枯草芽孢杆菌基因启动子的分离与鉴定

利用启动子探针型载体pSUPV4直接在大肠杆菌 (Escherichiacoli)中分离枯草芽孢杆菌 (Bacillussubtilis)WB6 0 0的基因启动子片段 ,获得 5 5个具有卡那霉素抗性的重组子。对 3个抗性最高的重组子pSU -Bs2 ,pSU -Bs4 ,pSU -Bs8进行序列测定和同源性分析发现 ,所克隆到的基因启动子片段均来自于枯草芽孢杆菌的基因组 ,并且具有枯草杆菌基因启动子的保守序列。对抗性最高的Bs2片段进一步研究表明 ,它可以在大肠杆菌中高效地启动来自于短小芽孢杆菌的碱性蛋白酶基因的表达 ,也能在枯草芽孢杆菌中启动卡那霉素抗性基因的表达。     

解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K⁺对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。     

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Cloning, Sequencing, and Disruption of the Bacillus subtilis psd Gene Coding for Phosphatidylserine Decarboxylase

The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of pss, the structural gene for phosphatidylserine synthase that catalyzes the preceding reaction in phosphatidylethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456–7461, 1994). Introduction of a plasmid containing the psd gene into temperature-sensitive Escherichia coli psd-2 mutant cells allowed growth at otherwise restrictive temperature. Phosphatidylserine was not detected in the psd-2 mutant cells harboring the plasmid; it accumulated in the mutant up to 29% of the total phospholipids without the plasmid. An enzyme activity that catalyzes decarboxylation of ¹⁴C-labeled phosphatidylserine to form phosphatidylethanolamine was detected in E. coli psd-2 cells harboring a Bacillus psd plasmid. E. coli cells harboring the psd plasmid, the expression of which was under the control of the T710 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The psd gene, together with pss, was located by Southern hybridization to the 238- to 306-kb SfiI-NotI fragment of the chromosome. A B. subtilis strain harboring an interrupted psd allele, psd1::neo, was constructed. The null psd mutant contained no phosphatidylethanolamine and accumulated phosphatidylserine. It grew well without supplementation of divalent cations which are essential for the E. coli pssA null mutant lacking phosphatidylethanolamine. In both the B. subtilis null pss and psd mutants, glucosyldiacylglycerol content increased two- to fourfold. The results suggest that the lack of phosphatidylethanolamine in the B. subtilis membrane may be compensated for by the increases in the contents of glucosyldiacylglycerols by an unknown mechanism.     

Molecular Cloning and Characterization of Fengycin Synthetase Gene fenB from Bacillus subtilis

A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed in Escherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25°C, an optimum pH of 4.5, a K_m value of 922 μM, and a turnover number of 236 s⁻¹. FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.     

原噬菌体PBSX阻遏基因及其温度敏感型等位基因的克隆分析

本研究利用聚合酶链式反应技术,成功地克隆了枯草芽孢杆菌缺陷型原噬菌体ＰＢＳＸ阻遏基因及其温度敏感型等位基因。核苷酸序列分析发现,野生型及其温度敏感型阻遏基因之间的碱基变异较大,但却存在几乎完全相同的开放读框,尤其是开放读框ｏｒｆⅠ,可能编码着１１３个氨基酸的阻遏蛋白,并且还推定了开放读框的启动区和核糖体结合位点。通过互补实验,证实了野生型阻遏基因的产物能够抑制温度诱导ＰＢＳＸ原噬菌体,表明克隆的基因有着正常的生物活性。     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Cloning and Sequence Analysis of the meso-Diaminopimelate Decarboxylase Gene from Bacillus methanolicus MGA3 and Comparison to Other Decarboxylase Genes

[This corrects the article on p. 2935 in vol. 59.].     

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as α_s-casein and BSA.     

Purification and Characterization of Gallic Acid Decarboxylase from Pantoea agglomerans T71

Oxygen-sensitive gallic acid decarboxylase from Pantoea (formerly Enterobacter) agglomerans T71 was purified from a cell extract after stabilization by reducing agents. This enzyme has a molecular mass of approximately 320 kDa and consists of six identical subunits. It is highly specific for gallic acid. Gallic acid decarboxylase is unique among similar decarboxylases in that it requires iron as a cofactor, as shown by plasma emission spectroscopy (which revealed an iron content of 0.8 mol per mol of enzyme subunit), spectrophotometric analysis (absorption shoulders at 398 and 472 nm), and inhibition of the enzyme activity by 2,2′-bipyridyl, o-phenanthroline, and EDTA. Another interesting feature of this strain is the fact that it contains a tannase, which is used together with the gallic acid decarboxylase in a two-enzyme resting cell bioconversion to synthesize valuable pyrogallol from readily available tannic acid.     

枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析

本文对33株枯草芽孢杆菌群菌株进行β-甘露聚糖酶活性筛选,其中的32株具有β-甘露聚糖酶活性,只有1株无β-甘露聚糖酶活性.通过基因克隆测序的方法获得33株枯草芽孢杆菌群菌株β-甘露聚糖酶基因编码区全序列,对酶基因进行同源性分析并构建系统发育树;在β-甘露聚糖酶基因系统发育树中,33株枯草芽孢杆菌群菌株聚为3个分支,分别是枯草芽孢杆菌分支、地衣芽孢杆菌分支和解淀粉芽孢杆菌分支;枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌β-甘露聚糖酶基因种内同源性大于91%,而种间同源性为60%69%.     

短小芽孢杆菌degQ基因的克隆与鉴定

degQ基因编码一个由46个氨基酸组成的多肽,能增强许多芽孢杆菌胞外酶基因的表达．以pMK4作克隆载体构建短小芽孢杆菌基因文库,并用DNA探针原位杂交法从中钓出degQ基因．对克隆基因的DNA序列进行了分析并证明克隆的短小芽孢杆菌degQ基因具有增强枯草杆菌蛋白酶和果聚糖蔗糖酶基因表达的能力．degQ基因克隆有助于研究芽孢杆菌的正调控机理并可望提高外源基因在芽孢杆菌中表达．     

Cloning and Expression of Thermostable alpha-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable alpha-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min.     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans

A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K_m of 2.9 ± 0.1 mM and a k_cat of 879 ± 73 min⁻¹. The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. The IS1071 transposon insertion sequence was found downstream of pdeA.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product from Bacillus subtilis

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.     

枯草芽孢杆菌耐盐突变株proA基因克隆及proBA基因渗透压调节功能研究

利用TaKaRaLAPCRTM试剂盒扩增枯草芽孢杆菌 931 5 1耐盐突变株proA基因的未知下游序列。根据测序结果 ,设计引物 ,克隆出发菌株和突变株全长proBA基因。将出发菌株和突变株的proBA基因分别转化大肠杆菌JM83(proBA- ) ,均能够与其功能互补。SDS PAGE分析其表达产物 ,有两条分子量分别约为 4 0kD和 4 5kD的新蛋白带出现。测定 4种转化子 (分别含有出发菌株和突变株proB基因的大肠杆菌 1 1 2 5 2转化子及proBA基因的大肠杆菌JM83转化子 )的耐盐能力。发现含有突变株proB或proBA基因转化子的耐盐能力 ,均比相应的含有出发菌株proB或proBA基因的转化子高。另外含有出发菌株和突变株的proBA基因转化子的耐盐能力 ,也均比相应的仅含proB基因的转化子高 ,表明枯草芽孢杆菌的ProA比大肠杆菌的ProA更为有效。测定所有JM83转化子胞内自由脯氨酸 ,发现其含量随盐浓度的上升而提高 ,其中含突变菌株proBA基因的转化子提高更为显著