• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied Microbiology >Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol
【24h】

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

机译：嗜热地热芽孢杆菌脂肪酶T6的随机诱变和结构指导共识进行蛋白质工程，以增强甲醇的稳定性

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.
机译：酶在有机溶剂的非天然环境中催化反应的能力为基于酶的工业过程提供了新的机会。但是，这种方法的主要缺点是大多数酶在极性有机溶剂中的稳定性有限。在这项研究中，我们采用蛋白质工程方法来生成脂肪酶，以增强甲醇的稳定性，这对于生物柴油的生产非常重要。两种蛋白质工程方法，即随机诱变（易错PCR）和结构指导的共识，被并行应用于来自嗜热脂肪芽孢杆菌T6的未探索的脂肪酶基因。在高甲醇浓度下孵育一段时间后，使用高通量比色筛选测定法评估脂肪酶活性。两种蛋白质工程方法都成功地在70％的甲醇中产生了半衰期值提高的变体。随机诱变文库的最佳变体Q185L表现出提高了23倍的稳定性，但与野生型相比，其甲醇分解活性却降低了一半。共有库中的最佳变体H86Y / A269T在甲醇中的稳定性提高了66倍，同时热稳定性（+ 4.3°C）提高了，大豆油中的脂肪酸甲酯的收率提高了2倍。基于计算机模拟，我们建议Q185L取代促进封闭的盖子构象，从而限制甲醇和过量底物进入活性位点。 H86Y / A269T增强的稳定性是形成新氢键的结果。这些改进的特性使该变体成为生产生物柴油的潜在生物催化剂。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号