苏云金杆菌以色列亚种几丁质酶基因的克隆及序列分析

从苏云金芽孢杆菌以色列亚种（Bacillus thuringiensis subsp israelensis)中提取基因组DNA,通过合成1对特异性引物,用touchdown PCR的方法扩增几丁质酶ichi基因序列（GenBank登录号：AF526379）。ichi序列全长为2570bp,含有1个2067bp的开放阅读框（ORF）,编码688个氨基酸,推测分子量为75.79kDa,等电点pI=5.90的几丁质酶前体。序列和结构比较分析表明：Ichi氨基酸序列与蜡状芽孢杆菌（Bacillus cereus)28-9几丁质酶CW、蜡状芽孢杆菌CH几丁质酶B及苏云金芽孢杆菌墨西哥亚种几丁质酶的同源性分别为97.24%、97.18%、97.63%,而与苏云金芽孢杆菌巴基斯坦亚种的同源性只有63.07%。Ichi编码区由分泌信号肽（46AA）、催化区（105AA)、粘蛋白Ⅲ型同源区（74AA）及几丁质结合区（40AA）组成。     

产几丁质酶的苏云金杆菌菌株筛选及酶合成条件研究

从本室保存的 64株苏云金芽孢杆菌中 ,筛选出一株几丁质酶活力较高的菌株WB 50。产酶条件研究表明 :在pH 7.0的基础培养基中添加 2 .0 %的细粉几丁质 ,1.0 %的酵母膏 ,2 2 0rpm 30℃下培养 72小时 ,几丁质酶的产出最大。     

苏云金杆菌几丁质酶新基因的筛选和全长基因的扩增

以煮沸冻融法制备PCR扩增模板,利用苏云金芽孢杆菌（Bacillus thuringiensis,Bt）几丁质酶基因特异引物进行15个Bt血清变种的扩增分析,获得9个几丁质酶全长基因扩增产物。经克隆和序列测定,从Bt serovar.entomocidus HD109、Bt serovar canadensis HD224、Bt serovar、alesti HD16和Bt serovar.toumanoffi HD201等4个菌株中分离了几丁质酶新基因。     

产几丁质酶侧孢短芽孢杆菌的筛选及其酶学性质研究

筛选并鉴定高产几丁质酶的芽孢杆菌菌株,旨在研究其酶学特性为高效利用几丁质酶资源奠定基础。分离纯化产几丁质酶芽孢杆菌,将目的菌株的几丁质酶基因异源表达并纯化后研究其酶学参数。考虑到菌株的生物安全性,选择侧孢短芽孢杆菌CDY64为进一步研究对象,将其几丁质酶基因表达于大肠杆菌BL21中。酶学研究表明,纯化后的几丁质酶最适反应温度为60℃,在p H6.0-8.0范围内均表现出良好的活性;使用胶体几丁质作为底物时Km与kcat值分别为5.85μmol/L和29.27 S-1。结果表明,侧孢短芽孢杆菌CDY64所产几丁质酶在高温下具有良好的催化能力,在体外对多种植物病原真菌表现出了良好的拮抗作用。     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

利用基因组挖掘技术分析短短芽孢杆菌菌株GZDF3全基因组几丁质酶基因家族

本研究目的是利用全基因组挖掘技术分析短短芽胞杆菌GZDF3菌株中的几丁质酶基因家族。首先利用Bio Edit软件本地搜索短短芽孢杆菌GZDF3全基因组数据库中几丁质酶基因家族,然后采用在线分析及相应软件分析预测几丁质酶基因氨基酸组成、理化性质、信号肽、疏水性、二级结构等,并构建系统发育树。结果显示,短短芽孢杆菌GZDF3全基因组中预测共存在6个几丁质酶基因,编码6种不同结构类型的几丁质酶。6种几丁质酶均属于糖苷键水解酶第18家族。其中2种几丁质酶N端有信号肽序列和信号肽酶切位点;有2种几丁质酶为碱性蛋白,4种为酸性蛋白,6种均为亲水性蛋白。本研究揭示了短短芽孢杆菌菌株GZDF3基因组中有结构多样的几丁质酶,分析结果将为后续的基因克隆及功能研究提供参考。     

地衣芽孢杆菌MY75菌株几丁质酶基因的异源表达及特性

【目的】地衣芽孢杆菌MY75菌株的几丁质酶基因的异源表达,并对表达蛋白的特性进行研究。【方法】制备MY75菌株培养上清粗蛋白,利用酶谱分析确定具有几丁质酶活的蛋白分子量。将该蛋白进行飞行时间质谱分析,确定其部分氨基酸序列,设计PCR引物对MY75菌株的几丁质酶基因进行克隆及异源表达。对表达蛋白的最适反应温度及pH,温度耐受性及金属离子对酶活力的影响等特性进行了研究,并测定了表达蛋白对真菌孢子萌发的抑制活性和对甜菜夜蛾幼虫的杀虫增效作用。【结果】酶谱分析证明MY75菌株培养上清液中仅含有一种55kDa的几丁质酶。将该编码基因chiMY克隆及序列分析后发现,基因长度为1797bp,编码599个氨基酸。在大肠杆菌中异源表达的几丁质酶ChiMY蛋白的分子量为67kDa。质谱分析证明,55kDa蛋白与67kDa蛋白序列相同。ChiMY最适pH和最适温度分别为7.0和50°C,为中性几丁质酶。Li+,Na+,和Mg2+离子对表达蛋白的酶活力具有促进作用,Mn2+,Cr3+,Zn2+和Ag+离子则能显著抑制酶活力,Cu2+和Fe3+离子完全抑制酶活性。生物测定的结果显示,异源表达的MY75几丁质酶能够抑制小麦赤霉及黑曲霉的孢子萌发,并且对苏云金芽孢杆菌的杀虫活力具有增效作用。【结论】地衣芽孢杆菌MY75菌株中仅有一种55kDa几丁质酶,其编码基因能够在大肠杆菌中大量表达,表达蛋白分子量与野生型蛋白之间有显著差异,由此证明MY75菌株中存在着几丁质酶的剪切加工过程。明确了地衣芽孢杆菌几丁质酶ChiMY具有抑制真菌活性及杀虫增效作用。上述全部研究结论在国内首次报道。     

苏云金杆菌entomocidus亚种几丁质酶基因的克隆与生物信息学分析

从苏云金杆菌(Bacillus thuringiensis,B t)entom oc idus亚种HD109菌株中克隆了几丁质酶基因chiA74-HD109,其序列全长为2 031 bp,编码676个氨基酸,GenBank登录号为AY455290。其编码产物与蜡状芽孢杆菌几丁质酶CHiB(AB041932)的相似性达97.9%,与B t kenyae亚种LB IT-82菌株几丁质酶chiA74(AF424979)的相似性达98.4%,与B t pak istan i亚种几丁质酶(U89796)的相似性为83.0%,与B t kurstak i亚种几丁质酶kchi(AY189740)的相似性达97.8%,与B t israe lensis亚种几丁质酶(AF526379)的相似性达96.6%,与B t几丁质酶(AY074882)的相似性达98.4%,与B t sotto亚种几丁质酶(AY129671)的相似性为97.3%。功能结构域分析显示其编码区包含信号肽、催化区、Ⅲ型粘蛋白同源区及几丁质结合区4个部分。     

海底沉积物中几丁质酶的筛选、分离及活性分析

目的:从海底沉积物中筛选得到一株几丁质酶活性较高的菌株,分离纯化菌株分泌的几丁质酶,并对其活性进行酶学分析。方法:以中国南海北部湾的沉积物为样本,结合透明圈法及DNS法筛选菌株。采用几丁质结合能力分析及多种层析方法分离纯化菌株分泌的几丁质酶,对其中一种几丁质酶进行酶学性质分析。结果:共筛选获得21株几丁质酶产生菌,其中分泌的几丁质酶活性最高的为蜡样芽孢杆菌B04(Bacillus cereus strain B04)。发现该菌共分泌6种含有几丁质结合域的蛋白。从蜡样芽孢杆菌B04发酵液中,分离纯化得到分子量为36 k Da的几丁质酶。研究发现,该酶是蜡样芽孢杆菌B04的一种主要的几丁质酶,其最适p H为4.0,最适反应温度为60℃,在p H 3.0~10.0范围内活性稳定,Co2+对其活力有明显促进作用,Ag+有显著的抑制作用。结论:为几丁质酶的工业化应用提供了基础。     

番茄灰霉病拮抗菌株初步鉴定及通过导入几丁质酶基因提高其生防效果

实验室保存菌Fh对番茄灰霉病有明显的抑制作用,通过形态特征、生理生化特征进行初步鉴定归属于芽孢杆菌属（Bacillus circulans）。从质粒pUC1965中得到含有几丁质酶基因的6.5kb DNA片段,将该基因片段与大肠杆菌 枯草芽孢杆菌穿梭质粒pBE2连接,获得重组质粒pBE2-chib。将重组质粒转入芽孢杆菌Fh中获得工程菌株Fh-chib。几丁质酶基因的PCR检测和几丁质平板实验表明几丁质酶基因被成功转入,工程菌株Fh-chib的原始粗酶液几丁质酶活为4.06U/ml。与野生菌相比,Fh-chib工程菌株对番茄灰霉病（Botrytis cinerea）抑菌效果提高34.46%。     

Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity

AIMS: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. METHODS AND RESULTS: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. CONCLUSION: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki

AIMS: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. METHODS AND RESULTS: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. CONCLUSIONS: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase 'structure-function' relation.     

Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti

Endogenous chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. The chitinase gene was cloned from B. thuringiensis serovar alesti strain HD-16, and the deduced 676 amino acid sequence showed a high degree of similarity with other Bacillus chitinases. Additionally, the deduced amino acid sequence showed that the protein contained an amino terminus signal peptide and consisted of a catalytic domain, a fibronectin type III domain and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase or cellulase sequences.     

Bt几丁质酶的基础表达及诱导合成的多态现象

多数微生物可以产生几丁质酶。一般认为几丁质酶基因表达受几丁质的诱导和葡萄糖抑制。但是苏云金芽胞杆菌Bacillus thuringiensis(简称Bt)几丁质酶的诱导表达方式是否与其他微生物相同,至今尚无定论。采用DNS法检测77株Bt在有或无诱导物培养基中的几丁质酶活力。研究了葡萄糖对4株不同表达类型菌株酶活力的影响,以及葡萄糖抑制与几丁质诱导之间的关系。研究发现在无几丁质诱导条件下,全部试验菌株都可以产生几丁质酶,保持一定量的基础表达,说明Bt能组成型合成几丁质酶,不需要诱导。添加诱导物之后,31株菌的酶活力没有任何变化,44株菌有不同程度的提高,但其中绝大部分诱导特性并不典型,酶活力提高不显著。许多Bt菌株几丁质酶表达兼具组成型和诱导型的特点。葡萄糖能够抑制几丁质的诱导作用,但是不能完全抑制Bt菌株几丁质酶的基础表达。比较组成型和诱导型菌株的几丁质酶基因chiA、chiB调节区域核苷酸序列,发现仅存在个别碱基的差异。     

Chitinase from Bacillus thuringiensis subsp. pakistani

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.