Engineering mammalian cytochrome P4502B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide
文献类型：期刊论文
| 作者 | Kumar, Santosh; Sun, Ling; Liu, Hong ; Muralidhara, B. K.; Halpert, James R.
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| 刊名 | PROTEIN ENGINEERING DESIGN & SELECTION
 |
| 出版日期 | 2006-12 |
| 卷号 | 19期号:12页码:547-554 |
| 关键词 | cytochrome P450 directed evolution heme accessibility random mutagenesis thermostability |
| ISSN号 | 1741-0126 |
| DOI | 10.1093/protein/gzl042 |
| 文献子类 | Article |
| 英文摘要 | The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H2O2-mediated substrate oxidation, has now been shown to exhibit a > 3.0-fold decrease in K-m,K-HOOH for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening > 3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained similar to 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T-m,T-app compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a > 2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent. |
| WOS关键词 | LABORATORY EVOLUTION ; SELF-SUFFICIENT ; BINDING ; HETEROGENEITY ; HYDROXYLATION ; BIOCATALYSTS ; TRANSITIONS ; MUTAGENESIS ; RESOLUTION ; IMIDAZOLE |
| 资助项目 | NIEHS NIH HHS[ES06676] ; NIEHS NIH HHS[ES03619] |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| WOS记录号 | WOS:000242717400003 |
| 出版者 | OXFORD UNIV PRESS |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/273439]  |
| 专题 | 中国科学院上海药物研究所 |
| 通讯作者 | Kumar, Santosh |
| 作者单位 | 1.Univ Texas, Med Branch, Dept Pharmacol & Toxicol, Galveston, TX 77555 USA 2.Chinese Acad Sci, Shanghai Inst Mat Med, Drug Discovery & Design Ctr, Shanghai 201203, Peoples R China |
| 推荐引用方式 GB/T 7714 | Kumar, Santosh,Sun, Ling,Liu, Hong,et al. Engineering mammalian cytochrome P4502B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide[J]. PROTEIN ENGINEERING DESIGN & SELECTION,2006,19(12):547-554. |
| APA | Kumar, Santosh,Sun, Ling,Liu, Hong,Muralidhara, B. K.,&Halpert, James R..(2006).Engineering mammalian cytochrome P4502B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide.PROTEIN ENGINEERING DESIGN & SELECTION,19(12),547-554. |
| MLA | Kumar, Santosh,et al."Engineering mammalian cytochrome P4502B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide".PROTEIN ENGINEERING DESIGN & SELECTION 19.12(2006):547-554. |
入库方式： OAI收割
来源：上海药物研究所
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