This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

中文翻译：

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新型工程土芽孢杆菌脂肪酶 GD-95RM 用于专注于脂肪酯清洁生产和家用洗涤产品配方的行业

本研究展示了一种新的微生物脂肪分解酶 GD-95RM，它使用先前表征的 GD-95 脂肪酶作为模板，通过随机诱变设计。GD-95 脂肪酶活性的提高是由 E100K、F154V 和 V174I 突变引起的。与 GD-95 脂肪酶相比，GD-95RM 脂肪酶的比活性增加了 1.3 倍（2000 U/mg），表现出对更高温度（75-85°C）的耐受性，对 p-NP 十二酸的 Vmax 增加了四倍，并显示p-NP 丁酸盐的 KM 降低了 2.5 倍。在与 25% 的乙醇一起孵育 20 天后，在 30 °C 和 55 °C 反应温度下水解短、中和长酰基链底物时，它保留了 > 50% 的脂肪分解活性。GD-95RM 还显示出对 5% NaCl、柠檬酸三钠、过硼酸钠、尿素、0.1% 硼酸、柠檬酸和 Triton X-100 的长期耐受性（40 天）。此外，油水解和酯交换结果揭示了 GD-95RM 脂肪酶通过使用广泛的植物油和鱼油、动物脂肪和不同醇作为底物的环保水解和酯交换反应生产脂肪酸或脂肪酸酯的能力。GD-95RM脂肪酶成功应用于油酸乙酯、油酸辛酯和油酸异戊酯的合成反应，无需额外的反应化合物或特殊的反应条件。