苏云金芽孢杆菌几丁质酶的研究进展

苏云金芽孢杆菌制剂作为无公害农药已经得到社会的认可,如果再开发其几丁质酶抑制真菌和杀虫增效功能,不仅可充分利用这一农业微生物菌种资源,也将给予传统生物农药以新的生命力。综述了苏云金芽孢杆菌几丁质酶方面研究的国内外最新进展。     

嗜热淀粉芽孢杆菌来源β-葡萄糖苷酶的重组表达与酶学性质

【背景】β-葡萄糖苷酶(EC 3.2.1.21,β-glucosidase),是纤维素分解酶系中的重要组成部分,目前工业上应用的β-葡萄糖苷酶多数来源于植物和真菌,来源于细菌的较少,且应用中还存在酶活力偏低、热稳定性差、反应条件适用范围窄、酶活力易受产物反馈抑制等问题,增加了经济成本。嗜热微生物具有特殊的遗传信息资源,极有可能从中挖掘到酶学性质优良的新型β-葡萄糖苷酶,从而解决工业难题。【目的】从嗜热淀粉芽孢杆菌(Bacillus thermoamylovorans)基因组中挖掘新型β-葡萄糖苷酶基因,通过基因重组、异源表达和蛋白纯化技术制备新型β-葡萄糖苷酶,并探究其酶学性质,为新型β-葡萄糖苷酶在纤维素水解等领域的应用奠定基础。【方法】人工合成新型β-葡萄糖苷酶基因bgl52,构建重组表达质粒pET22b-bgl52,并用电脉冲法转化到大肠杆菌BL21(DE3)中实现可溶性表达,利用Ni-NTA亲和层析纯化得到高纯度的β-葡萄糖苷酶Bgl52。【结果】实现重组表达质粒pET22b-bgl52在大肠杆菌BL21(DE3)中的可溶性表达,并获得β-葡萄糖苷酶Bgl52纯蛋白,蛋白分子量...     

长野芽孢杆菌普鲁兰酶基因在枯草芽孢杆菌中的表达及酶学性质研究

根据NCBI上报道的基因序列设计引物,以长野芽孢杆菌(Bacillus naganoensis)ATCC53909的染色体DNA为模板,PCR扩增普鲁兰酶编码基因pulB。将此基因与表达载体pWB980连接构建重组质粒pWB-pulB,并转化枯草芽孢杆菌WB600。SDS-PAGE结果显示,在100 kD处有特异性条带,经测定重组转化子粗酶液酶活力达10.94 U/mL。酶学性质分析表明,其最适反应温度为60℃,最适反应pH为5.0,且在温度30-60℃及pH4.0-6.0范围内稳定,适合淀粉加工行业的应用。     

碱性α-淀粉酶基因在巨大芽孢杆菌中的表达及酶学性质研究

将已克隆的碱性α-淀粉酶基因信号肽编码序列去除,用PCR的方法加入酶切位点,然后与表达载体pHIS1525连接转化大肠杆菌DH5α,筛选出阳性转化子DH5α-pHIS1525-JH,并提取质粒进一步转化巨大芽孢杆菌YYBm1原生质体,获得基因工程菌YYBm1 -pHIS1525-JH.SDS-PAGE分析表明该基因在巨大芽孢杆菌中得到了有效表达.酶学性质研究表明,该酶的最适温度与pH值分别为60℃与pH8.5,在pH7.0 - 10.5之间具有较好的稳定性,Km值为1.94 mg/mL,酶活力可达2516.5 U.     

紫色色杆菌苯丙氨酸羟化酶的异源表达及重组酶学性质研究

来源于紫色色杆菌(Chromobacterium violaceum)的苯丙氨酸羟化酶结构简单,性质更接近于人的苯丙氨酸羟化酶,具有潜在的医药应用价值。从紫色色杆菌基因组中克隆得到苯丙氨酸羟化酶基因pah。构建重组表达载体pET24a-pah,并在Escherichia coli BL21(DE3)中实现高效表达。离子层析纯化后,重组蛋白比酶活高达503.2 U/mg。酶学性质研究显示,该重组酶的最适温度为40℃左右,50℃时PAH的半衰期为15 min;最适pH在7.5左右,在pH6-8范围内较稳定。37℃,pH7.5条件下,Km值为1.5 mmol/L,Vmax为0.5 mmol/min,kcat为5.05/s,催化效率kcat/Km为3.37 L/mmol·s。     

内切葡聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质研究

通过PCR方法将已克隆的内切葡聚糖酶基因(GenBank No. DQ782954)信号肽编码序列去除, 然后与表达载体pHIS1525连接后转化大肠杆菌DH5a, 筛选出阳性转化子DH5 a -pHIS1525-G7并提取质粒进一步转化巨大芽孢杆菌WH320原生质体, 获得基因工程菌WH320-pHIS1525-G7。刚果红染色和SDS-PAGE分析表明该基因在巨大芽孢杆菌中得到了有效表达。基因工程菌经优化培养后, 胞外上清液中的酶活力可达889 U, 是出发菌株(即枯草芽孢杆菌C-36)的11.22倍。酶学性质研究表明: 该酶的最适反应温度与pH值分别为65℃与pH 6.0, 在pH 4.5~10.0范围内50℃保温30 min可保持在最高酶活的80%以上。     

枯草芽孢杆菌中性植酸酶的纯化和酶学性质

从土壤中分离到了产中性植酸酶的枯草芽孢杆菌菌株并对所产植酸酶进行了分离纯化。此中性植酸酶的反应最适 pH为 7 5,最适温度为 55℃ ,在 37℃下以植酸钠为底物的Km值为 0 1 9mmol/L ,植酸酶活性依赖Ca2 +的存在。酶蛋白的分子量大小约为 45kD ,纯酶蛋白N端序列为Lys His Lys Leu Ser Asp Pro Tyr His Phe Thr。     

枯草芽孢杆菌中怀植酸酶的纯化和酶学性质

从土壤中分离到了产中性植酸酶的枯草芽孢杆菌菌株并对所产植酸酶进行了分离纯化,此中性植酸酶的反应最适pH为7．5,最适温度为55度,在37度下以植酸钠为底物的Km值为0．19mmol/L,植酸酶活性依赖Ca^2 的存在,酶蛋白的分子量大小约为45kD,纯酶蛋白N端序列为Lys-His-Lys-Leu-Ser-Asp-Pro-Tyr-His-Phe-Thr。     

芽孢杆菌α-淀粉酶基因的克隆、表达和酶学性质分析

在仔猪结肠内容物中分离出一株能利用淀粉的芽孢杆菌Bacillussp.WS06,构建了全基因组DNA文库,从中筛选出α_淀粉酶基因amyF,分析测定了其核苷酸序列并进行了表达;其中amyF编码的蛋白有526个氨基酸、分子量为58.6kD;它与已报道的Bacillusmegaterium的α_淀粉酶序列有93%的同源性。经过氨基酸序列比较分析还发现,AmyF含有淀粉酶家族中4个高度保守的酶催化活性区。经多步纯化,重组酶的比活共提高了22.2倍,获得凝胶电泳均一的蛋白样品;经SDS_PAGE检测,AmyF酶分子量为57kD。该酶的最适反应温度为55℃～60℃,酶的最适反应pH为7.0,在温度不超过55℃时,酶活较稳定;AmyF能迅速降解淀粉生成麦芽寡糖,属于内切糖苷酶。     

四氢嘧啶羟化酶的克隆表达、酶学性质及其在羟基四氢嘧啶合成中的研究

四氢嘧啶羟化酶(EctD)是双加氧酶超家族的重要成员,其底物四氢嘧啶和产物羟基四氢嘧啶的应用广泛,因此EctD在生物制造领域具有重要的应用价值。从来源于新疆盐湖的需盐色盐杆菌(Chromohalobacter salexigens)中获得四氢嘧啶羟化酶基因(ectD),并在E.coli BL21(DE3)中进行表达,对异源表达的EctD酶学性质进行研究。结果表明：EctD的最适温度是30℃、最适pH是7.5,在30℃、pH 6.5～8.0条件下具有较好的稳定性;Fe²⁺、Ba²⁺、Mn²⁺、Mg²⁺、Fe³⁺和Ca²⁺离子可增强EctD的酶活,而Co²⁺、Zn²⁺和Cu²⁺离子明显抑制EctD的酶活;动力学参数为K_m=7.63 mmol/L、k_cat/K_m =1.01 1 L/(mmol·s)、V_max ...     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Characterization of the Bacillus thuringiensis strains isolated from Taiwan

Over 100 Bacillus thuringiensis (Bt) isolates which produced phase bright inclusions have been isolated from soil samples from different areas in Taiwan. Three types of crystal proteins were visualized by phase contrast microscopy. Among these isolates, only 14 different types of plasmid profiles have been observed. They all possess a variety of plasmids ranging from a few kb to around 250 kb in size. With respect to the crystal protein profiles, the plasmid profiles, and the shapes of crystal proteins, we found that the majority of our isolates (87%) were different from most of the known Bt strains. Our other two types of isolates (10 and 3%) resembled Bt var. kurstaki HD1 and Bt var. israelensis, respectively. Most of our isolates were active against Bombyx mori (Lepidoptera) and Aedes aegypti (Diptera). Most interestingly, two of our isolates, Nos. 82 and 96, were found highly toxic to Heliothis virescens, even compared with the standard strain, Bt var. kurstaki HD1. Using insecticidal crystal protein (ICP) gene probe from Bt var. aizawai HD-133 to probe the total DNA of our isolates, we observed that at least one plasmid from each of the tested strains reacted with the probe. A 10 kb plasmid from some of our isolates hybridized with the probe. This probably is the first evidence demonstrating that the ICP gene sequence can be found in a low molecular weight plasmid.     

苏云金杆菌生物农药生产和应用现状

苏云金杆菌（Bacillus thuringiensis,Bt）生物农药在全国乃至全世界都是被公认为最安全、最有效、工业化程度最高、最廉价因而也是应用范围最广、使用量最大的微生物杀虫剂,而中外专家和政府在设施害虫IPM计划和无公害农产品生产的推广杀虫剂首选就是Bt。因此,它在生物防治的作用和地位不可忽略。我国生产推广应用的Bt厂家一直对国内外Bt产品的应用状况十分关注。为此,本文就多年来国内Bt生产菌株及其防治对象,生产厂家及其生产能力、市场范围以及应用水平,存在问题以及发展前景等方面做综述。     

Characterization and pathogenic evaluation of Bacillus thuringiensis and Bacillus sphaericus isolates from Argentinean soils

Eighty soil samples of different origin (from urban, agricultural, forested and horticultural areas) which had not previously been treated with bioinsecticides, were collected and examined to investigate the presence of Bacillus thuringiensis and B. sphaericus. From a total of 1473 bacterial isolates examined by differential staining techniques and growth on nutrient agar with the addition of penicillin and streptomycin, 31 (2.1%) strains of Bacillus sphaericus and 25 (1.6%) strains of Bacillus thuringiensis were isolated. These strains were tested for their pathogenicity against Diptera (Culex quinquefasciatus) and Lepidoptera (Anticarsia gemmatalis and Spodoptera frugiperda). Seven strains of Bacillus thuringiensis subspecies kurstaki were found to be pathogenic to Spodoptera frugiperda and twenty-two strains showed a pathological effect against Anticarsia gemmatalis. None of the strains of Bacillus thuringiensis nor the Bacillus sphaericus investigated, showed pathogenic activity against Culex quinquefasciatus. The strains of Bacillus thuringiensis were characterized serologically as belonging to six serotypes (darmstadiensis, entomocidus, kurstaki, muju, sotto and xianguangiensis). One strain seemed to be a new serotype. The electrophoretic profiles of the strains of Bacillus thruringiensis showed bands of 130 kDa similar to those found in strains pathogenic against Lepidoptera. Some physicochemical characteristics were also studied in the soil samples, in order to relate them to the presence or absence of these Bacillus species.     

Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized. Received: 3 December 1999 / Accepted: 5 January 2000     

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1

Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.     

Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus

• 1.1. Spore coat extracts from Bacillus thuringiensis subspecies kurstaki and israelensis and Bacillus cereus T and B. cereus NRRL 569 were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by amino acid analysis.
• 2.2. Both B. cereus spore coats had similar electrophoretic profiles.
• 3.3. The B. thuringiensis spore coats contained crystal proteins as major components as well as lower mol. wt proteins.
• 4.4. B. thuringiensis subsp. israelensis had a unique coat protein profile which was different from B. cereus and B. thuringiensis subsp. kurstaki coats.
• 5.5. Insecticidal activity of spores against the tobacco hornworm, Manduca sexta, and the mosquito, Aedes aegypti, also was determined.
• 6.6. B. thuringiensis subsp. kurstaki spores were lethally toxic to the tobacco hornworm (Lepidoptera) larvae, whereas spores of the other subspecies were not.
• 7.7. Except for subspecies israelensis, none of the spores was effective against the mosquito (Diptera) larvae.

     

Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.     

苏云金芽胞杆菌spoIIID基因缺失突变株的特点

摘要：【目的】构建苏云金芽胞杆菌spoIIID基因缺失突变株,并研究其与出发菌株的表型及性质差异。【方法】采用基因同源重组技术敲除了苏云金芽胞杆菌HD-73菌株中的spoIIID基因,构建了spoIIID缺失突变株,测定生长曲线,并通过扫描电子显微镜观察,芽胞计数分析及SDS-PAGE 蛋白电泳比较突变株与出发菌株的差异。构建遗传互补菌株,观察菌株性状的回复情况。【结果】通过温敏载体同源重组敲除技术获得了苏云金芽胞杆菌HD-73菌株spoIIID基因缺失突变株,生长曲线测定表明,突变株较出发菌株在平稳期后期生长较缓和;扫描电子显微镜观察和芽胞计数分析显示,突变株基本丧失了形成芽胞的能力,但依然形成晶体。SDS-PAGE结果显示,在 SSM培养基中,突变株对伴胞晶体蛋白的形成量影响并不显著;在营养较富集的Luria-Bertani培养基中,突变株中伴胞晶体蛋白的形成量较野生型和互补株明显降低。利用载体pHT315携带spoIIID操纵子互补突变株,互补株恢复了产生晶体和芽胞的能力。【结论】本研究证明spoIIID基因是苏云金芽胞杆菌芽胞形成所必需,同时与晶体蛋白的表达相关。