共表达SHMT和TPase载体的构建及双酶法合成L-色氨酸
Construction of co-expression SHMT and TPase recombinant vector and dual-enzymatic synthesis of L-tryptophan
投稿时间:2009-11-18  
DOI:  
中文关键词:丝氨酸羟甲基转移酶,色氨酸酶,串联表达,双酶法合成
英文关键词:hydroxymethyltransferase, tryptophanase, co-expression, dual-enzymatic synthesis
基金项目:湖北省教育厅重点项目 (No. D200718002) 资助。
作者单位E-mail
李鑫 武汉工业学院生物与制药工程学院武汉 430023  
刘军 武汉工业学院生物与制药工程学院武汉 430023 Junliu85@yahoo.com.cn 
赵沁沁 武汉工业学院生物与制药工程学院武汉 430023  
徐爱才 武汉工业学院生物与制药工程学院武汉 430023  
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中文摘要:
      利用重组大肠杆菌表达丝氨酸羟甲基转移酶 (SHMT) 和色氨酸酶 (TPase),并利用双酶法合成L-色氨酸。采用PCR从大肠杆菌K12基因组中扩增上述两种酶的基因,利用pET-28a载体,构建单表达重组质粒pET-SHMT、pET-TPase和共表达重组质粒pET-ST。将上述3种重组质粒转入大肠杆菌BL21(DE3)进行表达。SDS-PAGE结果表明,单表达基因工程菌BL21(DE3)/pET-SHMT和BL21(DE3)/pET-TPase分别在47 kDa (SHMT) 和50 kDa (TPas
英文摘要:
      Hydroxymethyltransferase (SHMT) and tryptophanase (TPase) are key enzymes in biosynthesis of L-tryptophan. We constructed three recombinant plasmids, including pET-SHMT, pET-TPase, and pET-ST for over-expression or co-expression of SHMT and TPase in Escherichia coli BL21 (DE3). The SDS-PAGE analysis showed that the recombinant proteins of 47 kDa and 50 kDa were expressed of pET-SHMT and pET-TPase, respectively. As compared to the host stain, the enzyme activity of SHMT and TPase was increased by 6.4 and 8.4 folds, respectively. Co-expression of both recombinant proteins, 47 kDa and 50 kDa, was also successful by using pET-ST and the enzyme activities were enhanced by 6.1 and 6.9 folds. We designed two pathways of dual-enzymatic synthesis of L-tryptophan by using these recombinant strains as source of SHMT and TPase. In the first pathway, the pET-SHMT carrying strain was used to catalyze synthesis of L-serine, which was further transformed into L-tryptophan by the pET-TPase expressing strain. These two steps sequentially took place in different bioreactors. In the second pathway, the pET-ST carrying strain, in which two enzymes were co-expressed, was used to catalyze simultaneously two steps in a single bioreactor. HPLC analysis indicated a high yield of 41.5 g/L of L-tryptophan was achieved in the first pathway, while a lower yield of 28.9 g/L was observed in the second pathway. In the first pathway, the calculated conversion rates for L-glycine and indole were 83.3% and 92.5%, respectively. In the second pathway, a comparable conversion rate, 82.7%, was achieved for L-glycine, while conversion of indole was much lower, only 82.9%.
李鑫,刘军,赵沁沁,徐爱才.共表达SHMT和TPase载体的构建及双酶法合成L-色氨酸[J].生物工程学报,2010,26(9):1302~1308
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