枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理,获得了耐NaCl浓度达14％的突变株,同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株,其胞内自由脯氨酸的含量随着盐浓度的提高显著增加,说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因,得到一个约2.3kb的DNA片段,序列分析表明该片段含有一完整的proB基因和部分proA基因,与野生菌株的proB基因相比,突变株proB基因中有3个碱基发生了改变,其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr),另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株,能够与其功能互补。同时对部分proA基因序列分析发现,其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列,其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。     

亚硝酸钠体外随机突变枯草杆菌纤溶酶基因及其产物性质研究

通过对目的基因随机突变,希望获得纤溶酶活性提高的突变体。方法:采用一种简单方便的突变方法--亚硝酸钠直接突变含目的基因的质粒,然后转化受体菌获得突变体。在亚硝酸钠浓度为40mmol／L,温度为37℃,反应时间为1h条件下,突变带枯草杆菌纤溶酶基因的质粒pUBH,转化受体菌DB403,得到大量突变体。随机挑取约1600个转化子,用纤维平板法筛选。结果:获得酶活不同程度改变的突变体,其中有纤溶酶活性增加高达一倍的突变体;分离纯化了活性最高的突变酶,并证明其活性提高是与比活性提高相关的;序列分析该突变体发现碱基发生8处改变,而氨基酸只发生1处改变即V298A;和序列分析一致,SDS／PAGE和Westernblot检测结果显示其分子量和抗原性质没有改变。同时研究了诱变剂浓度、反应温度和作用时间对随机突变的影响。     

Properties of regeneration mutants of Bacillus subtilis

Abstract Higher regeneration mutants were isolated from Bacillus subtilis . Protoplasts from two out of four mutants regenerated at a 100% frequency on a semi-synthetic hypertonic medium. They conferred less autolytic productivity, and a revertant regained the parental levels of regeneration frequency and autolytic activities. This mutation ( rgn -1) expressed the other pleiotropic properties, i.e., nonmotility, phage PBS1 resistance and different cell morphology.     

β-葡聚糖酶高产菌株BS9418F的选育及其发酵条件的研究

经60 Coγ射线辐照处理获得的诱变菌株芽孢杆菌BS9418F ,其产酶活力比出发菌株提高 30 %以上。该菌株以大麦粉 7%、玉米粉 3%、豆粕 3%及适量无机盐为培养基最佳配比 ,其最适培养条件为 :培养基初始 pH 7.0 ,摇瓶装量 5 0mL/ 30 0mL三角瓶 ,种龄 16～ 2 0h ,接种量 2 %～ 3% ,培养温度 36～ 37℃ ,发酵周期 40h。在优化条件下 ,摇瓶发酵产 β 葡聚糖酶活力高达 5 5 0 0u/mL以上 ,比出发菌株初始发酵水平提高了 4倍以上     

产α-淀粉酶菌株的分离、鉴定及酶学性质研究

目的:筛选高产α-淀粉酶菌株,为工业生产α-淀粉酶提供储备菌株。方法:利用碘液显色法和摇瓶发酵法,从土壤中筛选产α-淀粉酶菌株;通过菌落形态、菌体特征观察和16S rDNA序列比对对菌种进行鉴定;发酵粗酶液经硫酸铵沉淀、透析脱盐后,对其酶学性质进行初步研究。结果:从土壤中筛选到一株高产α-淀粉酶菌株,枯草芽孢杆菌Bacillus subtilis XL-15。该菌株所产α-淀粉酶的最适反应温度为50℃,最适作用pH为6.5;Ca2 和Mn2 对酶有激活作用,而Cu2 、Zn2 和EDTA对酶有抑制作用;酶的动力学研究测出米氏常数Km值为1.726mg/mL。结论:该菌株是产α-淀粉酶的较好材料,且具有一定的应用前景。     

Stabilization of Bacillus subtilis Lipase A by increasing the residual packing

Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368～0.7433?kcal mol^?1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes.     

Stabilization of Bacillus subtilis Lipase A by increasing the residual packing

Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368∼0.7433 kcal mol^-1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes.     

枯草芽孢杆菌活菌体外拮抗6种肠道致病菌的研究

对枯草芽孢杆菌BS 3株在体外对 6种常见肠道致病菌 (肠产毒性大肠杆菌、肠侵袭性大肠杆菌、肠致病性大肠杆菌、鼠伤寒沙门菌、福氏志贺菌和宋内志贺菌 )的拮抗作用进行了研究。结果表明 ,枯草芽孢杆菌BS 3株对宋内志贺菌、肠致病性大肠杆菌及肠产毒性大肠杆菌拮抗作用较为明显。     

可控高突变率枯草芽孢杆菌工程菌株的构建及应用

[背景]适应性实验室进化是运用于菌株改良的定向进化方法之一.适应性进化已成功应用于扩大底物利用范围和提高底物(产物)耐受性.由于微生物存在错配修复机制,使得菌株自然突变率水平极低,实验室适应性进化周期长,容易导致传代过程染菌而失败.[目的]获得可控高突变率的枯草芽孢杆菌工程菌株.[方法]采用无痕等位基因置换系统,将枯草...     

添加物对枯草芽胞杆菌γ-聚谷氨酸合成代谢的影响

在枯草芽孢杆菌HCUL-B115代谢网络和发酵特性研究的基础上,通过添加适量的氨基酸、有机酸和维生素对聚γ谷氨酸(γPGA)发酵进行合成代谢进行研究。结果发现,大部分添加物对聚γ谷氨酸的积累都有一定的影响,特别是L谷氨酸、L苯丙氨酸、L精氨酸、L天冬氨酸、L缬氨酸、延胡索酸、草酸、丙二酸、烟酸、维生素B6和抗坏血酸等添加物对菌株HCUL-B115合成聚γ谷氨酸有明显促进作用,添加后产率比不添加任何物质提高20%左右。从代谢层面上分析,这些添加物除了促进菌体自身生长之外,同时防止了菌体对各添加物的过量合成,强化了菌株HCUL-B115合成聚γ谷氨酸的代谢途径。     

PspA外源表达对枯草芽孢杆菌168蛋白质分泌的影响

PspA同源物广泛存在于细菌和高等生物的组织中.在本研究中克隆了来源于地衣芽孢杆菌的PspA基因,并将其克隆于用于大肠-芽孢穿梭诱导表达载体pDG-StuI中构建重组质粒pDG-PspA.将构建的诱导表达型的重组质粒转化到Bacillus subtilis 168中,研究PspA的外源表达对该菌的生长,总蛋白分泌,以及Sec分泌途径中α-淀粉酶分泌的影响,结果表明,PspA基因的外源表达,在发酵过程后期能在一定程度上提高总蛋白的分泌量,在发酵过程后期能在一定程度上提高分泌的α-淀粉酶浓度.     

AMELIORATION OF THE DELETERIOUS PLEIOTROPIC EFFECTS OF AN ADAPTIVE MUTATION IN BACILLUS SUBTILIS

The deleterious pleiotropic effects of an adaptive mutation may be ameliorated by one of two modes of evolution: (1) by replacement, in which an adaptive mutation with harmful pleiotropic effects is replaced by one that confers an equal benefit but at less cost; or (2) by compensatory evolution, in which natural selection favors modifiers at other loci that compensate for the deleterious effects of the mutant allele. In this study, we have measured the potential of these two modes of evolution to ameliorate the deleterious pleiotropic effects of resistance to the antibiotic rifampicin in the soil bacterium Bacillus subtilis. One approach was to measure the fitness cost of a series of spontaneous rifampicin-resistance mutations from each of several strains. The potential for amelioration by the replacement mode was estimated by the variation in fitness cost among the mutants of a single strain. Another approach was to introduce a series of different rifampicin-resistance alleles into a diversity of strains, and to measure the fitness cost of rifampicin resistance for each allele-by-strain combination. The potential for amelioration by the replacement mode was estimated by the variation in fitness costs among rifampicin-resistance alleles; the potential for compensatory evolution was estimated by variation in the fitness cost of rifampicin resistance among strains. This study has shown that the cost of rifampicin resistance may be ameliorated by both the compensatory and replacement modes.     

Effect of host bacteria genotype on spontaneous reversions of Bacillus subtilis bacteriophage phi29 sus17 nonsense codon

Gene 17 of Bacillus subtilis bacteriophage Phi29 is an early gene playing a role in DNA replication. Its mutant sus17(112) carries the TAA nonsense triplet at the fifth codon of the gene. We isolated and sequenced 73 spontaneous revertants producing normal-size plaques on bacteria without an informational suppressor gene. In all revertants, the TAA triplet was changed by a one-base substitution and the sequences CAA, AAA, TTA, TAC and TAT were recovered at its place. The spectrum of these mutations was markedly influenced by the genotype of the bacteria in which the revertants arose. In agreement with the results described in Escherichia coli, the ratio of transversions to transitions (CAA being the only transition acceptable) was higher in strains harboring the functional allele recA(+) than in those with recA4. Our results support the idea that also in the Gram-positive B. subtilis, the spectra of spontaneous mutations are specifically modified by an SOS function. It is assumed that the single-stranded DNA chains generated in the course of phage DNA replication might act as an inducing factor.     

芽孢杆菌原生质体的形成,再生及种间融合的研究

原生质体融合技术不仅能使遗传基因高频率重组,而且可以集双亲优良遗传性状为一体.自Schaeffer等人成功地进行了微生物原生质体融合以来,这项技术便广为人们所接受.枯草芽孢杆菌分泌的抗菌蛋白能抑制多种植物病原菌的生长,苏云金芽孢杆菌生成的伴孢晶体蛋白可毒杀植物害虫.利用原生质体融合技术将这两种芽孢杆菌抑菌杀虫的遗传特性融为一体,选育出防治植物病虫害的新一代生防菌株成为可能,有关这方面的研究国内外尚未见报道.本文报道了抗菌蛋白产生菌TG26-10和晶体蛋白产生菌AS1.904-17原生质体的形成,再生及种间融合的影响因素,为进一步筛选目的融合子提供基础.