Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University, Jinju 52828, Republic of Korea.
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea.
J Microbiol Biotechnol. 2022 Jun 28;32(6):800-807. doi: 10.4014/jmb.2202.02017. Epub 2022 Apr 25.
Four genes encoding alkaline serine proteases from strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an shuttle vector. The ligation mixture was introduced into WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene () consisted of DNA mostly originated from either or in addition to some DNA from either or . Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. was overexpressed in BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from sp. for industrial applications.
四种来自 菌株的碱性丝氨酸蛋白酶基因被用作家族基因改组的模板基因。通过 DNA 酶 I 消化获得的改组基因，然后进行连续的无引物和常规 PCR 反应，与 pHY300PLK 连接，这是一种穿梭载体。连接混合物被引入到 WB600 中，一个转化体（FSM4）显示出更高的纤维蛋白溶解活性。DNA 测序证实，改组基因（）主要由来自 或 的 DNA 组成，此外还有一些来自 或 的 DNA。成熟 AprEFSM4（275 个氨基酸）与成熟 AprEJS2 在 4 个氨基酸和成熟 AprE176 在 2 个氨基酸处不同。AprEFSM4 通过使用 pET26b(+)在 BL21（DE3）中过表达，并纯化重组 AprEFSM4。AprEFSM4 的最适温度和 pH 与亲本酶相似。然而，与亲本酶相比，AprEFSM4 具有更好的热稳定性和纤维蛋白原水解活性。结果表明，DNA 改组可用于改善 sp. 的纤维蛋白溶解酶，以用于工业应用。
