黑曲霉C112脂肪酶基因的克隆及异源表达优化A novel lipase gene of Aspergillus niger C112 cloning, and heterologous expression optimization

• Published in: Zhongguo you zhi Vol. 50; no. 4; pp. 73 - 81
• Main Author: 徐鹏钧1,2，曾柏全1,2，肖志红3，李美群1,3，张轩1,3，李培旺3，赵茜彤1，颜丽安1 XU Pengjun1,2, ZENG Baiquan1,2, XIAO Zhihong3, LI Meiqun1,3, ZHANG Xuan1,3, LI Peiwang3, ZHAO Xitong1, YAN Li′an1
• Format: Journal Article
• Language: English
• Published: 中粮工科（西安）国际工程有限公司 01.04.2025
• Subjects: aspergillus niger c112; lipase; pichia pastoris; bioinformatics; cloning; 黑曲霉c112；脂肪酶；毕赤酵母；生物信息学；克隆
• ISSN: 1003-7969
• DOI: 10.19902/j.cnki.zgyz.1003-7969.240617

旨在为脂肪酶的改性及其工业化应用提供参考，基于黑曲霉C112菌株通过PCR扩增得到脂肪酶基因，对该基因进行了生物信息学分析，并运用基因工程技术构建了毕赤酵母GS115重组菌，对该重组菌诱导产生的脂肪酶进行了纯化，分析了该脂肪酶的酶学性质，并通过单因素实验和响应面实验对该重组菌产酶条件进行了优化。结果表明：该脂肪酶基因片段长度为894 bp，理论分子质量为31.79 kDa，其氨基酸序列中存在一个新的五肽基序RHSLG；该脂肪酶最适反应温度和pH分别为40 ℃和4，具有良好的热稳定性和pH稳定性；重组菌最佳产酶条件为YNB添加量10.50 mL（100 mL BMMY培养基中）、接种量4.3%、每隔24 h添加1%甲醇、培养时间2.8 d，优化条件下所得重组脂肪酶活性达到78.92 U/mL，是优化前的1.79倍。综上，经克隆、异源表达优化的重组脂肪酶稳定性强、酶活性高。To provide a reference for the modification of lipase and its industrial application, the lipase gene was amplified by PCR based on the Aspergillus niger C112 strain, and the gene was analyzed by bioinformatics. The recombinant Pichia pastoris GS115 strain was constructed by genetic engineering techniques,and the lipase induced by the recombinant strain was purified and its enzymatic properties were analyzed. In addition, the lipase production conditions with the recombinant strain were optimized through single factor experiment and response surface methodology. The results indicated that the length of the lipase gene fragment was 894 bp, and its theoretical molecular weight was 31.79 kDa. A new pentapeptide RHSLG was found in its amino acid sequence. The optimal reaction temperature and pH of the recombinant lipase were 40 ℃ and 4, respectively, which showed good thermal stability and pH stability. The optimal production conditions of the recombinant strain were 10.50 mL of YNB added in 100 mL BMMY medium, inoculation amount 4.3%, adding 1% methanol every 24 h, and a culture time of 2.8 d. Under the optimal conditions, the enzyme activity of the recombinant lipase reached 78.92 U/mL, which was 1.79 times that before optimization. In summary, the recombinant lipase after cloning and heterologous expression has strong stability and high enzyme activity.