Expression And Activity Determination Of Codon Optimized Antheraea Pernyi Lysozyme Gene In Pichia Pastoris

Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, as a defensive effector in innate immunity, plays an important role in combating microbial infections by rupturing the β-1,4-glycosidic bond between the alternating N-acetyl muramic acid and N-acetyl glucosamine residuals in bacterial peptidoglycan, the major bacterial cell wall polymer, thus damaging cell wall and causing bacterial cell lysis. Therefore, lysozyme plays a crucial role in the defense system of the most biological organism. In addition, Lysozyme can be used as a kind of important digestive enzyme for the growth of invertebrates by C and N elements decomposition bacteria. It can also inactivate the virus through combining with negative viral protein directly and DNA, RNA and apoprotein formation of salt. Lysozyme has many other functions, including reproduction, growth stimulation, antivirus, immunomodulatory, anti-inflammatory and anti-tumor. Therefore, lysozyme is widely used in the field of feed, food, medicine, biology and so on. At present, large amounts of lysozyme can be found in egg white. The traditional separation and purification method of lysozyme is crystallization. This method is not only low yield, and the raw material can not be recycled. This situation limits the development and application of lysozyme industry.Antheraea pernyi, as a unique economic insect to China, produces a variety of resistance components after inducing, which includes lysozyme. Just like egg white lysozyme, Antheraea pemyi lysozyme also belong to the lysozyme c gene family. The mature peptide of Antheraea pernyi lysozyme contains 120 amino acids, and the predicted molecular weight is 13987.8 Da. Evidence suggested that it contains four disulfide bond, and the activity center is Glu32 and Asp50. We optimized and synthesized the gene of Antheraea pernyi lysozyme on the basis of the preferred codon usage of Pichia pastoris. Otherwise, the gene was fused in frame with a string of nucleotides encoding six histidine residues at the 3’terminal. Antheraea pernyi lysozyme was successfully produced as a single major secreted protein in Pichia pastoris GS115. It was separated and purified by using the Ni-chelating affinity chromatography. At the same time, we determinated the antibacterial activity. 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 ℃ for 96 h. Under the optimized fermentation condition, the expression quantity reached 2.4 g/L and the specific activity of Antheraea pernyi lysozyme towards Micrococcus lysodeikticus was 22354 U/mg. Antheraea pernyi lysozyme exhibited antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck by the agar diffusion method. We also studied the optimum temperature and pH of Antheraea pernyi lysozyme. These showed that Antheraea pernyi lysozyme has a certain temperature and acid alkali resistance. These conclusions are proved that Antheraea pernyi lysozyme is a kind of widely used lysozyme.