粘质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析

通过生物信息学技术对Chi A基因序列进行分析预测,了解Chi A的基因结构及蛋白质性质。从自有菌株(粘质沙雷氏菌Serratia mareescens S68)中克隆到几丁质酶基因Chi A,利用相关软件对Chi A基因序列进行分析预测。Chi A基因全长1 714 bp,开放阅读框编码563个氨基酸,推测其编码的蛋白质分子量为60 983.8Da,等电点为6.35,是一种稳定的亲水性蛋白质。预测Chi A可能存在信号肽,切割位点在第23~24位氨基酸之间,1~23位氨基酸为其跨膜结构,其余肽链位于细胞外。Chi A主要存在3种二级结构元件,在二级、三级结构中都有体现。该Chi A是一种水溶性蛋白质,结构稳定且可以分泌到胞外。     

粘质沙雷氏菌氯霉素抗性基因的克隆及其性质分析

通过构建粘质沙雷氏菌KMR-3菌株的基因组DNA文库, 克隆到了与该菌的氯霉素抗性相关基因, 并对其部分特性进行了初步研究。结果表明: 克隆到的氯霉素抗性基因所编码的蛋白属于PRK10473蛋白, 由397个氨基酸编码, 与变形斑沙雷氏菌(Serratia proteamaculans 568) Bcr/CflA亚家族药物抗性转运蛋白同源性最高, 达到92%, 并对该基因的调控序列(启动子、终止子、SD序列及转录起始位点) 进行了分析。     

粘质沙雷氏菌氯霉素抗性基因的克隆及其性质分析

通过构建粘质沙雷氏菌KMR-3菌株的基因组DNA文库, 克隆到了与该菌的氯霉素抗性相关基因, 并对其部分特性进行了初步研究。结果表明: 克隆到的氯霉素抗性基因所编码的蛋白属于PRK10473蛋白, 由397个氨基酸编码, 与变形斑沙雷氏菌(Serratia proteamaculans 568) Bcr/CflA亚家族药物抗性转运蛋白同源性最高, 达到92%, 并对该基因的调控序列(启动子、终止子、SD序列及转录起始位点) 进行了分析。     

粘质沙雷氏菌的电镜观察

用扫描电镜和透射电镜对粘质沙雷氏菌(Serratia marcescens)的临界点干燥标本和负染标本作了观察,均可见到细胞表面常有一至二个直径为0.12～0.24μm的颗粒。在超薄切片中颗粒则有两种不同的结构:一种是外膜泡(Outer membrane vesicle);另一种是致密体(Dense body)。致密体可能是一种分泌性颗粒,它既不是内部的贮存物,也不似外部进来的异物。它们看来形成于细胞质内后分泌到细胞外。在细菌中这是一种罕见现象。有关致密体的化学性质和功能尚不清楚。文中指出粘质沙雷氏菌的纲胞表面也具有茂密的菌毛(Pili)。     

粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究

目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌B121(DE3)中实现高效表达,并对重组酶进行酶学性质研究.方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定.结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL.重组脂肪酶的最适反应温度为40～45℃,最适pH为7.0～7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失.该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力.结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础.     

粘质沙雷氏菌PL-06磷脂酶A1基因大肠杆菌优化表达

以粘质沙雷氏菌PL-06基因组为模板扩增得到磷脂酶A1基因plaA和含有辅助蛋白的磷脂酶A1基因plaB.plaA和plaB基因与pET-28a(+)连接后转入大肠杆菌BL21(DE3)表达,得到基因工程菌AP28和BP28.AP28最佳的诱导表达条件为诱导初始OD600值0.5,IPTG浓度为0.2mmol/L,诱导温度为37℃,诱导时间为4h,优化后磷脂酶A1蛋白表达水平从32％上升至46％,包涵体复性的磷脂酶A1酶活从10.8U/ml上升到12U/ml,相对于BP28工程菌,目的蛋白的表达对宿主细胞毒性小,而且得到的蛋白容易纯化.因此通过优化磷脂酶A1基因plaA的诱导条件,使磷脂酶A1以大量的包涵体形式表达,从而得到较高活性的磷脂酶A1并避免其对宿主细胞的毒性是可行的,而且可以得到大量纯化的磷脂酶A1蛋白,方便下一步的研究.     

粘质沙雷氏菌产几丁质酶发酵条件的研究

目的：通过对粘质沙雷氏菌发酵条件的优化,提高其产几丁质酶的能力。方法：以实验室保存菌种粘质沙雷氏菌S418为对象,通过单因素试验和三因素三水平正交试验筛选出了菌株S418产几丁质酶的最佳培养基配方及培养条件。结果：该菌种产酶的最佳发酵条件：0.2%（w/v）胶体几丁质,1%蛋白胨,0.05%KH2PO4,在28℃、pH7.0、接种量6%,培养72h,酶活达到5.49U/mL。结论：优化后菌株S418产几丁质酶的条件。     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

粘质沙雷氏菌发酵生产D-乳酸的研究

本文对粘质沙雷氏菌发酵生产D-乳酸进行了研究。以粘质沙雷氏菌G1(Serratia marcescens G1)为出发菌种,摇瓶试验确定了发酵培养方式:前12 h为菌体生长阶段,有氧培养,温度28℃,pH值7.0;后36 h为D-乳酸合成积累阶段,无氧培养,温度44℃,pH值6.0。且发现使用葡萄糖为碳源时更有利于D-乳酸的合成积累。采用缺失2,3-丁二醇合成能力的基因工程菌株R1为出发株,经筛选后得到耐受较高浓度乳酸盐的菌株R150,以R150为发酵菌种,在3.7 L发酵罐上采用两阶段发酵法,并通过增加起始菌体浓度的方法,发酵生成的D-乳酸浓度达到83.5 g/L,光学纯度达到98.9%。本研究成果为使用粘质沙雷氏菌发酵生产D-乳酸的深入研究打下了基础。     

一株新海洋粘质沙雷氏菌产灵菌红素发酵条件优化

灵菌红素是由微生物产生的一种红色次级代谢产物,因其具有抗菌、抗癌和抗疟疾等功效,受到了生物医药领域的广泛关注。微生物发酵是当前产灵菌红素的主要方法,分离筛选高产灵菌红素的微生物、优化发酵条件是提高灵菌红素产率的重要途径。本研究从深圳湾筛选出一株含有红色色素的菌株,并基于16S rRNA基因序列对该菌株进行了系统发育分析和物种鉴定。紫外可见光全波长扫描和HPLC-MS图谱分析证明,该菌株所产红色色素为灵菌红素。进一步通过单因素试验和正交优化法优化了该菌产灵菌红素的发酵条件和发酵培养基组分。系统发育分析表明,该菌株为一株海洋粘质沙雷氏菌（Serratia marcescens）,并将其命名为S. marcescens SOCE 001。产灵菌红素的最佳发酵条件为：温度28 ℃、振荡培养转速220 r/min、培养基pH 7。发酵培养基最佳组合为：果糖添加量2 g/L、蛋白胨添加量10 g/L,MgSO₄添加量2 g/L。优化发酵条件后,经24 h培养, 灵菌红素产量可达2.468 g/L。     

粘质沙雷氏菌几丁质酶chiB基因的克隆与序列分析

采用改进的方法提取粘质沙雷氏菌基因组DNA,通过PCR扩增,得到大小为1 500 bp特异性DNA片段(chiB基因),以pUC18质粒构建了pUC-ch iB克隆载体,转化至感受态细胞E.coliDH5α培养,并筛选出重组质粒。经测序分析,证明克隆片段与文献报道相一致。     

一株高产PLC的CW-W-90-3菌的鉴定

198 9年 ,筛选了 1株高产 phospholipaseC(PLC)的CW W 90 3菌株[1,2 ] ,据其形态特征、生理生化反应 ,初步将其归于弧菌科气单胞菌属[3 ] ,由于该菌株的许多生理生化特性与粘质沙雷氏菌相同。但其极生单鞭毛和无色素及少许生理生化特性与粘质沙雷氏菌相异。后经AutomatedBacteriaIdentificationSystem BiologMicroStationSystem检测 96种C源和N源的利用及其个体群体发育 ,说明其与粘质沙雷氏菌 (Serratiamarcescens)相符 ;并在基因组水平上研究该菌株的系统发育 ,从分子水平上对该菌株进行 16SrRNA序列分析煌同源性比较。根据CW W 90 3菌株 16SrRNA与GeneBank数据库中Serratiamarcescens的 16SrRNA的序列具有 99%的同源性 ,终将CW W 90 3菌株鉴定为粘质沙雷氏菌武汉株 (SerratiamarcescensWuhanstrain)。     

木瓜凝乳酶基因的克隆及序列分析

通过设计一对特异性的引物,采用RT－PCR方法从未成熟的木瓜组织中扩增得到木瓜凝乳酶基因,并将其重组到pPIC9K载体中,转化大肠杆菌并筛选阳性克隆,序列测定并利用BLAST软件进行核酸及氨基酸序列相似性分析,结果表明：通过序列组成及特征结构分析,扩增得到的基因为木瓜凝乳酶基因。     

X-Ray Analysis of the Magnesium-containing Endonuclease from Serratia marcescens

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescenswas refined at the resolution of 1.07 Å to Rfactor of 12.4% and R _freefactor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg²⁺ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N-and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg²⁺ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O¹atom of Asn119 were shown to be within the range of 2.01–2.11 Å. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 Ų, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 Ų, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.     

Comparative studies of chitinases A,B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.     

Comparative studies of chitinases A, B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.