High-level Expression Of Human Lysozyme In Pichia Pastoris

As a natural antibacterial agent,human lysozyme(hLYZ)has potential application value in many fields.However,raw materials and purification cost limited its application.This thesis focused on using Pichia pastoris expression system to produce hLYZ,to reduce the operation cost of hLYZ and to solve the above probelms.The main resluts were as follows:(1)The codons of a-factor signal and hLYZ gene were optimized as a whole and a recombinant expression vector pPICZ-OptaF+hLYZ was constructed.By transforming,recombinant K1 was obtained.In shaking flasks,the total protein concentration and enzyme activity reached 260 mg·L-1 and 12,937 U·mL-1,respectively.In 5 L fermentor,the total protein concentration and enzyme activity reached was 1.71 g·L-1 and 262,152 U·mL-1.(2)Based on the optimization,39 nucleotides were inserted into a-factor signal sequence,another expression vector pPICZ-Ehn?F+hLYZ was constructed and recombinant K4 was obtained.In shaking flasks,total protein concentration and enzyme activity were 320 mg·L-1 and 16,974 U·mL-1,respectively,and both of indexes were higher that those obtained by K1 strain.In 5 L fermentor,the total protein concentration reached 2.54 g·L-1,which was 48.5%higher than that of K1.However,total enzyme activity was only 258,712 U·mL-1,which was slightly lower than that of K1 strain.(3)Investigating the expression performance of K4 under high cell density(?100 g-DCW·L-1)induction.Inducing K4 at methanol concentration of 5 g·L-1,total protein concentration reached 3.02 g·L-1 after 54 h induction.However,severe protein degradation declined the concentration to 2.07 g·L-1 after 68 h induction.Using methanol/sorbitol co-feeding strategy while controlling methanol concentration at 5 g·L-1,and ingoring dissolved oxygen concentration(DO).After 68 h induction,total protein concentration reached 2.88 g·L-1,no protein degradation occurred.Under the two induction strategies,the highest total enzyme activity levels were 324,072 U·mL-1 and 290,438.8 U·mL-1,respectively.(4)Using Bgl ? and BamH ?,multiple tandem of hLYZ gene expression vectors were designed.Recombinant strains of K-2-2 and K-4-4 were successfully obtained.In shaking flasks,the highest total protein concentrations of K-2-2 and K-4-2 were 193.0 mg·L-1 and 185.3 mg·L-1,respectively.The corresponding total enzyme activities were 5,256 U·mL-1 and 5,424.U-mL-1.In 5 L fermentor,K-2-2 was induced for 73 h when initating the induction at lower cell density(?50 g-DCW·L-1)and 65 h when intinating induction at higher cell density(?100 g-DCW.L-1),the total protein concentrations were only 0.79 g·L-1 and 0.93 g·L-1,respectively.The total enzyme activities were 70,333 U·mL-1 and 71,529 U·mL-1,respectively.The protein concentration and enzyme activity were only 50%of K1.After inducing expression for 10 h,K-4-2 lost its metabolic activity and fermentations failed.The hLYZ expression performance of recombinants which transformed by the multiple tandem of hLYZ gene was low.