基于异源表达和甲基化修饰制备抗自切重组胰蛋白酶

Preparation of an anti-autolysis recombinant trypsin based on heterologous expression and methylation

当前广泛应用的胰蛋白酶制剂,其主要来源为动物胰脏提取,具有原料受限、成本高和纯度低等缺点。此外,胰蛋白酶的自切降解也影响了其在储存和应用中的稳定性。【目的】通过毕赤酵母异源表达和甲基化修饰制备新型抗自切重组胰蛋白酶。【方法】通过基因重组技术实现猪胰蛋白酶酶原在毕赤酵母中的异源表达;通过单因素试验对酶原激活的温度、pH和时间进行考察和优化;通过探索甲基化修饰方法提高重组胰蛋白酶的抗自切性能。【结果】成功构建了胰蛋白酶酶原的毕赤酵母工程菌株,优化了酶原的肠激酶激活条件,考察了胰蛋白酶浓度、甲基化试剂添加量和反应时间对甲基化效果的影响,在胰蛋白酶浓度10 mg/mL,甲基化试剂添加量30μL,反应时间3h条件下,所获甲基化酶在酶活损失仅22%情况下,自切6h酶活保留率提高达到了79%,相比对照提高了约3.4倍,大幅提高了重组胰蛋白酶的抗自切性能。【结论】本研究基于异源表达和甲基化修饰成功制备了新型抗自切重组胰蛋白酶,该成果有助于提高我国胰蛋白酶的生产和应用水平。

The widely used trypsin is mainly extracted from animal pancreas,which has the disadvantages of limited raw materials,high costs,and low purity.In addition,the autolysis of trypsin affects its stability in storage and application.[Objective] To obtain an anti-autolysis recombinant trypsin by heterologous expression and methylation.[Methods] We employed gene recombination to realize the heterologous expression of porcine trypsinogen in Pichia pastoris.Furthermore,we conducted single factor experiments to investigate and optimize the temperature,pH,and time of enzyme activation and improved the anti-autolysis performance of the recombinant trypsin by methylation.[Results] The engineered strain of P.pastoris expressing trypsinogen was successfully constructed.Under the trypsin concentration of 10 mg/mL,methylation reagent addition of 30 μL,and reaction time of 3 h,the methylated trypsin showed the activity loss of only 22% and the relative activity of 79% after autolysis for 6 h,which was about 3.4 times higher than that of the control,suggesting that the anti-autolysis performance of the recombinant trypsin was greatly improved.[Conclusion] This study successfully produced a novel anti-autolysis recombinant trypsin by heterologous expression and methylation,which can improve the production and application of trypsin in China.