College of Light Industry Science and Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing, 210018, China.
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, 30 Puzhu South Road, Nanjing, 211800, China.
Microb Cell Fact. 2018 Mar 19;17(1):45. doi: 10.1186/s12934-018-0885-z.
In industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type.
A novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8.5. It was a cold-adapted enzyme and retained approximately 40% of its maximum activity at 0 °C. A mutant, with higher activity and thermo-stability was obtained by random mutagenesis. Kinetic analysis indicated that the mutant Val29Ala/Tyr193Cys shown 43.5% decrease in K , 2.6-fold increase in K , and 4.7-fold increase in K /K relative to the wild type. Single mutants V29A and Y193C were constructed and their kinetic parameters were measured. The results showed that the values of K , K , and K /K of V29A were similar to those of the wild type while Y193C showed 52.7% decrease in K , 2.7-fold increase in K , and 5.6-fold increase in K /K compared with the wild type. The 3-D structure and docking analysis revealed that the replacement of Tyr by Cys could enlarge the binding pocket. Moreover Y193C also showed a better thermo-stability for the reason its higher hydrophobicity and retained 67% relative activity after incubation for 3 h at 50 °C.
The superior quality of modified esterase suggested it has great potential application in extreme conditions and the mutational work recommended that important information for the study of esterase structure and function.
在工业中，脂肪酶反应在高温等不敏感条件下发生，因此具有独特性质的新型坚固酯酶吸引了工业应用。蛋白质工程是获得所需酶特性的工具。从南海分离到一种新型酯酶基因，并进行随机诱变和定点突变，以获得比野生型更高的活性和热稳定性。
一种新型酯酶对 p-硝基苯乙酸酯（pNPA，C2）表现出最高的水解活性，最佳活性在 40°C 和 pH8.5。它是一种耐冷酶，在 0°C 时保留了其最大活性的约 40%。通过随机诱变获得了一种具有更高活性和热稳定性的突变体。动力学分析表明，突变体 Val29Ala/Tyr193Cys 的 K 降低了 43.5%，K 增加了 2.6 倍，K /K 增加了 4.7 倍。构建了单突变体 V29A 和 Y193C，并测量了它们的动力学参数。结果表明，V29A 的 K 、K 和 K /K 值与野生型相似，而 Y193C 的 K 降低了 52.7%，K 增加了 2.7 倍，K /K 增加了 5.6 倍。与野生型相比。3-D 结构和对接分析表明，用半胱氨酸取代酪氨酸可以扩大结合口袋。此外，Y193C 的疏水性更高，在 50°C 孵育 3 小时后，相对活性保留了 67%，因此具有更好的热稳定性。
改良酯酶的优异品质表明它在极端条件下具有很大的应用潜力，突变工作为酯酶结构和功能的研究提供了重要信息。
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