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.     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Molecular Cloning of the Transglutaminase Gene from Bacillus subtilis and Its Expression in Escherichia coli

We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis. The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300. The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp. or from mammals. The -10 and -35 regions of a putative promoter resembled the consensus sequence for the σ^K-dependent promoter. In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region. These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation. Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage. Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.     

Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGC-GGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

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.     

大蒜花叶病毒外壳蛋白基因cDNA的克隆和序列分析

我们从自然发病的大蒜中分离得到了大蒜花叶病毒。以其基因组ＲＮＡ为模板合成了３＇末端部分ｃＤＮＡ。从中选出一批插入片段在２．０ｋｂ以上的重组克隆,经Ｎｏｒｔｈｅｒｎ点杂交分析证实了所选克隆与基因组ＲＮＡ同源。通过对若干个克隆的插入片段两端部分序列的测定,选出一个克隆ｐＧＭ４９５,其插入片段的长度约为２．４ｋｂ,３′末端存有一个Ｐｏｌｙ（Ａ）结构,它应包含了编码该病毒外壳蛋白全部序列。序列测定的结果表明,这个ｃＤＮＡ片段全长为２３７９ｂｐ,其中含有与酶切图谱分析结果相符的ＥｅｏＲＩ、ＰｓｔＩ及ＢａｍＨＩ酶切位点。第一个终止密码子ＴＡＡ与３′ｇ末端相距２６４ｂｐ,我们根据碱基序列推定的氨基酸序列与其它已发表的Ｐｏｔｙｖｉｒｕｓ的外壳蛋白氨基酸序列以及外壳蛋白翻译后加工的蛋白酶专一切点相比较后推测,编码该病毒外壳蛋白序列可能起始于３′末端上游的１１７０ｂｐ处,共编码３０２个氨基酸,其分子量为３６ｋＤ,略大于ＳＤＳ－ＰＡＧＥ所测定的３３ｋＤ,非编码区域长２６４ｂｐ,富含ＡＴ,并有多个终止密码子的存在。趾３′末端３２～２７ｂｐ处有一个ＡＡＴＡＡ序列。     

纳豆激酶基因的克隆及其在枯草杆菌中的表达

为研究纳豆激酶（nattokinase,NK)的生物化学性质,以纳豆芽胞杆菌基因组DNA为模板,PCR扩增包含启动子及3‘端非翻译区的纳豆激酶全长基因序列。经鉴定后构建大肠杆菌－枯草杆菌穿梭表达质粒pBLNK,转化蛋白缺陷型枯草杆菌,筛选表达菌株。表达株培养15h后收集培养上清液,SDS－PAGE分析证实表达产物后,用超滤浓缩、分子筛、离子交换等步骤分离纯化重组NK,每升发酵液得纯度达95％的重组NK约100mg,比活性达12000u/mg。     

Molecular Cloning and Nucleotide Sequence of the Porphobilinogen Deaminase Gene,hemC, from Chlorobium vibrioforme

We previously reported the DNA sequence and expression of the Chlorobium vibrioforme glutamyl-tRNA reductase (hemA) gene (Majumdar et al., Arch Microbiol 156:281, 1991). The sequence downstream of the hemA gene indicated homology to Escherichia coli and Bacillus subtilis porphobilinogen deaminase (hemC) gene. The Chlorobium gene was confirmed to be the porphobilinogen deaminase gene, and complete sequence of the structural gene was obtained. A 2.8-kb DNA fragment containing the 1.3-kb hemA gene of Chlorobium was cloned into a hemC auxotroph (Sz16) of Bacillus subtilis, and complementation of the auxotroph to prototrophy was achieved. DNA sequence data showed a single open reading frame of 840 bp coding a protein of 279 amino acid residues. The deduced amino acid sequence of the Chlorobium porphobilinogen deaminase revealed 39% to 46% homology with the corresponding prokaryotic and eukaryotic sequences. Received: 20 September 1996 / Accepted: 26 October 1996     

Molecular Cloning,Nucleotide Sequence,and Expression of the Structural Gene for Alkaline Serine Protease from Alkaliphilic Bacillus sp. 221

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Isolation and Nucleotide Sequence of Rat Cu/Zn Superoxide Dismutase cDNA Clones

Superoxide dismutase (SOD: EC 1.15.1.1) catalyzes the dismutation of oxygen radicals and is thought to protect cells against free radical damage. We have isolated and sequenced cDNA clones of the rat Cu/Zn SOD, and have used these clones to map the rat genomic sequences coding for this enzyme. Rat Cu/Zn SOD is coded for by a single copy gene which is transcribed into an mRNA species of approximately 800 bases. Comparison of the nucleotide sequences of rat and human SOD cDNAs shows that they are homologous over 83% of the coding sequences and that in the 3′-untranslated region the extent of homology drops to 66%. The predicted rat SOD amino acid sequence is very similar to that of other eukaryotic SODs, showing 70% homology with the SODs of other mammals. Sequence conservation is particularly high in domains believed to be of functional importance.     

Molecular Cloning and Sequence Analysis of Two Distinct Halotolerant Extracellular Proteases from Bacillus subtilis FP-133

We cloned the genes encoding the two distinct extracellular halotolerant proteases of Bacillus subtilis FP-133 Expro-I and Expro-II, which were classified as alkaline serine and neutral proteases respectively. Three-dimensional modeling suggested that acidic and polar amino acid residues located on the surface stabilize protein structure in the presence of relatively high NaCl concentrations.     

西伯利亚蓼中PsMnSOD基因的克隆及其抗逆性检测

采用RACE技术从西伯利亚蓼中克隆了锰超氧化物歧化酶基因PsMnSOD的cDNA（GenBank登录号FJ848572）。长为792bp的PsMnSODcDNA序列含有编码234个氨基酸的705bp的开放读码框、57bp的5＇非翻译区和30bp的3＇非翻译区。构建了PsMnSOD基因的酵母表达载体pYES2-PsMnSOD并将其转化到野生型酵母菌株中。分析PsMnSOD基因在酵母中的表达对酵母SOD酶活性的影响及其对盐胁迫、PEG胁迫、高温和低温胁迫下抗逆性影响的结果表明,PsMnSOD基因在酵母中表达后酵母SOD酶活性提高,酵母对盐渍、PEG、高温和低温等非生物胁迫的抗逆性也增强。     

Cloning and Expression in Escherichia Coli of a Gene Encoding Superoxide Dismutase from Listeria Ivanovii

A chromosomal DNA fragment from the gram-positive bacterium Listeria ivanovii (ATCC 19119) encoding a superoxide dismutase (SOD) gene has been cloned in Escherichia coli QC779 (sodAsodB) using the plasmid vector pTZ19R. The DNA fragment inserted into the plasmid showed-high structural instability in E. coli QC779 (recA⁺). but turned out to be a stable 1.95 kbp DNA fragment when transformed into E. coli DHSa (recA-). The gene is expressed in both of these E. coli strains at high levels. Preliminary studies showed that the activity of the recombinant SOD within E. coli DHSα was up to 13-times the combined activity of both E. coli SODs. The recombinant SOD forms active hybrid SODS with both E. coli SODs in vivo.     

超氧化物歧化酶(SOD)的分子生物学

本文主要从分子生物学角度概述了超氧化物歧化酶基因的克隆,表达,分子调控,及转基因研究的情况。     

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.     

枯草杆菌启动子－信号肽序列的克隆及序列分析

利用含红霉素抗性基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢｏｍＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ的双脱氧链终止法测定了１０个克隆片段的ＤＮＡ顺序,结果表明,克隆的片段都含有启动子、核糖体结合优点及信号肽序列。克隆片段可以在大肠杆菌和枯草杆菌中恢复氨苄青霉素抗性的表型。β－内酰胺酶活力测定结果证明：大肠杆菌的酶活力主要积累在周质空间内而枯草杆菌的酶活力主要分泌到胞外。     

Nucleotide Sequence of the Gene That Encodes a Neopullulanase from an Alkalophilic Bacillus

A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.