Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, People's Republic of China
Appl Environ Microbiol. 2018 Jan 2;84(2). doi: 10.1128/AEM.02129-17. Print 2018 Jan 15.
lipase (RML), as a kind of eukaryotic protein catalyst, plays an important role in the food, organic chemical, and biofuel industries. However, RML retains its catalytic activity below 50°C, which limits its industrial applications at higher temperatures. Soluble expression of this eukaryotic protein in not only helps to screen for thermostable mutants quickly but also provides the opportunity to develop rapid and effective ways to enhance the thermal stability of eukaryotic proteins. Therefore, in this study, RML was engineered using multiple computational design methods, followed by filtration via conservation analysis and functional region assessment. We successfully obtained a limited screening library (only 36 candidates) to validate thermostable single point mutants, among which 24 of the candidates showed higher thermostability and 13 point mutations resulted in an apparent melting temperature ([Formula: see text]) of at least 1°C higher. Furthermore, both of the two disulfide bonds predicted from four rational-design algorithms were further introduced and found to stabilize RML. The most stable mutant, with T18K/T22I/E230I/S56C-N63C/V189C-D238C mutations, exhibited a 14.3°C-higher [Formula: see text] and a 12.5-fold increase in half-life at 70°C. The catalytic efficiency of the engineered lipase was 39% higher than that of the wild type. The results demonstrate that rationally designed point mutations and disulfide bonds can effectively reduce the number of screened clones to enhance the thermostability of RML. lipase, whose structure is well established, can be widely applied in diverse chemical processes. Soluble expression of lipase in provides an opportunity to explore efficient methods for enhancing eukaryotic protein thermostability. This study highlights a strategy that combines computational algorithms to predict single point mutations and disulfide bonds in RML without losing catalytic activity. Through this strategy, an RML variant with greatly enhanced thermostability was obtained. This study provides a competitive alternative for wild-type RML in practical applications and further a rapid and effective strategy for thermostability engineering.
脂肪酶（RML）作为一种真核蛋白催化剂，在食品、有机化学和生物燃料等行业发挥着重要作用。然而，RML 的催化活性在 50°C 以下保留，这限制了其在较高温度下的工业应用。该真核蛋白在 中的可溶性表达不仅有助于快速筛选耐热突变体，还为开发提高真核蛋白热稳定性的快速有效方法提供了机会。因此，在本研究中，使用多种计算设计方法对 RML 进行了工程改造，然后通过保守性分析和功能区域评估进行了过滤。我们成功获得了一个有限的筛选文库（仅 36 个候选物），以验证耐热单点突变体，其中 24 个候选物表现出更高的热稳定性，13 个点突变导致明显的熔点（[Formula: see text]）至少提高 1°C。此外，从四个合理设计算法预测的两个二硫键进一步被引入并发现稳定了 RML。最稳定的突变体 T18K/T22I/E230I/S56C-N63C/V189C-D238C 突变，[Formula: see text]提高了 14.3°C，在 70°C 时半衰期增加了 12.5 倍。工程化脂肪酶的催化效率比野生型高 39%。结果表明，合理设计的点突变和二硫键可以有效地减少筛选克隆的数量，从而提高 RML 的热稳定性。脂肪酶的结构已经很好地建立，它可以广泛应用于各种化学过程中。在 中的可溶性表达为探索提高真核蛋白热稳定性的有效方法提供了机会。本研究强调了一种策略，该策略结合了计算算法来预测 RML 中的单点突变和二硫键，而不会失去催化活性。通过该策略，获得了一种热稳定性大大提高的 RML 变体。本研究为实际应用中野生型 RML 提供了一种有竞争力的替代方案，并进一步提供了一种快速有效的热稳定性工程策略。
