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

Abstract:

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 ofP.pastoris expressing trypsinogen was successfully constructed.Under the trypsin concentration of10 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.

Key words:trypsin;methylation;anti-autolysis