蛋清溶菌酶基因的密码子优化及其在毕赤酵母中的分泌表达

作为一种天然的抑菌物质,溶菌酶对抗生素耐药菌有较强的杀伤作用，具有无耐药性、无残留等特点，被认为在诸如动物饲料、药品等领域可有效代替抗生素，改善滥用抗生素所引起的一系列问题，市场前景巨大。通过微生物发酵大规模生产重组溶菌酶符合市场发展趋势，由于目前生产的菌株表达量偏低，限制了溶菌酶的应用和产业化发展。为提高蛋清溶菌酶的发酵表达量，根据毕赤酵母密码子的偏爱性,对其编码基因进行密码子优化，并构建了真核分泌表达载体pPIC9K-coEWL，经遗传霉素G418抗性筛选后，获得了一株酵母转化子(G-p-coEWL)，其对G418的抗性浓度高达15 mg·mL^-1。在28℃、pH 6.0、转速240 r·min^-1和甲醇浓度1%的诱导条件下,酵母重组子G-p-coEWL经摇瓶培养72 h，实现了蛋清溶菌酶的分泌表达。在发酵上清液中，总蛋白浓度为 607 mg·L^-1，酶活达到677 U·mL^-1。此外，本实验还比较了葡聚糖凝胶柱Sephadex G-50和强酸性阳离子交换柱SP-Sepharose FF两种方法对目的蛋白的分离效果,得到 Sephadex G-50的分离效果更好，并在14.4 kDa处得到单一的溶菌酶条带。

Codon optimization of egg white lysozyme gene and its secretory expression in Pichia pastoris

As a natural antibacterial substance, lysozyme has a strong killing effect on antibiotic-resistant bacteria, without drug resistance and antibiotic residues in the environment. It is considered to be an alternative to antibiotics in certain fields, such as animal feed and medicines, which can effectively improve a series of problems caused by antibiotic abuse. The large-scale production of recombinant lysozyme by microbial fermentation is a trend in the future, because of the current expression level of the production bacteria is low which limits the application of lysozyme and the development of industry. In order to improve the expression level of egg white lysozyme(EWL) , the gene of which was optimized according to the preferred codon of Pichia pastoris， and the secretory expression vector pPIC9K-coEWL was constructed successfully. The restructuring vector is cut by SalI and transformed into Pichia pastoris. A yeast transformant which can grow in the presence of 15 mg·mL^-1 G418 is obtained after screening and confirmed by colony PCR. At 28 °C, pH 6.0, rotation speed of 240 r·min^-1 and methanol concentration of 1%, the culture of G-p-coEWL was carried out for 72 hours. The protein fraction of the fermentation supernatant is 607 mg·L^-1, and the enzyme activity reached 677 U·mL^-1. The egg white lysozyme is purified by Sephadex G-50 and SP-Sepharose FF respectively. The purification effect of Sephadex G-50 is better, and a single lysozyme band is obtained at 14.4 kDa.