Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50 and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca²⁺ and Cu²⁺ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca²⁺ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches. The K _m and V _max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis.     

Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C

Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76?%, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62?kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by ??-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50?°C and pH?7.0, respectively. The purified CMcellulase was stable with about 60?% activity in broad pH ranges from 5 to 10 and temperature of 40 to 60?°C. However, purified CMcellulase was stable at about 70?% at 70?°C and also stable overall at 78?% for surfactants. CMcellulase activity was inhibited by ions such as HgCl₂, followed by CuSo₄, FeCl₂, and MnCl₂, while CoCl₂ activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.     

Molecular Cloning,Characterization, and Dye-Decolorizing Ability of a Temperature- and pH-Stable Laccase from Bacillus subtilis X1

Laccases from fungal origin are typically unstable at high temperatures and alkaline conditions. This characteristic limits their practical applications. In this study, a new bacterial strain exhibiting laccase activity was isolated from raw fennel honey samples and identified as Bacillus subtilis X1. The CotA-laccase gene was cloned from strain X1 and efficiently expressed in Escherichia coli in a biologically active form. The purified recombinant laccase demonstrated an extensive pH range for catalyzing substrates and high stability toward alkaline pH and high temperatures. No loss of laccase activity was observed at pH 9.0 after 10 days of incubation, and approximately 21 % of the initial activity was detected after 10 h at 80 °C. Two anthraquinonic dyes (reactive blue 4 and reactive yellow brown) and two azo dyes (reactive red 11 and reactive brilliant orange) could be partially decolorized by purified laccase in the absence of a mediator. The decolorization process was efficiently promoted when methylsyringate was present, with more than 90 % of color removal occurring in 3 h at pH 7.0 or 9.0. These unusual properties indicated a high potential of the novel CotA-laccase for industrial applications.     

Purification and Characterization of the Enzyme Cholesterol Oxidase from a New Isolate of Streptomyces sp.

An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.     

Cloning of Haloacid Dehalogenase Gene from Bacillus Cereus Local Strain with the Addition of Restriction Sites

Previous studies had successfully isolated and characterized the haloacid dehalogenase gene from Bacillus cereus local strain, namely bcfd1 gene. In the further research, this gene would be sub cloned into expression vector in order to analyse its expression. However, this gene could not be directly sub cloned because it doesn’t have suitable restriction sites that facilitate correct orientation of cloning. Therefore, the addition of suitable restriction sites at both end of the gene was necessary. The research is started by designing specific pair of primers to amplify the bcfd1 gene from Bacillus cereus chromosome by adding EcoRI on forward primer and HindIII on reverse primer. The 870 bp of bcfd1 gene code for haloacid dehalogenase with suitable restiction sites has been successfully cloned into the pGEM-T Easy cloning vector. The recombinant clone that was obtained from screened by ampicillin resistant and ß-galactosidase activity was confirmed by size screening, restriction analysis, and re-PCR. This clone already to be performed for the further sub cloning process in order to get the right cloning direction.     

Characterization of Novel EGs Reconstructed from Bacillus subtilis Endoglucanase

Bacterial cellulases have taken on satisfactory application performance and economic value in detergent industry. Neutral endoglucanase (EG1) gene was cloned from Bacillus subtilis and expressed Pichia pastoris in our previous study. Redesigned endoglucanases enhanced cellulase domain, added and deleted carbohydrate-binding module (CBM), named EG2, EG3, and EG4, respectively, were constructed in this study. The redesigned EG genes were expressed in P. pastoris, and their characters were also discussed. The optimal temperature and pH value of the all EGs was 65 °C and 6.0, respectively, where their enzymatic activities in P. pastoris cultivation supernatant reached 867, 651, 966, and 881 U/mL. EG2 showed 24.9 % enzymatic activity loss compared to natural endoglucanase. EG4 showed specific activity 30 % loss and thermostability decrease compared to EG1, which suggested CBM played an important role in improving the catalytic power and heat stability of cellulase family which attached. The specific activity of EG2 and EG3 showed similar to EG1, which suggested neither enhancement of CD nor CBM to endoglucanase can improve its catalytic power, which might rest with its intact topologic structure.     

催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用

从枯草芽孢杆菌(Bacillus subtilis 168)基因组中的ORF32230出发,通过氨基酸序列分析推测其可能为酰基氨肽酶基因,并与典型的脯氨酸寡肽酶家族成员一致,含有2个独立的结构域,活性中心由催化三联体丝氨酸-天冬氨酸-组氨酸(Ser-Asp-His)组成.将BSU32230的基因片段与p ET-21a载体相连,转入BLP(DE3)表达菌中,在0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)存在及20℃条件下诱导表达该蛋白.利用硫酸铵沉淀与Ni亲和层析对BSU32230蛋白进行纯化,并通过实验证明该蛋白同时具有酯酶和肽酶2种活性.该酶最佳反应温度为50℃,最佳p H值为8.0,40℃下半衰期约29 h,在p H=4~10范围内稳定.该酶能够在有机相中催化不对称Aldol加成反应,且反应产物的立体选择性较好(84.6%).     

Biochemical Characterization and Antitumor Study of l-Glutaminase from Bacillus cereus MTCC 1305

l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na⁺, K⁺) and phosphate ion activated the enzyme activity, while divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, Pb²⁺, Ca²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cu²⁺) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K _m, V _max, K _cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22?×?10² M^?1s^?1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC₅₀ value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).     

Production, Purification, and Biochemical Characterization of a Fibrinolytic Enzyme from Thermophilic Streptomyces sp. MCMB-379

Production of the fibrinolytic enzyme was carried out using 2.5-L glass fermentor, culture of thermophilic Streptomyces sp., and glucose yeast extract peptone medium of pH 8.0. Five successive batches were carried out under controlled fermentation conditions viz., agitation 140 rpm, aeration 0.5 vvm, 55 °C, and 18 h. The total protein extracellularly produced in the cell-free broth was ~300-500 mg/L. The enzyme belongs to serine endopeptidase type. Studies on the fibrin degradation indicate that the enzyme degrades the fibrin into small molecular weight products as seen from HPLC profile. Phase-contrast microscopic structure of fibrin showed that enzyme cleaves the fibrin filaments. The ex vivo activity of the actinokinase was compared with 500 IU of urokinase and 350 IU of streptokinase. The ex vivo clot lysis was found to be faster as compared to the commercial available enzymes.     

Lipopeptides from the Banyan Endophyte, Bacillus subtilis K1: Mass Spectrometric Characterization of a Library of Fengycins

Mass spectrometric analysis of a banyan endophyte, Bacillus subtilis K1, extract showing broad spectrum antifungal activity revealed a complex mixture of lipopeptides, iturins, surfactins, and fengycins. Fractionation by reversed-phase high performance liquid chromatography (HPLC) facilitated a detailed analysis of fengycin microheterogeneity. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric studies permitted the identification of several new fengycin variants. Four major sites of heterogeneity are identified: (1) N-terminus ??-hydroxy fatty acid moiety, where chain length variation and the presence of unsaturation occur, (2) position 6 (Ala/Val/Ile/Leu), (3) position 10 (Val/Ile) within the macrocyclic ring, and (4) Gln to Glu replacement at position 8, resulting in fengycin variants that differ in mass by 1?Da. Diagnostic fragment ions provide a quick method for localizing the sites of variation in the macrocycle or the linear segment. Subsequent establishment of the sequences is achieved by MS/MS analysis of linear fengycin species produced by hydrolysis of the macrocyclic lactone. Unsaturation in the fatty acid chain and the presence of linear precursors in the B. subtilis K1 extract are also established by mass spectrometry. The anomalous distribution of intensities within isotopic multiplets is a diagnostic for Gln/Glu replacements. High resolution mass spectrometry facilitates the identification of fengycin species differing by 1?Da by localizing the variable position (Gln₈/Glu₈) in the fengycin variants.     

Cloning and Characterization of a Heme Oxygenase-2 Gene from Alfalfa (Medicago sativa L.)

Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H(2)O(2) (especially) treatment, respectively.     

絮凝基因的克隆及其絮凝形态表征

分离出一株具有强絮凝特性的芽孢杆菌Bacillus sp. F2, 其絮凝率达到84%, 并构建了絮凝基因组文库, 从中筛选并获得表达絮凝活性的大肠杆菌阳性克隆子FC2. 絮凝试验测定FC2的絮凝率为90%, 稍高于原絮凝菌F2, 高于受体菌JM109(6.9%), 说明FC2絮凝性状遗传于原絮凝菌F2. 采用轻敲模式下的原子力显微镜成像技术、光学显微镜、ζ电位等对加入FC2与F2及不加入絮凝剂的絮凝微观形貌等进行了测定. 原子力显微成像显示, 加入克隆菌FC2发酵液的高岭土悬浮液形成的絮凝胶体出现大而紧密的球形颗粒结构, 且表面积粗糙, 凹凸程度大, 具有大的比表面积和吸附液体悬浮颗粒的能力. 向高岭土悬浮液中加入克隆菌FC2发酵液后, 絮凝颗粒由松散的不定形结构转变为密集分布、 水平尺寸均匀的球形结构, 表明克隆菌FC2发酵液中的凝集素容易以高岭土悬浮颗粒为中心吸附在其表面, 而且絮凝率达到90%, 进一步证实了克隆菌FC2发酵液的除污染效能. ζ电位测定结果表明, 离子键作用强度不同, 致使絮凝形态存在差异, 为研究生物絮凝剂的絮凝机理提供了有力的依据.     

Characterization of a Heavy Metal Translocating P-Type ATPase Gene from an Environmental Heavy Metal Resistance Enterobacter sp. Isolate

Heavy metals are common contaminants found in polluted areas. We have identified a heavy metal translocating P-type ATPase gene (hmtp) via fosmid library and in vitro transposon mutagenesis from an Enterobacter sp. isolate. This gene is believed to participate in the bacterium’s heavy metal resistance traits. The complete gene was identified, cloned, and expressed in a suitable Escherichia coli host cell. E. coli W3110, RW3110 (zntA::Km), GG48 (ΔzitB::Cm zntA::Km), and GG51 (ΔzitB::Cm) were used to study the possible effects of this gene for heavy metal (cadmium and zinc in particular) resistance. Among the E. coli strains tested, RW3110 and GG48 showed more sensitivity to cadmium and zinc compared to the wild-type E. coli W3110 and strain GG51. Therefore, strains RW3110 and GG48 were chosen for the reference hosts for further evaluation of the gene’s effect. The results showed that expression of this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms.     

Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery

Surfactin produced by Bacillus subtilis (ATCC 21332) was used to examine the effect of altering salt concentration, pH, and temperature on surfactin activity (as measured by reductions in surface tension). These parameters are some of the conditions that define oil reservoir characteristics and can affect the application of surfactants. The Biotechnology for Oilfield Operations research program at the Idaho National Engineering and Environmental Laboratory (INEEL) has successfully produced surfactin from potato process effluents for possible use as an economical alternative to chemical surfactants for improved oil recovery. Surfactants enhance the recovery of oil through a reduction of the interfacial tension between the oil and water interfaces, or by mediating changes in the wettability index of the system. We investigated changes in surfactin activity under a range of conditions by measuring surface tension. Surface tension was determined using video image analysis of inverted pendant drops. Experimental variables included NaCl (0–10%), pH (3.0–10.0), and temperature (21–70°C). Each of these parameters, as well as selected combinations, resulted in discrete changes in surfactin activity. It is therefore important to consider the exploration of the studied surfactin as an enhanced oil recovery agent.