几种细菌杀虫剂的菌种鉴定

以鉴定为目的,对国内广泛使用的几种昆虫病原性产伴孢晶体芽孢杆菌：青虫菌,杀螟杆菌及140杀虫菌,荆菌II号进行了形态、培养特性、生化反应、外毒素产生、抗原分析及酯酶分析等研究。上述各菌的生化反应、鞭毛抗原分析(140菌无鞭毛抗原)、菌体抗原分析、酯酶电泳图型等特性,均与苏云金杆菌蜡螟变种(Bacillus thuringensis var. galleriae)相同。以微生物学方法测定上述各菌均能产生β-外毒素。故青虫菌、杀螟杆菌、荆菌II号均鉴定为Bacillus thttringensis var．Galleriae血清型H5酯酶型5 galleriae;140菌鉴定为Bacillus thuringensis var．Gulleriae(无鞭毛菌株)酯酶型5 galleriae。对生化反应、抗原分析及酯酶分析的特点进行了比较和讨论,认为抗原分析具有特异性高及快速的优点。并认为菌体抗原同样具有变种特异性,也能用于该菌群的鉴定工作。     

苏芸金杆菌的酯酶分析

用聚丙烯酰胺凝胶电泳分析了Bacillus thuringtcnsis 20个变种21个菌株的酯酶图型。试验证明,酯酶的电泳图型与H抗原密切相关,除了个别血清型外,苏芸金杆菌的每一个血清型都表现出一个不同的酯酶图型。本试验采用的方法优于淀粉凝肢电泳方法,因而分离的酯酶区带比国外报道的要多,其图型也有变化。文中还首次报道了 B. t. var. Kurstaki、B. t. var.Ostriniae、B．T．Var．Dsrmstadiensis、B. t. var toumanoffi B. t. var thompsoni、B. t. var．Pakistani、B. t. var. israelensis和B．T．Var．Yunnanensis的酯酶图型,并将20个变种定为17个酯酶型。文中讨论了酯酶型与生化特征及血清型之间的关系。改进了的这一方法可用于苏芸金杆菌的快速鉴定中,本试验得到的各种酯酶图型可为苏芸金杆菌的酯酶分析提供参考。     

苏云金杆菌印第安变种和九州变种的酯酶分析

为了使苏云金杆菌各变种的酯酶图型趋于完善,我们采用了聚丙烯酰胺凝胶电泳的方法,对没有分析过的苏云金杆菌印第安变种(Bacillus thuringiensis var.indiana)和九州变种(B.t.var.kyushuensis)进行了分析。     

金针菇品系间酯酶同工酶标记筛选研究

采用聚丙烯酰胺凝胶垂直板状电泳,研究了不同生长发育期,不同组织对金针菇酯酶同工酶电泳表型的影响,筛选出不受生长发育期及常规培养条件等影响的酯酶标记区带。标记区带分所有品系的出现的基本带和部分品系出现的识别带。酯酶同工酶标记区带电泳表型显示出多态性。     

三种杜拉属（Drawida）蚯蚓的酯酶同工酶的初步研究

应用聚丙烯酰胺凝胶电泳的方法，对寡毛纲杜拉属三种蚯蚓进行酯酶同工酶酶谱分析。其结果表明，同属蚯蚓具有几条相似的酶谱区带，而不同种蚯蚓又具有各自独特的酶谱带，种间酶谱区带数目、泳动率及染色强度都有明显区别。雅和杜拉蚓酶谱图和天锡杜拉蚓相似，而与管状杜拉蚓相差较远，表明同属蚯蚓在进化水平上的亲缘关系。     

分离纯化质粒DNA方法的比较

质粒的研究在遗传工程上占有重要地位。其分离纯化是一项费工费时和得率甚微的工作。一般常用的方法有制备性电泳,平衡等密度超离心和速度区带超离心等,这些方法各有特点,作者对它们进行了比较。质粒DDNA从苏芸金杆菌(Bacillus thuri-ngiensis)变种 isvaelensis 4Q_2-22和变种     

三种蜘蛛酯酶同工酶的比较研究

应用聚丙烯酰胺凝胶电泳对狼蛛科的拟水狼蛛、蟹蛛科的三突花蛛和肖蛸科的鳞纹肖蛸3种蜘蛛的酯酶同工酶进行了比较研究。结果表明,不同科的蜘蛛酯酶同工酶种问差异性大并有着明显的种簇特异性,推测它们的酯酶同工酶酶谱中的区带组受不同的基因位点控制,且各自的基因位点数不等;同种蜘蛛的雌蛛和雄蛛之间也有各自的酯酶同工酶谱型,但差异小,其控制基因位点大体相同。这样,我们从分子的水平上讨论了酯酶同工酶的差异性可以用来作为识别物种的附加指标。     

一种快速简易的苏芸金杆菌变种鉴别法——菌落碘色反应法

一般认为,用血清学方法鉴定苏芸金杆菌变种的方法较好,但也要参考生理生化、酯酶型等方法。我们用菌落碘色反应法经过几年的反复试验表明,对苏芸金杆菌变种来说,该法可做为用其它方法鉴定前的初始鉴别方法。     

蜜蜂酯酶同工酶的研究

本实验用聚丙烯酰胺凝胶等电聚集电泳分析了意蜂和中蜂的酯酶同工酶。意蜂和中蜂的酶谱有明显的种间差别。意蜂的酶谱可分为四组区带:酯酶Ⅰ、Ⅱ、Ⅲ、Ⅳ。意蜂不同发育阶段的酶谱也有不同。酯酶Ⅳ的含量在蛹期有很大的变化。酯酶Ⅳ表现出多态现象,对多态现象的分析可推测种群的杂合程度。     

新颖深海微生物酯酶EstC11的酶学性质及其在手性拆分乙酸苏合香酯中的应用

【背景】手性乙酸苏合香酯是重要的手性香料产品,在食品及精细化工等领域都有重要的应用。酶催化不对称合成手性乙酸苏合香酯产品具有极好的工业应用前景。【目的】研究酯酶EstC11的基本酶学性质及其在制备手性乙酸苏合香酯中的应用。【方法】对来自西太平洋深海热液口芽孢杆菌Bacillus sp.CX01中的新颖微生物酯酶基因EstC11进行克隆、表达及酶学性质鉴定。通过对p H、温度、有机溶剂等反应条件的优化提高酯酶手性拆分乙酸苏合香酯的光学纯度。【结果】酯酶EstC11的最适反应p H为8.5,最适温度为25°C,一些金属离子和有机溶剂对酯酶EstC11的水解活性具有不同程度的抑制作用。通过对反应条件的优化,在最适反应条件下(p H 9.0 50 mmol/L Tris-HCl,20°C,50 mmol/L底物浓度)反应3 h后,(R)-乙酸苏合香酯的光学纯度达98%,得率为39%。【结论】通过对酯酶拆分条件的优化,手性拆分乙酸苏合香酯生成(R)-乙酸苏合香酯的光学纯度明显提高,为酯酶EstC11在工业化上的应用奠定了基础。     

The effect of growth temperature on esterase patterns in psychrotrophic Bacillus species and other Gram-positive genera

The growth temperature of Bacillus stearothermophilus was previously reported to affect the esterase band pattern obtained after gel electrophoresis. In this study gel electrophoresis of esterases was done on a group of Antarctic bacterial strains cultivated at different temperatures to investigate whether band shift due to a change in growth temperature was a general phenomenon or limited only to select groups. Most strains studied were in the Bacillus genus. Standard strains of known Bacillus species and other Gram-positive genera were examined for comparative purposes. A change in the esterase band pattern was observed as a result of variation in growth temperature. Two major esterase band were dominant in this diverse group of species.     

枯草芽胞杆菌乙酰木聚糖酯酶的鉴定及诱导剂对酯酶活力的影响

以枯草芽胞杆菌CICC 20034为研究对象,对其分泌的高相对分子质量酯酶进行鉴定,并考察诱导剂对其活力的影响。结果表明:枯草芽胞杆菌CICC 20034可分泌一种相对分子质量为1.07×105的酯酶,经蛋白质质谱鉴定为乙酰木聚糖酯酶,单体分相对子质量为3.56×104。在发酵培养基中添加乙酸乙酯和木糖可以显著的促进乙酰木聚糖酯酶的活力,而三丁酸甘油酯和大分子诱导剂——木聚糖、玉米芯粉和壳聚糖对酯酶的活力几乎无促进作用。枯草芽胞杆菌CICC 20034以乙酸乙酯为诱导剂时最高比酶活为0.62 U/mL,为已知报道的野生细菌乙酰木聚糖酯酶的最高酯酶活力。     

An organic-solvent-tolerant esterase from thermophilic Bacillus licheniformis S-86

A thermophile, halotolerant and organic-solvent-tolerant esterase producer Bacillus sp. S-86 strain previously isolated was found to belong to Bacillus licheniformis species through morphological, biochemical, 16S rRNA gene sequence analyses and rDNA intergenic spacers amplification (ITS-PCR). The strain can grow at 55 degrees C in presence of C2-C7 alkanols (log P=-0.86 to 2.39), and NaCl concentrations up to 15% (w/v). This bacterium showed optimal growth and esterase production at 50 degrees C. Two different molecular weight esterase activities were detected in zymographic assays. PMSF inhibited type I esterase activity, showing no inhibitory effect on type II esterase activity. B. licheniformis S-86 was able to grow in presence of hydroxylic organic-solvents like propan-2-ol, butan-1-ol and 3-methylbutan-1-ol. At a sub-lethal concentration of these solvents (392 mmoll(-1) propan-2-ol; 99 mmol l(-1) butan-1-ol, 37 mmol l(-1) 3-methylbutan-1-ol), adequate to produce 50% cell growth inhibition at 50 degrees C, an increment between 1.9 and 2.3 times was observed in type I esterase production, and between 2.2 and 3.1 times in type II esterase production.     

Analysis of the Crystal Antigens of Bacillus thuringiensis by Gel Diffusion

S ummary . The antigenic patterns of the crystal protein inclusions of Bacillus thuringiensis were determined. No specific antigenic patterns associated with previously described subgroups of this species were found. A larger number of categories of crystal antigen than of flagellar antigen or esterase type were found. In some cases isolates indistinguishable in classifications based on flagellar antigens or esterase types could be subdivided by their crystal antigenic pattern. The use of crystal antigens in classification is discussed.     

Extracellular esterase activity from Bacillus stearothermophilus

Bacillus stearothermophilus has been reported to produce an extracellular esterase with molecular weight of 42–47 kDa. Extracellular esterase activity in Bacillus stearothermophilus (NCIB 13335) was found to reside in protein with a molecular weight less than 10 kDa. This small esterase was responsible for all the esterase activity observed in this strain under the conditions studied.     

六种鼠组织中过氧化物酶和酯酶变异的比较

本文研究姬鼠属（Ａｐｏｄｅｍｕｓ）２种鼠和鼠属（Ｒａｔｕｓ）４种鼠的心组织过氧化物酶和肝组织酯酶同工酶等位基因的变异。结果表明,６种鼠心组织过氧化物酶皆为４条区带,其中社鼠和白腹巨鼠的酶带泳动速度略快于褐家鼠和黄毛鼠的酶带;鼠属４种鼠的酶带间距大致相同,而姬鼠属的酶带间距略大于鼠属,这种谱型的区别就所分析的６种鼠而言,可以认为是该同工酶表现的属间区别。肝组织酯酶区带较多,其酶带数和泳动速度的差异具有明显的种间特征;泳动最快的酶带在姬鼠中是２条,在鼠属中是１条,可以认为是属间特征的区别。从９项生化指标的异同作动物配对比较,表明褐家鼠与黄毛鼠亲缘上较近,社鼠与白腹巨鼠亲缘上也较近,而且白腹巨鼠在进化上可能要比其余３种鼠属动物更接近于姬鼠属     

恙螨酯酶同工酶的比较研究

本实验采用聚丙烯酰胺凝胶垂直板型电泳法分析了五种恙螨成虫及微红纤恙螨各虫期酯酶同工酶的变化.在酶谱上,五种恙螨成虫均有区别,酶带数和酶活性强弱均有不同,其差异的大小符合它们现有的种属关系的远近,即与它们所处的分类地位相符.因此在以幼虫为主的恙螨分类中,成虫的同工酶分析可起到辅助和补充的作用.在微红纤恙螨各虫期的酶谱中亦存在差异,酶带数的趋势是从卵至成虫由少渐多.此结果表明酯酶同工酶与个体发育过程中的体内变化有关,可成为指示恙螨个体发育过程中的生化指标之一.     

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.     

Molecular characterisation of the gene encoding an esterase from Bacillus licheniformis sharing significant similarities with lipases

An esterase gene from the moderate thermophilic strain Bacillus licheniformis LCB40 was cloned and expressed in Escherichia coli. Comparison of the amino acid sequence of the esterase with those of known lipases and esterases showed the presence of the well-conserved Gly-X-Ser-X-Gly pentapeptide, with an alanine replacing the first glycine. This substitution has never been reported for an esterase but it is present in the lipases from Bacillus subtilis, Bacillus pumilus and Galactomyces candidum. The amino acid sequence showed similarities with lipases and with mammalian lecithin-cholesterol acyltranferases and no similarities with esterases. The enzyme activity of a crude extract from a recombinant Escherichia coli strain showed hydrolysis of p-nitrophenyl caprylate (pNPC8) as for esterases, but not of p-nitrophenyl palmitate (pNPC16) or olive oil such as for lipases. Thus, the enzyme displays the original property of associating the activity of an esterase with a primary sequence showing high similarity with lipases.     

Effective extracellular production of Bacillus stearothermophilus esterase by pH-stat modal fed-batch culture of recombinant Bacillus brevis

An automated two-component substrate feeding strategy with a pH-stat modal fed-batch culture using a high pH limit was developed to effectively porduce esterase from a hyperprotein exreting Bacillus brevis HPD31 harboring a plasmid pHSC131 which carries a Bacillus stearothermo philus esterase gene. First, the effect of single- and multi-substrate feedings on the growth and activity of the excreted esterase was investigated. Then a two-component (polypepton + glucose) feeding using different feed rates was studied. Highest activity of the excreted esterase (34 U/mL) was obtained when the concentrations of poly-pepton and glucose in the nutrient feed solution were 250 and 41.60 g/L respectively. The absence and excessive amount of glucose in the nutrient feed solution was ineffective for the exracellular esterase formation because without glucose the increase in cell concentration was minimum while excessive amount of glucose favored cell growth at the expense of the esterase production. It is believed that the mechanism of enzyme excretion is growth dependent and that a higher cell growth of the host is in effect unfavorable for the enzyme production. The feed rate, automatically controlled by the direct signal of the pH change, at 0.30 mL/pulse was found optimum for the esterase production while lower (0.15 mL/pulse) and higher (0.67 mL/pulse) feed rates did not produce good results. The activity of the excreted esterase was increased more than eight times from 4 U/mL obtained in the conventional batch culture to 34 U/mL obtained in this study. The esterase productivity was likewise increased more than threefold. (c) 1992 John Wiley & Sons, Inc.