Department of Microbiology and Biotechnology, Institute of Biosciences, Life Sciences Center, Vilnius University, Sauletekis Avenue 7, Vilnius, 10257, Lithuania.
Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekis Avenue 7, Vilnius, 10257, Lithuania.
World J Microbiol Biotechnol. 2020 Feb 27;36(3):41. doi: 10.1007/s11274-020-02816-3.
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 V towards p-NP dodecanoate and showed 2.5-fold lower K 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.
本研究通过对先前表征的 GD-95 脂肪酶进行随机突变设计，提出了一种新的微生物脂肪酶 GD-95RM。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 脂肪酶的比活性提高了 1.3 倍（2000 U/mg），表现出对较高温度（75-85°C）的抗性，对 p-NP 十二烷酸的 V 值增加了四倍，对 p-NP 丁酸的 K 值降低了 2.5 倍。在 30°C 和 55°C 反应温度下，用 25%乙醇孵育 20 天后，GD-95RM 在水解短链、中链和长链酰基链底物时，其脂肪酶活性仍保留>50%。GD-95RM 还能耐受 5%NaCl、柠檬酸三钠、过硼酸钠、尿素、0.1%硼酸、柠檬酸和 Triton X-100，在 5%浓度下长期耐受（40 d）。此外，油脂水解和转酯化结果表明，GD-95RM 脂肪酶通过使用各种植物油、鱼油、动物脂肪和不同醇作为底物的环保水解和转酯化反应，具有生产脂肪酸或脂肪酸酯的能力。GD-95RM 脂肪酶成功地应用于油酸乙酯、辛醇油酸酯和异戊醇油酸酯的合成反应中，无需使用额外的反应化合物或特殊的反应条件。
