定点突变提高土曲霉Aspergillus terreus脂肪酶的催化活性
Improvement of catalytic activity of Aspergillus terreus lipase by site-directed mutagenesis
投稿时间:2018-01-24  
DOI:  10.13345/j.cjb.180040
中文关键词:土曲霉脂肪酶,毕赤酵母,同源建模,定点突变,催化活性,分子对接
英文关键词:Aspergillus terrus lipase, Pichia pastoris, homologous modeling, site-directed mutagenesis, catalytic activity, molecular docking
基金项目:国家自然科学基金 (No. 31671799),江苏省“六大人才高峰”项目 (No. NY-010),江苏省“333工程”项目 (No. BRA2015316),国家高技术研究发展计划 (863计划) (No. 2012AA022207) 资助。
作者单位E-mail
张晓凤 江南大学 生物工程学院 工业生物技术教育部重点实验室江苏 无锡 214122  
喻晓蔚 江南大学 生物工程学院 工业生物技术教育部重点实验室江苏 无锡 214122 bioyuxw@aliyun.com 
徐岩 江南大学 生物工程学院 工业生物技术教育部重点实验室江苏 无锡 214122  
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中文摘要:
      本研究旨在利用理性设计的方法来提高来源于土曲霉Aspergillus terreus的酸性脂肪酶ATL的催化活力。通过同源比对,选择脂肪酶盖子区域和底物结合口袋域中的位点进行定点突变,得到8种ATL的突变脂肪酶。结果发现,盖子区域突变酶ATLLid与底物结合口袋域突变酶ATLV218W的催化活性显著提高。ATLLid和ATLV218W对底物对硝基苯酚月桂酸酯p-nitrophenyl laurate (p-NPL) 的催化活性最高,kcat值较ATL分别提高了39.37倍和50.79倍,kcat/Km值较ATL分别提高了2.85倍和8.48倍。与ATL相比,ATLLid和ATLV218W的热稳定性略有下降,最适pH为5.0,pH 4.0–8.0具有较好的稳定性,说明突变未对ATL的嗜酸耐酸特性产生影响。通过同源建模模拟及分子对接技术分析底物p-NPL与酶分子间的相互作用,解析了ATLLid和ATLV218W催化活性提高的机理。
英文摘要:
      The catalytic activity of Aspergillus terreus lipase (ATL) was improved by rational design. According to the sequence analysis and homologous modeling, several amino acids involved in the lid domain and substrate binding pocket domains of the acidic lipase ATL were mutated by site-directed mutagenesis, and eight mutants were constructed. These mutants and the wild type lipase ATL were expressed in Pichia pastoris GS115 and the enzymatic properties were characterized. The mutants ATLLid and ATLV218W exhibited higher hydrolytic activity than ATL towards p-nitrophenyl laurate. The kcat values of ATLLid and ATLV218W towards p-nitrophenyl laurate were 39.37- and 50.79-fold higher, and the kcat/Km values were 2.85- and 8.48-fold higher than the wild type, respectively. Although thermostability of these mutants decreased slightly, ATLLid and ATLV218W still exhibited the maximum activity at pH 5.0 and high stability in a broad range of pH (4.0–8.0), which were similar to the wild type. Using homologous modeling and molecular docking technology the mechanism for the improvement of catalytic activity was analyzed. These findings not only shed light on the relationship between the lid domain/substrate binding pocket domain and catalytic activity but also provided comprehensive scheme for further engineering to gain more efficient lipases.
张晓凤,喻晓蔚,徐岩.定点突变提高土曲霉Aspergillus terreus脂肪酶的催化活性[J].生物工程学报,2018,34(7):1091~1105
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