解脂耶氏酵母脂肪酶LIP4和LIP5在毕赤酵母中的异源表达及酶学性质

Heterologous expression and characterization of Yarrowia lipolytica lipase 4 and lipase 5 in Pichia pastoris

【目的】克隆解脂耶氏酵母(Yarrowia lipolytica)脂肪酶LIP4和LIP5的cDNA序列,研究其基因结构,并实现其在毕赤酵母中的功能表达,以探讨其酶学性质。【方法】利用反转录PCR首次扩增LIP4和LIP5的编码基因,用SignalP 3.0分析其基因序列,然后分别构建胞内表达载体pPIC3.5K-Lip4、pPIC3.5K-Lip5和胞外表达载体pPIC9K-Lip4、pPIC9K-Lip5,将其转入毕赤酵母GS115中表达,以NTA树脂纯化酶蛋白,研究其酶学性质。【结果】cDNA序列测序结果显示两者均不含内含子,酶蛋白的氨基酸序列中含有典型脂肪酶的活性三联体结构和五肽保守区;酶学性质研究表明,两者的最适底物均为癸酸(C8)对硝基苯酚酯,最适pH为7.0,最适温度为40℃,但LIP4对pH和温度更敏感;两者均能被Ca2+激活,且LIP5还能为Mg2+激活,但均被Hg2+、乙二胺四乙酸(EDTA)和苯甲基磺酰氟(PMSF)强烈抑制。【结论】首次克隆了解脂耶氏酵母脂肪酶LIP4和LIP5编码基因,实现了其在毕赤酵母中的活性表达,并初步研究了其酶学性质,为上述脂肪酶的应用及进一步深入研究解脂耶氏酵母脂肪酶家族奠定了基础。

[Objective] To clone cDNA sequences of lipase 4（LIP4） and lipase 5（LIP5）,analyze gene structures and express them in Pichia pastoris so as to investigate their enzymatic characteristics.[Methods] We first cloned cDNA sequences of LIP4 and LIP5 by reverse transcription PCR and analyzed their gene structures by SignalP 3.0.Then,intracellular expression vectors pPIC3.5K-Lip4,pPIC3.5K-Lip5 and inducible secretion vectors pPIC9K-Lip4,pPIC9K-Lip5 were constructed.All vectors were transformed into Pichia pastoris GS115 by electroporation,resulting in a series of engineered strains.After fermentation and NTA-Ni resin purification,the enzymatic properties of LIP4 and LIP5 were examined.[Results] The cloned cDNA sequences revealed that there was no intron in both of Lip4 and Lip5.The two lipases both contained catalytic triads and conserved GHSLG motifs.Their optimal substrate,pH,temperature were respectively pNP-caprylate（C8）,7.0 and 40°C.The activities of LIP4 and LIP5 were 10.16 U/mg and 5.1 U/mg,respectively.It was found that LIP4 was more sensitive to the variations of pH and temperature than LIP5.LIP4 and LIP5 could both be stimulated by Ca2＋,besides LIP5 could also be activated by Mg2＋.They were both strongly inhibited by Hg2＋,Phenylmethanesulfonyl fluoride（PMSF） and Dithiothreitol（DTT）.[Conclusion] The cloning of Lip4 and Lip5,expression in P.pastoris and characterization of their properties would offer a solid basis for their large-scale production and future application.In addition,the results also enriched the data for a systematic research on the lipase gene family of Y.lipolytica.