密码子优化的人溶菌酶基因在毕赤酵母中的分泌表达及抗菌活性分析

人溶菌酶（human lysozyme，hLYZ）是一种天然活性蛋白质，具有抗菌性，目前主要应用于食品添加剂、动物饲料、药物治疗领域。根据毕赤酵母表达偏爱密码子特点，通过优化hlyz基因密码子，人工合成目的基因，构建了分泌表达载体pPIC9K-hlyz。通过电击转化法将pPIC9K-hly质粒重组至毕赤酵母GS115染色体中，经筛选得到重组毕赤酵母GS115-pPIC9K-hlyz。在28 ℃、pH 6.0、转速240 r·min^-1、1%甲醇诱导下表达120 h，发酵液上清酶活达到最高，为（2 414.9±108.1）U·mL^-1，蛋白浓度为166.7 μg·mL^-1。发酵液上清经过Sephadex G50纯化，获得较纯的hLYZ，经SDS-PAGE分析，在近14.7 kDa处显示单一条带。抗菌实验结果表明，重组hLYZ对革兰氏阳性菌的抗菌效果大于革兰氏阴性菌；重组hLYZ的抗菌效果是hLYZ标准品的10~20倍。

Secreted expression of codon-optimized human lysozyme gene in Pichia pastoris and antibacterial activity analysis

Human lysozyme (hLYZ) is widely used in food preservation, animal feed, biomedicine for its antibacterial properties. In this paper,lysozyme gene codon was optimized according to the codon bias of Pichia pastoris. Then pPIC9K-hlyz was transformed into GS115 chromosome through homologous recombination. Recombinant Pichia pastoris G-P-hlyz was successfully screened by 6.0 mg·mL^-1 G418 and colony PCR. At 28 ℃, pH 6.0, 240 r·min^-1,1% methanol, induced expression for 120 h, the recombinant protein was secretly expressed, the hLYZ activity of the fermentation broth reached (2 414.9±108.1) U·mL^-1, and the protein content was 166.7 μg·mL^-1. After the supermatant of fermentation broth was purified by Sephadex G50, hLYZ was purified as a single protein with a molecular weight of approximately 14.7 kDa which is verified by the SDS-PAGE analysis results. In addition, in vitro antibacterial experiments, hLYZ has better antibacterial effect on gram-positive bacteria than that on gram-negative bacteria. More importantly, the antibacterial activity is 10-20 times higher than the standard product.