Expression and activity determination of codon optimized Antheraea pernyi lysozyme in Pichia pastoris

Abstract:

[Objective] In order to improve the expression of Antheraea pernyi lysozyme in Pichia pastoris and determinate the lysozyme activity. [Methods] According to the Pichia pastoris codon preference, we optimized the codons of Antheraea pernyi lysozyme gene and added six histidine residues at the 3′ terminal of the gene. The optimized gene cloned into pPIC9K vector. Then we transfered it into Pichia pastoris GS115 through the electroporation method. High copy transformants were screened by using different concentration gradient of G418 and realized the secretory expression by methanol induction. We separated and purificated Antheraea pernyi lysozyme by ammonium sulfate precipitation and Ni-chelating affinity chromatography. Then we determinated the Antheraea pernyi lysozyme activity by turbidimetric method and detected antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck by the agar diffusion method. [Results] The expression conditions were optimized, and the highest expression level of Antheraea pernyi lysozyme occurred when the recombinant strain was induced with 0.75% methanol under pH 7.0 at 25 °C for 96 h. The expression quantity reached 2.4 g/L and the specific activity of Antheraea pernyi lysozyme towards Micrococcus lysodeikticus was 23 970 U/mg. [Conclusion] Codon optimized Antheraea pernyi lysozyme was successfully produced as a single major secreted protein in Pichia pastoris GS115. Antheraea pernyi lysozyme exhibited antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck.