Directed evolution of e-coli alkaline phosphatase towards higher catalytic activity
文献类型：期刊论文
| 作者 | Xu, HF; Zhang, XE; Zhang, ZP; Zhang, YM; Cass, AEG |
| 刊名 | Biocatalysis and biotransformation
 |
| 出版日期 | 2003-02-01 |
| 卷号 | 21期号:1页码:41-47 |
| 关键词 | Directed evolution Error-prone pcr Dna shuffling E. coli alkaline phosphatase Catalytic activity Non-active site |
| ISSN号 | 1024-2422 |
| DOI | 10.1080/1024242031000087493 |
| 通讯作者 | Zhang, xe() |
| 英文摘要 | Directed evolution was used to enhance the catalytic activity of e. coli alkaline phosphatase (eap). through two rounds of error-prone pcr and one round of dna shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. in 1.0 m tris buffer, the specific activity of the most active eap variant s2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the d101s parent enzyme and similar to40 times more active than the wild-type ear at the same time, the k-m value of the s2163 variant decreased to 1491 mum from the 2384 mum of the d101s. as a result, the k(cat)/k-m ratio of this variant showed a 5.8-fold enhancement over that of d101s parent enzyme. three activating amino acid substitutions, k167r, g180s and s374c, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. possible explanations for the improvement of activity were analyzed. |
| WOS关键词 | ESCHERICHIA-COLI ; IN-VITRO ; SITE ; RECOMBINATION ; FRAGMENTATION ; MUTAGENESIS ; INHIBITION ; MECHANISM ; MUTATIONS ; KINETICS |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS类目 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| WOS记录号 | WOS:000182131700006 |
| 出版者 | TAYLOR & FRANCIS LTD |
| URI标识 | http://www.irgrid.ac.cn/handle/1471x/2376138 |
| 专题 | 武汉病毒研究所 |
| 通讯作者 | Zhang, XE |
| 作者单位 | 1.Chinese Acad Sci, Wuhan Inst Virol, Wuhan 430071, Peoples R China 2.Univ London Imperial Coll Sci Technol & Med, Dept Sci Biol, London SW7 2AY, England |
| 推荐引用方式 GB/T 7714 | Xu, HF,Zhang, XE,Zhang, ZP,et al. Directed evolution of e-coli alkaline phosphatase towards higher catalytic activity[J]. Biocatalysis and biotransformation,2003,21(1):41-47. |
| APA | Xu, HF,Zhang, XE,Zhang, ZP,Zhang, YM,&Cass, AEG.(2003).Directed evolution of e-coli alkaline phosphatase towards higher catalytic activity.Biocatalysis and biotransformation,21(1),41-47. |
| MLA | Xu, HF,et al."Directed evolution of e-coli alkaline phosphatase towards higher catalytic activity".Biocatalysis and biotransformation 21.1(2003):41-47. |
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来源：武汉病毒研究所
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