用于生产L－苯丙氨酸的基因工程菌的构建  

Construction of Recombinant E. coli Strain for Production of L-Phenylalanine

作　　者：史燕东[1] 吴梧桐[1] 周艳 张玉彬 龚韬 

机构地区：[1]中国药科大学生物技术中心

出　　处：《药物生物技术》1994年第1期8-13,共6页Pharmaceutical Biotechnology

摘　　要：本文报道用含ｔｙｒＢ基因（在大肠杆菌中编码芳香族氨基酸转氨酶）的质粒ｐＫＢ７转化不同的突主菌，筛选出ＣＴＢ２菌（ｐＫＢ７转化ＪＭ１０７得到）表达的芳香族氨基酸转氨酶活力最高。进一步研究证明ＣＴＢ２菌在菌浓度为２０ｍｇ／ｍｌ，温度３０℃，反就６０分钟．能催化苯丙酮酸加氨生成大量的Ｌ－苯内氮酸，而没有付产物酪氨酸生戊。以从ＣＴＢ２菌中提取纯化出的质粒为模板，经ＰＣＲ扩增反应可以得到约长１．２ｋｂ的ｔｙｒＢ基因片段，ＳＤＳ－聚丙烯酰胺凝胶电泳实验表明ＣＴＢ２菌可溶性蛋白在分子量约４００００，有一条明显加深的蛋白区带，这与理论估算的芳香族氨基酸转氨酶的分子量４３０００基本一致。而ＪＭ１０７和含ｐＫＫ２３３－２空质粒的ＪＭ１０７在该位点的区带则没有加深，该加深区带的蛋白含量占到总溶性蛋白的２０％左右。he recombinant plasmid pKB7 contains E. coli tyrB gene which codes for aromatic transaminase.Atransformant,JM107 with pKB7,was selected from different transformants with pKB7.This strain isnamed CTB2.After CTB2 mixed with phenylpyruvic acid and L-aspartic acid at the optimum cell con-centration 20 mg/ml,and at 30℃ for about 60 mintites,a large amount of L-phenylalanine withoutby-product tyrosine was observed.After reutilizing CTB2 for seven times,we found that there stillwas 50%of the transaminase activitv remained;Restilts showed that there were no remarkably differ-ences between the phenylpyruvic acids made in China and by Sigma Corp.for CTB2.While the plas-mid extracted from CTB2 was used as DNA template,a 1.2Kb tyrB fragment could be observed byPCR. SDS-PAGE demonstrated that the band at 40,000 dalton of CTB2 is darker than the same bandsof JM107 and CTB9(Jm107 with pKK23-2).The molecular weight of this darker band consistswith the theoretical molecular weight of E,coli aromatic transaminase;The amount of protein in thisdarker band is about 20%of the total dissoluble proteins.

关 键 词：L-苯丙氨酸 CTB2菌 tyrB基因 基因工程 

分 类 号：R977.4[医药卫生—药品] TQ464.7[医药卫生—药学]