| 题名 | 真菌杀虫剂菌种库的建立及部分相关代谢产物特性 |
| 作者 | 杨革 |
| 学位类别 | 博士 |
| 答辩日期 | 2004 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 陈洪章 |
| 关键词 | 虫生真菌 菌种库 球抱白僵菌 金龟子绿僵菌 几丁质酶 拮抗毒素蛋白 纯化 特征工作一 气-液-固三相流化床多尺度模拟 |
| 其他题名 | Establishment of storehouse of microorganism strains producing fungus insecticide and the part characteristics of related metabolites |
| 学位专业 | 生物化工 |
| 中文摘要 | 进行了大量的虫生真菌菌种筛选工作，将62份不同来源的害虫样品进行平板初筛后，得到80余株几丁质酶产生菌。对初筛菌种分别进行最适pH值、最适作用温度、产抱量和毒力的实验，得到32株希望菌株。从结果可知，这些菌株有的耐高温，有的耐”酸，也有的同时耐高温和耐酸，寄主谱较广，因而建立的是一个小型但比较有特色的菌种库。应用He-Ne激光对球抱白僵菌Beauveriabassiana75A进行辐照处理。研究了不同剂量激光辐照对菌体生长的影响，0.48mW·cm-2、15min的剂量利于菌体的诱变。激光辐照方式有两种：一是对用生理盐水制成的抱子悬液进行辐照；二是对用液体培养基制成的抱子悬液进行辐照。这两种激光辐照方式中，“生理盐水菌悬液”辐照方式诱变效果较好。经发酵产抱试验和菌体生物量测定，发现He-Ne激光对球抱白僵菌具有明显的生物刺激效应和诱变作用，并初步筛选到产抱量有较大变化的辐照变异菌株；同时，通过对变异菌株的细胞外、胞周间、细胞内三位区的RNA、DNA分析，进一步证实了He-Ne激光对球抱白僵菌的诱变作用。金龟子绿僵菌（Metarhiziumanisopliae）是一种重要的昆虫寄生真菌。几丁质酶合成的最佳碳源和诱导物为几丁质。在一定范围内增加培养基中几丁质浓度，葡萄搪浓度，微量元素盐浓度和碳氮比都能提高几丁质酶活。胆汁酸和吐温80作为表面活性剂能显著提高几丁质酶活。通过摇瓶试验得到优化培养基和培养条件。根据优化条件在摇瓶和3．7L发酵罐中分别进行产酶试验，实验结果表明，几丁质酶活分别达到0.231IU．mL-1，和0.273IU．mL-1。从自然催病死亡的金龟子体内分离到一株金龟子绿僵菌（Metarhiziumanisopliae），它在几丁质的诱导下能产生较高活性的几丁质酶。发酵液经硫酸按盐析、DEAE纤维素柱层析、PhenylSepharoseT‘6FastFlow疏水柱层析等方法，得到电泳纯的几丁质酶。用SDS-PAGE测得该酶分子量为61.5KD，而经质谱分析为57.14KD。最适反应温度为55℃，最适反应PH值为6.0，酶的等电点pI为4.02，其N末端序列为VIGPAAPL，用硫酸一酚法测得其含糖量为56.2％。水解几丁质的Kn为14．5μmol／L。该酶在斗soC，pH3.0-9.5较为稳定。Zn2+、Ca2+、Ba2+和Mn2+离子对”几T质酶活性有明显的促进作用，而Hg2+、Co2＋和Fe2＋离子完全抑制几丁质酶的活性。此酶还可被EDTA所抑制，表明金属离子为其活性所必需。PMSF试剂对几丁质酶的活力影响比较大，丝氨酸可能是酶活力的必需基团。金龟子绿僵菌（Metarhiziumanisopliae）M-6菌株对多种作物害虫有良好的防效。本文研究了M一6菌株产生的拮抗毒素蛋白的纯化及其部分特性。该菌株的发酵液经过酸沉淀和有机溶剂抽提，所得的拮抗物粗提液对热稳定，对胰蛋白酶不敏感，对蛋白酶K、碱性蛋白酶部分敏感，对氯仿部分敏感，其作用的活性pH范围低至4，高至12以上，比较耐碱性。粗提液经phenyl-SepharoseCL-4B柱层析、DEAE-Sephacel柱层析和FPLC的Superdex76llR10／30柱层析等步骤后，拮抗物质得到纯化，硅胶薄层层析显色为单点。该物质在275nm处有吸收峰，对蛋白酶有一定耐受性，荀三酮反应呈阴性，但酸水解后，荀三酮反应呈阳性，双缩脉反应也呈阳性。氨基酸组分分析结果表明，该物质由谷氨酸、天冬氨酸、甘氨酸、酪氨酸、苏氨酸、脯氨酸、亮氨酸、异亮氨酸、色氨酸和一种异常氨基酸组成。推测该物质为一种环状多肤，命名为MP-1。紫外光照射处理后，MP-1的抗菌活性损失不大。昆虫活性抑制试验结果表明，MP-1对10种供试昆虫的活性有抑制作用，其完全抑制浓度因昆虫种类不同而有差异。 |
| 英文摘要 | More than eighty microbial strains producing high-activity chitinase were isolated from many pests. Morphologic and enzymological characters of part of these strains were investigated. Strain Bb 75A and M-6 with high chitinase activity were identified as Beauveria bassiana and Metarhizium anisopliae,respectively. On the basis of above experiments, a small library of microorganism stains producing chitinase with various characteristics was established. The influences of He-Ne irradiation on the mutation and growth of Beauveria bassiana 75A strain was investigated. The optimal He-Ne laser irradiation dosage was 0.48mW ? cm" for 15min. Among the two ways of He-Ne laser irradiation, irradiation on bacteria suspension in physiological salt water appeared more effective in activation than in liquid medium. Based on the analysis of the sporulation fermentation and microbal biomass, it was found that He-Ne laser could give rise to an markedly biological stimulating effect and certain mutagenesis effect. Several strains that produced sporulation abnormally were screened. By the contrast of RNA and DNA contents from the extracell, intracell and periplasmic between mutant and original strains, further evidence that the He-Ne laser beam caused mutagenesis of Beauveria bassiana was concluded . Metarhizium anisopliae is an important parasite of plant insect. Metarhizium anisopliae M-6 could secrete high amounts of extracellular chitinase(0.126IU.mL'1) with 0.5% chitin as sole carbon source.The best carbon source and inducer for chitinase production was chitin. Chitinase biosynthesis was repressed by N-acetylglucosamine rather than glucose. Chitinase activity increased with chitin concentration, glucose concentration, trace element concentration and C/N ratio within the range tested. A fungus producing chitinase was isolated from the dead body of Anisopliae. A chitinase was isolated from the culture of Metarhizium anisopliae and purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation, DEAE-cellulose and hydrophobic interaction column chromatography. Its molecular weight was estimated to be about 61.5kD by SDS-PAGE, and 57.14kD by mass spectrum. The optimal reaction temperature and pH of the enzyme activity were 55°C and 6.0 respectively. The isoelectric point was 4.02 . Its N-terminal sequence was VIGPAAPL. The carbohydrate content was 56.2% obtained by phenol-sulfuric acid assay. Michaelis constant of the enzyme was 14.5//mol/L. The enzyme activity was stable at 45°C and in the pH range of 3.0—9.5. The activity was enhanced by Zn2\ Ca2\ Ba2'' and Mn2+, and was strongly inhabited by Hg2+- Co2+ and Fe2+. EDTA inhibited the enzyme activity. Ser may be essential residue for enzyme activity. This bacterium was shown strong inhibition to several plant diseases. In this paper, the purification of the anti-insect substance produced by Metarhizium anisopliae M-6 and the substance properities were studied. After the steps of acid precipitation, methanol and ether extraction, an antagonistic insects protein was isolated from Metarhizium anisopliae M-6. Crude extract obtained by precipitation of the cell-free culture of M-6was thermostable, resistant to trypsin, partial sensitive to proteinase K, ala-proteinase. Its active pH range was wide, from 4.0 to more than 12.0, relative more stable at high pH. After the crude extract M-6 was chromatographed by Phenyl-Sepharose CL_4B, DEAE-Sephacel and FPLC Superdex 75 HR 10/30 column chromatography, the anti-insect material was purified. The purified material had absorption peak at 275nm and was exhibited negative in biuret color reaction. However, after hydrolyzed with HCI, this substance showed positive in the same reaction. Amino acid analysis to the hydrolysate of MP-1 showed that MP-1 was composed of Glu, Asp, Tyr, Gly, Thr, Pro,Leu, lie, Trp and an unknown amino acid. Considering with its partial resistance to proteinases, it was suggested that this ant-insect material was a cyclic peptide. This peptide, named MP -1, had strong inhibition on many insects and showed high stability against ultraviolet radiation. MP -1 may have potential role in plant diseases biological control. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 60 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1388]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 杨革. 真菌杀虫剂菌种库的建立及部分相关代谢产物特性[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004. |
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