Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution
文献类型：期刊论文
| 作者 | Martinez, Ronny1,2; Jakob, Felix1; Tu, Ran3; Siegert, Petra4; Maurer, Karl-Heinz4; Schwaneberg, Ulrich1 |
| 刊名 | BIOTECHNOLOGY AND BIOENGINEERING
 |
| 出版日期 | 2013-03-01 |
| 卷号 | 110期号:3页码:711-720 |
| 关键词 | thermostability low temperature alkaline protease directed evolution |
| 英文摘要 | Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15 degrees C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58 degrees C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (Kcat) at 15 degrees C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15 degrees C) and over 100 times prolonged half-life at 60 degrees C (224min compared to 2min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711720. (c) 2012 Wiley Periodicals, Inc. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Biotechnology & Applied Microbiology |
| 研究领域[WOS] | Biotechnology & Applied Microbiology |
| 关键词[WOS] | COLD ADAPTATION ; PSYCHROPHILIC ENZYMES ; RANDOM MUTAGENESIS ; SERINE-PROTEASE ; SUBTILISIN BPN' ; MOLECULAR-BASIS ; THERMOSTABILITY ; STABILITY ; PROTEINS ; SEQUENCE |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000313806400005 |
| 源URL | [http://124.16.173.210/handle/834782/1276]  |
| 专题 | 天津工业生物技术研究所_进化代谢工程和分子进化研究组 王钦宏_期刊论文 |
| 作者单位 | 1.Rhein Westfal TH Aachen, Lehrstuhl Biotechnol, D-52074 Aachen, Germany 2.Univ Bremen, D-28359 Bremen, Germany 3.Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Tianjin, Peoples R China 4.Henkel AG & Co KGaA, Dusseldorf, Germany |
| 推荐引用方式 GB/T 7714 | Martinez, Ronny,Jakob, Felix,Tu, Ran,et al. Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution[J]. BIOTECHNOLOGY AND BIOENGINEERING,2013,110(3):711-720. |
| APA | Martinez, Ronny,Jakob, Felix,Tu, Ran,Siegert, Petra,Maurer, Karl-Heinz,&Schwaneberg, Ulrich.(2013).Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution.BIOTECHNOLOGY AND BIOENGINEERING,110(3),711-720. |
| MLA | Martinez, Ronny,et al."Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution".BIOTECHNOLOGY AND BIOENGINEERING 110.3(2013):711-720. |
入库方式： OAI收割
来源：天津工业生物技术研究所
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