with Lys generated variants with a dramatic loss of LAP activity. Kinetic analysis of Asn-373 variants with p-leucine-nitroanilide as the substrate revealed an increase in k_cat with no significant change in K_m, leading to a more than 2-fold increase in the catalytic efficiency. Thermostability assays showed that replacement of Asn-335, Asn-341, and Asn-373 by aspartic acid markedly increased the half-life of the enzyme at 70 °C, indicating that the deamination of these residues may have a deleterious effect on LAPII.     

Histidines 345 and 378 of <Emphasis Type="Italic">Bacillus stearotheromophilus</Emphasis> leucine aminopeptidase II are essential for the catalytic activity of the enzyme

The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the K_m value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in K_cat/K_m for H227L was partly caused by a 3.9-fold increase in K_m value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.     

Properties of immobilized lipase from Bacillus stearothermophilus MC7. Acidolysis of triolein with caprylic acid

Extracellular bacterial lipases are promising biocatalysts for industry, because they are stable and active enzymes from easily available sources. A lipase from Bacillus stearothermophilus MC7 was immobilized on four polymer carriers by physical adsorption: chitosan, DEAE-cellulose, polypropylene, and polyurethane. The four biocatalysts differ in their hydrolytic activity against long-chain and short-chain triglycerides. Lipase MC7 immobilized on polypropylene (PP-MC7) stands out with its high activity against tributirin. According to the preliminary data, all four preparations were suitable for application in the test reaction of acidolysis of triolein with caprylic acid. The highest degree of conversion of the initial triolein was achieved in the presence of PP-MC7 preparation—50%. But variation of the reaction conditions did not lead to synthesis of the target di-substituted product (dicapryloyl-oleoylglycerol, COC). Reaction proceeds as a selective mono-substitution in the glycerol backbone.     

Toward the smallest active subdomain of a TIM-barrel fold: insights from a truncated α-amylase

AmyTM is a truncated mutant of the α-amylase of Bacillus stearothermophilus US100. It has been derived from the wild type amylase gene via a reading frame shift, following a tandem duplication of the mutant primer, associated to an Adenine base deletion. AmyTM was composed of 720 nucleotides encoding 240 amino acid residues out of 549 of the wild type. The AmyTM protein was devoided of the three catalytic residues but still retains catalytic activity. It is Ca-independent maltotetraose producing amylase, optimally active at pH 6 and 60 °C, under monomeric or multimeric forms.AmyTM is the smallest functional truncated TIM barrel. It contains the βαβα unit as the minimal subdomain associated to an enzymatic function. The enzymatic activity can, until now, be attributed to the presence of the whole domain B, in the structure of AmyTM. This mutant revealed, for the first time, the regeneration of a catalytic site after its abolition. This fact may be considered as the restoration of a primitive active site, which was lost in the course of evolution toward more stable domains.     

Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 °C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be␣divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could␣be discriminated from the mesophilic by the two PCR-based methods used to characterize B.␣weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.     

Transformation of synthetic pyrethroid insecticides by a thermophilic Bacillus sp

Employing a mineral salts medium containing Tween 80 as the primary carbon source, a strain of Bacillus stearothermophilus was isolated which was able to hydrolyse selected second and third-generation pyrethroids to non-insecticidal products. Of a range of pyrethroid insecticides the trans-isomer of permethrin was the most readily transformed by this microbial isolate, whilst flumethrin was the least. 3-Phenoxybenzoic acid and the respective halovinyl or haloacid moieties were detected as the major hydrolytic products of the pyrethroids. It is believed that 3-phenoxybenzoic acid was formed from 3-phenoxybenzyl alcohol which was not however detected as an intermediate in these systems. 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoic acid. A potential metabolic pathway has been described.     

Dynamics of heat destruction of spores: A new view

Summary Discrepancies between actual, viable spore populations and those predicted by a classical model during heat sterilization of food and pharmaceutical products have long concerned food engineers and scientists as they pursue new sterilization techniques, including ultra-high temperature processes. Among potential causes of those discrepancies, activation of dormant spores is significant, and models addressing that factor were developed recently. This paper reviews historic and current views on the biology and models of microbial spore populations during heat sterilization. Activation and inactivation of viable spores are emphasized, with each viewed as a first-order reaction. Rate constants of those reactions may differ significantly, inactivation rates of dormant and activated spores may differ, and variations of all rate constants with temperature appear to be well described by Arrhenius equations. Model-based analyses show how categories of survivor response curves observed during isothermal heat treatments can arise from simultaneous activation and inactivation of spores in an overall population. Effects of different distributions of initial subpopulations, different distributions of rate constants, and heat shock for homogenizing an indicator population are shown. The complexity of new, multiple process models has not increased greatly, but the potential for accurate, dynamic prediction of product safety after prescribed sterilization has. The relevant biology is understood and accounted for more thoroughly, and it is anticipated that the new models will aid design and evaluation of new and improved sterilization processes for food and pharmaceuticals.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.     

一株高产淀粉酶的解淀粉芽胞杆菌的分离鉴定及其产酶条件优化

目的从土壤中分离产淀粉酶相对较高的菌株,并对其产酶条件进行优化,以期获得更经济、易得的高活力淀粉酶。方法用平板稀释涂布法分别从温州及其周边地区多个淀粉含量高的土壤、污水中采集的样品中筛选产淀粉酶菌株,通过菌落形态、生理生化反应和16S rDNA序列比对对菌株进行鉴定;并从碳源、氮源、离子浓度及紫外诱变等产酶条件对高产淀粉酶菌株进行优化;利用碘-淀粉法等对粗酶液酶学性质进行初步研究。结果从采集到的26个样品中共筛选到19株酶活较高、产酶稳定的野生型菌株。其中编号为Z19的菌株酶活最高,达到351.78 U/mL。经形态和生理生化鉴定,结合16S rDNA序列分析,将该菌株鉴定为解淀粉芽胞杆菌。对菌株Z19的液体发酵条件研究表明,最佳条件为玉米粉1.0%,明胶1.5%,淀粉含量0.5%,NaCl 0.5%,pH7.0,发酵温度37℃,72 h,酶活达到1 360.92 U/mL;在固体培养基上,直接用麸皮,接种量为0.1%,含水量为60%,pH7.0,120 h时,酶活达到1 504.2 U/mL。其最适酶反应pH为6.5,反应温度55~60℃,底物浓度为0.5%。结论从土壤中分离得到了产淀粉酶相对较高的解淀粉芽胞杆菌Z19菌株,产酶条件适用于工业生产,具有一定的应用前景。