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    杨帆, 刘晓, 王之可, 李素霞. Kex2蛋白酶K291突变体性质和动力学研究[J]. 华东理工大学学报(自然科学版), 2021, 47(6): 699-705. DOI: 10.14135/j.cnki.1006-3080.20201009001
    引用本文: 杨帆, 刘晓, 王之可, 李素霞. Kex2蛋白酶K291突变体性质和动力学研究[J]. 华东理工大学学报(自然科学版), 2021, 47(6): 699-705. DOI: 10.14135/j.cnki.1006-3080.20201009001
    YANG Fan, LIU Xiao, WANG Zhike, LI Suxia. Properties and Kinetics of Kex2 Protease Mutants K291[J]. Journal of East China University of Science and Technology, 2021, 47(6): 699-705. DOI: 10.14135/j.cnki.1006-3080.20201009001
    Citation: YANG Fan, LIU Xiao, WANG Zhike, LI Suxia. Properties and Kinetics of Kex2 Protease Mutants K291[J]. Journal of East China University of Science and Technology, 2021, 47(6): 699-705. DOI: 10.14135/j.cnki.1006-3080.20201009001

    Kex2蛋白酶K291突变体性质和动力学研究

    Properties and Kinetics of Kex2 Protease Mutants K291

    • 摘要: 利用毕赤酵母GS115表达系统,成功表达并纯化获得了两种Kex2蛋白酶突变体Kex2-K291L和Kex2-K291H。对比研究了天然Kex2蛋白酶和这两种突变体的酶学性质和稳定性。实验结果表明,与天然Kex2蛋白酶相比,Kex2-K291H和Kex2-K291L两种突变体的稳定性得到了明显的改善。两种突变体的最适pH和最适温度与天然Kex2蛋白酶保持一致,均为pH 9.0和37 ℃,两种突变体的温度稳定性也与天然Kex2蛋白酶基本一致。与天然Kex2蛋白酶相比,突变体Kex2-K291H的pH值稳定性范围从5.0~6.0扩大至5.0~7.0。蛋白酶促反应动力学研究表明,突变体Kex2-K291H和Kex2-K291L的Kcat/Km值分别为天然Kex2蛋白酶的1.85倍和2.05倍。突变体Kex2-K291H和Kex2-K291L在改善天然Kex2蛋白酶自降解问题的同时,提高了pH稳定性和催化效率。

       

      Abstract: Two kinds of Kex2 protease mutants, Kex2-K291L and Kex2-K291H were successfully expressed in Pichia pastoris, which were induced by methanol and purified with anion exchange chromatography (Q-FF). Finally, the enzymatic characteristics of these Kex2 proteases were characterized. Compared with the wild-type Kex2, the degradation of the two mutants Kex2-K291H and Kex2-K291L was significantly improved. The wild-type Kex2 protease was degraded during the purification, and a non-single band appeared, while the mutants were not degraded during the purification, and it was still a single band. The optimum pH and temperature of these two mutants were the same as those of the wild-type Kex2. Their optimal pH and temperature were pH 9.0 and 37 ℃, respectively. Compared with the wild-type Kex2 protease, the pH stability range of the mutant Kex2-K291H was expanded, from the range of pH 5.0 to 6.0 to the range of pH 5.0 to 7.0. Compared with the wild-type Kex2, Kex2-K291H was more stable at the temperature range of 4 ℃ to 37 ℃. Enzymatic reaction kinetics studies showed that the Kcat/Km values of mutant Kex2-K291H and Kex2-K291L were 1.85 and 2.05 fold higher than that of the wild-type Kex2 protease, respectively.

       

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