人溶菌酶基因在乳酸克鲁维酵母中的表达

人溶菌酶是一种天然广谱抑菌物质,在食品和医药工业有潜在应用前景。为获得高活性的人溶菌酶制剂,采用乳酸克鲁维酵母表达系统,对经密码子优化的人溶菌酶基因(h LYZ)进行分泌表达。将人工合成h LYZ插入到乳酸克鲁维酵母表达载体p KLAC1,构建重组载体p KLAC1-h LYZ,并用电脉冲法将SacⅡ线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过全细胞PCR鉴定,最后获得了一株多拷贝整合的基因工程菌h LYZ1。工程菌可以分泌表达分子量约14 ku的目的蛋白质,与预期大小相符。摇瓶发酵培养128 h,酶活最高达到1430 U/mL。抗菌活性检测结果显示,重组人溶菌酶对溶壁微球菌、大肠杆菌和枯草芽孢杆菌有较好的溶菌活性。本研究成功地在乳酸克鲁维酵母中表达了重组人溶菌酶,表达的蛋白具有较高的酶活性,试验结果为利用乳酸克鲁维酵母表达系统规模化生产重组人溶菌酶奠了基础。

Expression of the Human Lysozyme Gene in Kluyveromyces lactis

Human lysozyme, an enzyme with natural broad-spectrum antimicrobial activity, has potential applications in the food and pharmaceutical industries. To obtain human lysozyme preparations with high activity, a Kluyveromyces lactis yeast expression system was used for soluble expression of the human lysozyme gene (hLYZ) with optimized codons. An artificial hLYZ gene was cloned into the K. lactis expression vector pKLAC1, resulting in the recombinant vector pKLAC1-hLZY, which was then linearized with SacII and transformed into K. lactis GG799 by electroporation. Following whole-cell PCR, a multicopy recombinant strain (named hLYZ1) was obtained. The engineered strain secreted a target protein of approximately 14 ku in molecular weight, which agreed with that of the expected product. The maximum activity of hLZY in the culture supernatant reached 1430 U/mL after 128 h of culture. Antibacterial activity testing confirmed that the recombinant human lysozyme was effective against Micrococcus luteus, Escherichia coli, and Bacillus subtilis. In conclusion, a recombinant hLYZ with high enzyme activity was successfully expressed in K. lactis GG799, thereby providing a basis for studies on the large-scale production of recombinant human lysozyme using the K. lactis expression system.