High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction

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• 作者：Jun-hui Zhang^a ; Ying Lin^a ; Yu-fei Sun^a ; ^b ; Yan-rui Ye^a ; Sui-ping Zheng^a ; Shuang-yan Han^a ; ^{syhan@scut.edu.cn}
• 关键词：High-throughput screening ; pH indicator ; Rhizomucor miehei lipase ; Synthetic activity ; Thermostability ; Yeast surface display
• 刊名：Enzyme and Microbial Technology
• 出版年：2012
• 期刊代码：26_01410229
• 类别：ce
• 出版时间：10 May, 2012
• 卷：50
• 期：6-7
• 页码：325-330
• 文件大小：632 K

摘要

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants鈥?lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1%residual activity after being heated at 70 掳C for 5 h comparing to 51.0%of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.