2,2-二甲基环丙腈水合酶产生菌的氮离子注入选育及突变株产酶条件研究  

The Breeding of 2,2-Dimethylcyclopropanecarboxnitrile Hydratase-Producing Mutant Strain by N^+ Implantation and Optimization of Fermentation Conditions

作　　者：吴玉峰[1] 郑裕国[1] 沈寅初[1] 

机构地区：[1]浙江工业大学生物工程研究所,杭州310032

出　　处：《高校化学工程学报》2007年第6期971-976,共6页Journal of Chemical Engineering of Chinese Universities

基　　金：国家973计划项目(2003CB716005);国家教育部博士点专项基金(20051033701);国家863计划项目(.2006AA02Z241)

摘　　要：离子注入技术被广泛应用于微生物的品种改良。利用N+离子注入法对本实验室筛选并保藏的产2,2-二甲基环丙腈水合酶菌种Pseudomonas sp.ZJUT0509进行选育,以提高2,2-二甲基环丙甲酰胺的产量,为进一步的手性拆分提供更多的原料。结果表明,氮离子注入诱变Pseudomonas sp.ZJUT0509的存活率曲线符合离子注入诱变的"马鞍型"特征曲线。通过离子注入获得的突变株的腈水合酶活性有显著提高,其中突变株F60-4的酶活达30.1 U·mL-1,约为原始菌株的9倍。对突变株F60-4进行产酶条件的优化考察,获得了培养条件。F60-4菌株产酶的最佳碳源是葡萄糖(2g·L-1),最佳氮源是酵母粉(10g·L-1),最适初始pH值为6.5,最适发酵温度为20℃,最佳诱导剂为1.0g·L-1的己内酰胺。与原始菌株Pseudomonas sp.ZJUT0509相比,突变株F60-4对有机氮源的利用率更高。最佳诱导剂由己内酰胺代替了原来的2,2-二甲基环丙甲腈,使实验操作简便。突变株F60-4的发酵周期为36h,比原始菌株缩短了12h。As a method used in the breeding of microorganism, the technology of ion implantation has gained more and more attention, A strain, Pseudomonas sp, Z JUT0509, isolated from soil and capable of producing nitrile hydratase was bred by N^＋ implantation to improve its activity of nitrile hydratase and enhance the productivity of 2,2-dimethylcyclopropanecarboxamide, which is a key intermediate for cilastatin production. The results show that, after irradiation, the survival rate curve of Pseudomonas sp, Z JUT0509 consists with the ＂saddle＂ character of ion implantation, The activity of strain F60-4, which is the best one of the mutants from Pseudomonas sp. ZJUT0509, reaches 30.1 U.mL^-1, which is about 9 times higher than that of its original strain, The optimum conditions for enzyme production by mutant F60-4 was obtained as follows： 2.0 g.L^-1 of glucose, 10 g.L^-1 of yeast extract, 6.5 of initial pH of broth, 20℃ of fermentation temperature and 1.0 g.L^-1 of caprolactam used as inducer. Comparing with the original strain, the mutant strain F60-4 could use the organic nitrogen source much better, and instead of 2,2-dimethylcyclopropanecarboxnitrile, the optimum inducer used with it is caprolactam, The fermentation time of mutant F60-4 can be shorten to 36 h, it is 12 h less than that of the original strain.

关 键 词：氮离子注入 腈水合酶 2 2-二甲基环丙甲腈 2.2-二甲基环丙甲酰胺 

分 类 号：TQ925.9[轻工技术与工程—发酵工程] Q556.4[生物学—生物化学]