Study On Recombinant Engineered Yeast Of Expressing Human Lysozyme And Activepowder Preparation

Lysozyme is a alkalic protein that can hydrolyze cell walls and has a great value in clinical medicine and food preservation because of strengthening the immune system and antibacterial effects.According to different source,Lysozyme is able to be categorized into chicken egg-white lysozyme、goose egg-white lysozyme and phage lysozyme.Human lysozyme belongs to c-lysozyme and encodes 148 amino acids.The mature peptide contains 130 amino acids and the quality of the relative molecular weight is 14.7 kD.In this experiment,we extracted human lysozyme gene from human placenta and removaled the signal peptide,then constructed a new expression vector pPICZaA-hLYZ and transformed it into Pichia pastoris X-33.The recombinant yeast was induced by methanol for 48 hours,then we detected the protein expression by SDS-PAGE and activity by Lywallzyme Micrococcus antibacterial test.We mutated leucine and glutamic acid into proline and histidine by site-directed mutagenesis and it was found that the protein expression and stability of enzyme activity was increased.This result showed that the mutation maybe conducive to cut the carrier signal peptide and transport protein to cross cell membranes.In recent years,there are a lot of production in yeast secretory expression system reported but few in yeast intracellular expression system.Our study aims to construct a new type engineered yeast of expressing lysozyme by intracellular expression to develop new animal feed additive so that avoiding the high cost and unstable activity of using lysozyme preparations directly.The method is that cloning human lysozyme gene into the intracellular expression plasmid pPICZA,then transforms the recombinant vector into Pichia pastoris X-33 by electronic transformation,subsequently the recombinant yeast is cultured in BMMY flask to induce the expression by methanol and detect enzyme activity.Single factor experiment and orthogonal experiment can determine the medium component and cultural condition optimization.The result shows that we have succeed in expressing protein and the activity is 1920 U.The cost of BMGY/BMMY is too high to apply in industrial production,so we optimized culture medium ingredients to save production cost.The experiment selected wort and silkworm chrysalis powder as yeast culture to provide necessary carbon source and nitrogen source,and nutrition salt provide trace elements which was essential for the growth of yeast.Orthogonal experiment result showed that the order of R value:pH in growth phase was more important than methanol concentration in induction phase,whereas the influence of inoculate quantity was minimal,the optimum culture condition was initial pH of cell growth 6.0,initial methanol inducing concentration 1%and initial inoculate quantity 3%.We made the engineered yeast into active powder by Vacuum Freezing and Drying Technology and the lysozyme activity of powder was 1320U and the yeast survival rate was 88.1%.The research was prepared for the development and utilization of new type feed additive.