【作者】 姚国玉；

【导师】 陈强； 姚斌；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 本研究通过对菌种新颖性和产酶能力评估确定了链霉菌S27（Streptomyces sp.S27）为本实验的研究对象。链霉菌是一种资源丰富的拮抗微生物,在工业、农业和环境科学等方面有着广泛的用途。链霉菌S27分离自新疆吐鲁番盆地的火焰山。利用生物信息学的方法,对链霉菌来能源的糖苷水解酶16家族的β-1,3-葡聚糖酶序列进行比对设计简并引物,通过简并PCR扩增得到来源于链霉菌S27编码的β-1,3-葡聚糖酶的基因片段,再结合基因组文库筛选和TAIL PCR的方法最终获得基因的全序列并成功地在大肠杆菌BL21（DE3）中得到活性表达。bglS27基因全长1,359bp,编码453个氨基酸,理论分子量42.7kDa。成熟蛋白包含一个糖苷水解酶16家族的催化域,一个短的Gly连接域和一个第13家族的糖基结合域。纯化的重组酶ManS27以内切方式特异性地切割β-1,3-葡聚糖苷键。它在65℃和pH5.5活性最高,热稳定好,其比活和K_m值分别为236.0 Umg^-1和1.89 mg ml^-1。抑菌试验表明,BglS27可以抑制产毒素真菌（如:Fusarium Graminearum,Fusarium crookwellense,Paecilomyces variotii）和致病真菌（Rhizoctonic solani）的生长。BglS27具有的良好性质使其在食品和饲料保藏、植物保护和其它生物技术领域有潜在的应用前景。根据已知的链霉菌S27来源的β-甘露糖苷酶基因片段序列设计引物,通过筛选链霉菌S27基因组文库的方法克隆得到β-甘露糖苷酶基因manS27。该基因全长2,496bp,编码832个氨基酸和一个终止密码子,理论分子量为92.5 kDa。将manS27以正确阅读框架克隆到表达载体pET-22b（+）上,并在大肠杆菌BL21（DE3）中诱导表达。纯化的MartS27酶学性质分析表明,其最适温度为50℃,最适pH值为7.0,比活和K_m值分别为35.3 Umg^-1和13.75 mg ml^-1。实验结果表明,ManS27具有转糖基作用。本研究从链霉菌S27克隆得到β-1,3-葡聚糖酶和β-甘露糖苷酶的基因,通过构建表达载体并转化大肠杆菌获得了表达生物活性产物的重组子,这些重组子的获得为下一步高产商品化菌株的构建及其酶的分子生物学研究奠定了良好的基础,具有一定的应用价值。更多还原

【Abstract】 Streptomyces is an important resource-rich antagonistic microorganism and is widely used in industry, agriculture and environmental sciences.In this study,Streptomyces sp.S27 was selected as the research objects based on the evaluation of species-novelty and enzyme production capacity.Streptomyces sp.S27 was isolated from the Flaming Mountain in the Turpan Basin of Xinjiang Uygur Autonomous Region, China.By using bioinformatical methods,the amino acid sequence of Streptomyces producedβ-1,3-glucanase from glycoside hydrolase（GH） family 16 were aligned and analyzed.A set of degenerate primers were designed based on the conserved motifs.Gene fragments ofβ-1,3-glucanase from Streptomyces was cloned by using degenerated PCR method.The full length gene bglS27 was obtained by combinating with genomic library screening and TAIL PCR,and successfully expressed in Escherichia coli BL21（DE3）. The bglS27 gene contains 1,362 bp and encodes a protein of 453 amino acids with a calculated molecular mass of 42.7 kDa.The mature protein comprises a catalytic module of glycosyl hydrolase（GH） family 16, a short glycine linker region,and a family 13 carbohydrate-binding module（CBM）.The purified recombinant enzyme showed optimal activity at 65℃and pH 5.5,and preferentially catalyzed the hydrolysis of glucans withβ-1,3-linkage with an endolytic mode of action.Specific activity and Km value of BglS27 for laminarin was 304.2 U mg^-1 and 1.89 mg ml^-1,respectively.In antifungal assay,BglS27 had ability to inhibit the growth of some mycotoxin-producing fungi,such as Fusarium Graminearum, Fusarium crookwellense,and Paecilomyces variotii,and phytopathogenic fungus Rhizoctonic solani.These favorable properties make BglS27 a good candidate for use in feed and food preservation,plant protection, and other biotechnological applications.A pair of primers were designed based on the gene fragment ofβ-mannosidase from Streptomyces sp. S27 to clone the full length gene,manS27,by screening the genomic library from Streptomyces sp.S27. The gene manS27 contains 2,496 bp and encodes a protein of 832 amino acids with a calculated molecular mass of 92.5 kDa.The gene manS27 was inserted into the expression vector of pET-22b（+） and transformed into Eschetichia coli BL21（DE3） to express the recombinant protein ManS27. Characterization of ManS27 indicated that the optimum temperature and pH were 50℃and pH7.0,and the specific activity and Km value of ManS27 were 304.2 U mg^-1 and 1.89 mg ml^-1,respectively.The result of transglycosylatic experiment showed that ManS27 had the activity of transglycosylation.In this study,aβ-1,3-glucanase gene and aβ-mannosidase gene were cloned from Streptomyces sp. S27.By constructing and transformating expression vector to Eschetichia coli,we have obtained recombinants which are promising in applications of constructing the commercialized high-yielding strains and molecular biological research on enzymy.更多还原