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.     

嗜碱细菌NTT36嗜碱基因的亚克隆及其在大肠杆菌和农杆菌中的表达

将大肠杆菌ＨＢ１０１嗜碱转化子中质粒ｐＧＣＡ所携带的嗜碱基因亚克隆至双元载体ｐＢＩ１２１质粒中,构建了植物表达载体ｐＬＧＣ重组质粒。用其转化大肠杆菌ＨＢ１０１获得了能在碱性和卡那霉素抗性平板上生长的转化子,再通过三亲交配法将亚克隆质粒ｐＬＧＣ转化进农杆菌ＬＢＡ４４０４,又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子,Ｓｏｕｔｈｅｒｎ杂交结果表明ＨＢ１０１转化子亚克隆质粒ｐＬＧＣ是由来自于嗜碱芽孢杆菌ＮＴＴ３６染色体ＤＮＡ和双元载体ｐＢＩ１２１组成,且农杆菌ＬＢＡ４４０４转化子含有来自大肠杆菌亚克隆转化子的ｐＬＧＣ质粒。     

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.     

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.     

来源于Bacillus subtilis的中性植酸酶基因的克隆及在大肠杆菌中的表达

通过ＰＣＲ的方法从Bacillus subtilis基因组中克隆了中性植酸酶基因nphy,ＤＮＡ全序列分析表明其结构基因全长１１５２个核苷酸（编码３８３个氨基酸）,５′端有一编码２６个氨基酸的信号肽序列。去除信号肽编码序列的nphy克隆到大肠杆菌ＩＰＴＧ诱导表达载体pTYB40上,在大肠杆菌中得到了高效表达,表达量达到大肠杆菌可溶性蛋白的４０％以上,表达产物具有生物学活性,证实了克隆到的中性植酸酶的基因有正常的生物学功能。     

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 (TGTAAGCGGTTAACC). 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.     

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.     

枯草芽孢杆菌寡聚—1，6—葡萄糖苷酶基因的克隆及其在大肠杆菌中的表达

本研究用鸟枪法构建了枯草芽孢杆菌（Bacillus subtilis)HB002的基因组文库,经平板法筛选得到了六株能水解合成底物对－硝基苯－α-D－葡萄糖吡喃糖苷的阳性克隆,经鉴定均含克隆了寡聚－1,6－葡萄糖苷酶基因的重组质粒（命名为pHBM001-pHBM006)。选择pHBM003,对其插入片段测序分析,此片段内有一编码561个氨基酸的开放阅读框,该 蛋白质的计算分子量为65.985kD。HB002的寡聚－1,6－葡萄糖苷酶的氨基酸序列与Bacillus sp.和凝结芽孢杆菌（Bacillus coagulans)的寡聚－1,6－葡萄糖苷酶的氨基酸序列一致性分别为81％、67％,相似性分别为89％、79％。从pHBM003中扩增出寡聚－1,6－葡萄糖苷酶基因,克隆到pBV220上,转化大肠杆菌（Escherichia coli)DH5α,得到三个能水解对－硝基苯－α－D－葡萄糖吡喃糖苷的阳性克隆HBM003－1～HBM003－3,将此三个菌株热诱导表达,SDS－PAGE电泳可检测到特异表达的蛋白质,其中HBM003－1、HBM003－2表达的蛋白约66kD,为完整的寡聚－1,6－葡萄糖苷酶,而HBM003－3表达的蛋白质偏小;表达的蛋白质均有寡聚－1,6－葡萄糖苷酶活性。     

枯草芽孢杆菌(Bacillus subtilis)碱性脂肪酶基因在毕赤酵母中的表达

将来自枯草芽孢杆菌的碱性脂肪酶基因经密码子优化,全基因合成后克隆到pPICZαA载体,构建了pPICZαA-bsl分泌型重组质粒,该重组质粒经限制性内切酶PmeI线性化后使用LiCl法转化到毕赤酵母X-33,经过筛选获得分泌表达碱性脂肪酶的重组毕赤酵母X-33/pPICZαA-bsl。摇瓶发酵液上清酶活最高可达4.78 U/mL,初步研究了该脂肪酶的酶学性质,其最适作用温度为40-60℃,最适pH9.0,且具有高度耐碱的特性。该重组脂肪酶对旧新闻纸具备较明显的脱墨能力。     

枯草芽孢杆菌碱性蛋白酶基因的克隆和表达

目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。     

Cloning of the Thermostable Cellulase Gene from Newly Isolated Bacillus subtilis and its Expression in Escherichia coli

A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.     

野油菜黄单胞菌S-152内切葡聚糖酶基因在大肠杆菌中的克隆与表达

以质粒pUC9为载体,大肠杆菌JM83为宿主菌,用鸟枪法克隆到纤维素降解细菌野油菜黄单胞菌S-152的内切葡聚糖酶(CMCase)基因,克隆到的CMCas e基因位于2.7kb的HindⅢ片段上,该重组质粒命名为pUC9H-1。Southern印迹杂交分析结果显示所克隆到的内切葡聚糖酶基因与野油菜黄单胞菌染色体DNA有亲和性。对克隆株的酶学性质分析表明,其CMCase活力为0.310μmol Glu/mg蛋白·分钟,最适作用pH为6.4,最适作用温度为55℃。 Abstract:Using plasmid pUC9 as vector,Escherichia coli JM83 as host strain,an endoglucanase(CMCase)gene has been cloned by Shoot-gun method from cellulose-degrading bacteria Xanthomonas campestris S-152,The recombinant plasmid pCU9H-1 was isolated from the positive transformant producing CMCase.The CMCase gene located in a 2.7kb HindIII DNA fragment.The result of Southern hybridization indicated that the CMCase gene cloned has affinity to chromosome DNA of Xanthomonas campestris S-152.The analysis of enzymatic activity of CMCase positive clone JM83(pUC9H-1)was carried out and the result indicated that the CMCase activity was about 0.310μmol Glu/mg pro·min,the optimum pH was 6.4 and the optimum temperature was 55℃.     

Characterization of an Endoglucanase from Pseudomonas fluorescens subsp. cellulosa Produced in Escherichia coli and Regulation of the Expression of Its Cloned Gene

Several enzymatic properties of an endoglucanase produced in Escherichia coli by a gene from Pseudomonas fluorescens subsp. cellulosa were investigated. Gel filtration revealed a single peak of M_r 36,000 with endoglucanase activity. The pH optimum of the enzyme was 7.0. Carboxymethyl cellulose and barley β-glucan (mixed β-1,3 and 1,4 linkages) were good substrates, but not laminarin (β-1,3 linkages), amylose, filter paper, microcrystalline cellulose (Avicel), or cellotriose. The mode of action was typical of an “endo”-acting enzyme. Taken together, these properties do not correspond to those of any of the endoglucanases described in P. fluorescens subsp. cellulosa. Consequently, the gene was designated egIX. The enzyme was sensitive to end-product inhibition by cellobiose but was only moderately inhibited by glucose. The enzyme was formed constitutively in E. coli throughout the growth phase. Urea had no effect on endoglucanase synthesis, but glucose acted as a catabolite repressor. The formation of the enzyme in E. coli was partially dependent on cyclic AMP.     

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.     

枯草杆菌中性蛋白酶基因在大肠杆菌中的表达

蛋白酶是枯草杆菌(Bacillus subtilis)产生的具有重要工业价值的水解酶。对蛋白酶基因的分离与高效率表达一直是基因工程研究领域的重要内容之一^[1-4]。蛋白酶基因的筛选可采用不同的方法,如“免疫法”、“DNA 杂交法”、“遗传互补法”等。大肠杆菌(Escherichia coli)是基因工程中最常用的宿主菌, 若能以E．Coli作为筛选蛋白酶基因的宿主苗,那么使用E．Coli的常规载体,便可直接获得完整的蛋白 酶基因。枯草杆菌的蛋白酶基因能否在大肠杆菌中表达．则是实现这一目标的关键。Koide等人^[5]报道过枯草杆菌的胞内丝氨酸蛋白酶基因在大肠杆菌中的表达。转化细胞在含有脱脂牛奶的平板上可产生十分微弱的水解圈。Ikeraara等人^[6]将Subtilisin E(枯草杆菌蛋白酶E)插人大肠杆菌的表达载体,具有活性的Subtilisin E便可分泌到大肠杆菌的细胞周质中。吴汝平撰文指出^[7]。克隆的枯草杆菌蛋白酶基因不能在大肠杆菌中表达。是因为大肠杆菌不能转录枯草杆菌的促使生长调节基因。Wang等人^[8]则认为,在大肠杆菌中观察不到野生型的中性蛋白酶基因E(nprE)的表达。是因为nprE的表达产物对大肠杆菌有致死作用．除去该基因上的核糖体结合位点,nprE便能在大肠杆菌中低水平表达,并能将表达产 物分泌至胞外。由上可知．枯草杆菌的蛋白酶基因能否在大肠杆菌中表达以及表达的位置仍然是一个众说纷纭的问题,这一问题也正是能否用大肠杆菌作为宿主菌筛选蛋白酶基因的关键。