【作者】 张搏；

【导师】 闫云君； 杨江科；

【作者基本信息】 华中科技大学 ， 生物化工， 2007， 硕士

【摘要】 从武汉市九峰森林公园附近采集含油污土样18份,经平板粗筛、摇瓶发酵复筛,获得一株脂肪酶酶活力为7.0U/mL的菌株,编号为26-2。经表型和16S rRNA序列分析（GenBank登录号:DQ789596）及系统发育分析将其鉴定为荧光假单胞菌（Pseudomonas fluorescens）26-2。应用单因子试验和正交试验对荧光假单胞菌26-2产脂肪酶发酵条件进行了快速优化。首先应用单因子试验确定荧光假单胞菌26-2产脂肪酶最适碳源和最适氮源分别为淀粉和酵母提取物。在此基础上通过L9 （34）正交试验,考察了淀粉、酵母提取物、橄榄油和硫酸镁等4个因素对产酶的影响,获得优化的培养基组成。最后通过单因子试验确定最适发酵温度和最适起始pH。试验获得的优化培养条件为:淀粉2.0%,酵母提取物3%,硫酸镁0.05%,K₂HPO₄0.2%,橄榄油0.2%,pH7.0,发酵温度30℃,培养60h后,酶活达到15.0U/mL。荧光假单胞菌26-2脂肪酶的最适pH为9.0,最适温度为50℃;Ca2+对酶活具有促进作用,而EDTA等则明显抑制酶活。通过PCR方法克隆了荧光假单胞菌26-2的脂肪酶基因。该基因开放式阅读框（ORF）为1854bp,编码617个氨基酸残基。与邻近种属脂肪酶基因序列的多重比较表明,该基因属于脂肪酶家族I.3。经EcoRI/NotI酶切,将其克隆到酵母表达载体pPIC9K上,构建成质粒pPIC9K-lipA,并导入毕赤酵母GS115中,初步获得脂肪酶诱导表达的基因工程菌。该菌在摇瓶发酵条件下,3d后发酵液酶活可达5.U/mL。更多还原

【Abstract】 By plates screening with Rhodamine B as indicator and flask fermentation, a strain with the lipase activity higher than 5.0U/mL were obtained, and identified as Pseudomonas fluorescens 26-2 by phenotypic characteristics and partial 16S rRNA sequence.Lipase production condition of P. fluorescens 26-2 was optimized using monofactorial experiment and orthogonal test. The optimum carbon source and nitrogen source for P. fluorescens 26-2 screened through monofactorial experiment were soluble starch and yeast extract, respectively. Four factors, soluble starch, yeast extract, olive and MgSO4 were investigated by orthogonal test to obtain optimal medium composition. The effect of pH and temperature on the production of the lipase was also examined with the optimized medium by monofactorial experiment. The optimized condition was: yeast extract 3%, starch 2.0%, K₂HPO₄ 0.2%, MgSO₄ 0.05%, olive oil 0.2%; the initial pH of fermentation was pH7.0, and the termperature was 30°C; After 60 h, the lipase activity reached the maxium15 U/ml. The optimal pH and temperature of enzymes was 9.0 and 50℃, respectively. The Ca2+ can enhance the activity of the enzyme, while the EDTA can inhibit the activity.The lipase gene of P. fluorescens 26-2 was cloned by PCR. The Open Reading Frame （ORF） of the lipase gene is 1854bp and encodes 617 amino acids. Digested with EcoRI/NotI, the lipase gene was cloned into vector pPIC9K, and transformed into Pichia pastoris GS115 by lithium chloride transformation method. The inducible expression of the lipase in recombinant yeast was checked by Rhodamin B plates and the enzyme activity up to 5.0 U/mL in fermentation liquid.更多还原