Molecular Modification And High-efficiency Expression Of Human Lysozyme

Human lysozyme（human lysozyme,h LYZ）has attracted extensive attention from all walks of life in recent years due to its natural and efficient antibacterial ability and wide range of uses.However,h LYZ has limited sources and low extraction activity,which cannot meet the growing market demand.Using bioengineering methods to improve h LYZ’s enzyme activity and yield can effectively solve these problems.In this study,the lysozyme molecule was modified to enhance its enzyme activity,and to achieve high-efficiency expression in the Pichia pastoris expression system,the main results are as follows:（1）Exploring the potential mutation sites of h LYZ.Through the molecular docking simulation of human lysozyme HZM（2R-K）and the substrate（NAG）₄,the transformation potential of Val110 that hinders docking to Ser110 was found and the mutant V110S was designed.Sequence comparison of HZM（2R-K）and similar lysozyme proteins revealed that HZM（2R-K）has a low utilization rate of amino acid selection at 4 positions in other sequences.The mutants A47N,L79I,N88D,and R122S were designed with the amino acids that were highly adopted and well conserved as the mutational orientation,respectively.（2）Construction of h LYZ mutant strains and expression in shake flasks.h LYZ recombinant strain X33-p PICZ-HZM（2R-K）was constructed and subjected to expression at shake flask level with the highest level of enzyme activity expression being 1226 U·m L^-1.Five mutant recombinant strains（A47N,L79I,N88D,V110S and R122S）were constructed and expressed at shake flask levels,and the best performing mutant N88D showed enzyme activity of 3066 U·m L^-1,which were 2.50 times that of the original strain.Different combinations of single mutation sites were constructed into multilocus mutants,the mutant strain X33-p PICZ-HZM（2R-K）-N88D/V110S-2 with the highest viability was obtained,and the enzyme activity was 6213 U·m L^-1,which was 2.03-fold that of the N88D mutant and 5.07-fold that of the original strain.The shake flask expression supernatants from the optimal strain of all double mutants were tested for inhibition plate,and it was found that there was clear inhibition zones in Staphylococcus aureus and Micrococcus lysodeikticus,and the size of inhibition zone was positively correlated with the enzyme activity.（3）Expression and optimization of h LYZ in a fermenter.X33-p PICZ-HZM（2R-K）-N88D/V110S-2 was amplified and expressed in a 3 L fermenter,and the final protein content was 0.95 mg·m L^-1,and the final enzyme activity was 152000 U·m L^-1,24.46 times the expression in shake flasks.After the methanol induction medium was replaced with the dual carbon source induction medium,the final protein content reached 1.18 mg·m L^-1,and the final enzyme activity reached 222666 U·m L^-1,which was 0.3 times higher than before optimization;The induced OD value was optimized,and the effect was the best when the induced OD₆₀₀=150,the final protein content reached 1.53 mg·m L^-1,and the final enzyme activity reached304000 U·m L^-1,which was twice that before optimization.（4）Co-expression of molecular chaperones to improve h LYZ expression level.Hac1 and Pdi chaperones were constructed and fused into X33-p PICZ-HZM（2R-K）-N88D/V110S-2 for co-expression.The optimal strain X33-p PICZ-HZM（2R-K）-N88D/V110S-2-Hac1-4 fused with Hac1 had an enzyme activity of 6833 U·m L^-1,which was 5.57 times that of the original strain.The enzyme activity of the optimal strain X33-p PICZ-HZM（2R-K）-N88D/V110S-2-Pdi-2shake flask was 6400 U·m L^-1,which was 5.22 times that of the original strain.The tandem expression of Hac1 and Pdi does not work well,which highest enzyme activity was only4866 U·m L^-1.In the inducible expression in 3 L fermenter,the expression level of X33-p PICZ-HZM（2R-K）-N88D/V110S-2-Hac1-4 was increased,and the final protein content reached1.65 mg·m L^-1,The final enzyme activity was 330666 U·m L^-1,which was 2.18 times that of the initial tank activity and 53.22 times that of the shake flask expression,which further increased the expression level of h LYZ.