【摘要】 以猪源胰蛋白酶作为研究对象,尝试利用删除猪源胰蛋白酶活性位点邻近?-loop的方法来提高其热稳定性和活性。首先通过Gromacs v4.5.5分子动力学模拟软件,预测出了胰蛋白酶的柔性区Fragment 41~46(即?-loop所在区域)。然后根据删除?-loop结构中不同的氨基酸数目,确立了三个突变R1Q、K3Q及C5Q;通过重叠延伸PCR技术构建突变菌株,发酵表达后,进行了酶热稳定性和活性分析。结果表明,R1Q、K3Q及C5Q的活性在一定程度上均有提高,在热稳定性上R1Q和K3Q有所提高,C5Q有所下降,其中R1Q突变体酶的最高活性较野生型提高了1.42倍,同时,失活半衰期和最适反应温度较野生型也分别提高了213.3 min和11.8℃。实验结果证明,在删除该?-loop中的异亮氨酸后,得到了突变型R1Q酶,成功实现酶双功能的共同进化。该策略有潜力应用于其他工业酶分子的热稳定性和活性改造,为推动生物酶的工业化应用奠定基础。更多还原

【Abstract】 Thermostability and activity of porcine trypsin were studied by deleting ?-loop near active sites. Molecular dynamics software Gromacs v4.5.5 was used to predict Fragment 41~46 at the flexible region of porcine trypsin(which is the ?-loop region). Subsequently, three mutations R1Q, K3Q and C5Q were obtained by deleting different amounts of amino acids in the ?-loop. The results demonstrate that the activities of R1Q, K3Q and C5Q are all improved, and the maximum activity of R1Q is improved by 1.42 times compared to that of the wild type. Meanwhile, the inactivation half-life period and the optimal reaction temperature of R1Q are increased by 213.3 min and 11.8℃, respectively. Moreover, thermostability is improved for R1Q and K3Q, while it is decreased for C5Q. The results indicate that R1Q with isoleucine deleted from the ?-loop shows both thermostability and activity improvements. This study provides a promising method to improve thermostability and activity of other industrial enzymes.更多还原