Cloning, Expression and Activity Analysis of Lipase Gene from Rhizomucor miehei

Abstract

Abstract: Lipase gene cDNA fragment from Rhizomucor miehei(RML)was amplified by RT-PCR method. Expression vector RML-pPIC9K containing lipase gene was constructed and the gene was expressed in His4 mutant Pichia pastoris yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression product of RML gene was analysis by SDS-PAGE, and molecular weight of the expressed protein was estimated to be 39 kD. The activity of lipase was up to 84 U/mL determined by the method of titration of NaOH. The result indicated that RML gene was functionally overexpressed in Pichia pastoris.

Key words: Rhizomucor miehei, Lipase Pichia pastoris, Gene clone, Gene expression

Miao Changlin, Luo Wen, Liu Shuna, Lü Pengmei, Li Huiwen, Yang Lingmei, Yuan Zhenhong, Jiang Jianchun. Cloning, Expression and Activity Analysis of Lipase Gene from Rhizomucor miehei[J]. Biotechnology Bulletin, 2013, 0(6): 122-127.

References

[1] 张开平, 惠明, 田青, 等.微生物脂肪酶的应用领域及研究进展[J].河南工业大学学报:自然科学版, 2012, 33(1):90-94.
[2] 杨姣, 耿红冉, 江凌, 等.脂肪酶催化性能强化研究进展[J].现代化工, 2011, 31(9):23-27, 29.
[3] Torres C, Otero C. Part III. Direct enzymatic esterification of lactic acid with fatty acids [J]. Enzyme and Microbial Technology, 2001, 29(1):3-12
[4] Alcántara AR. Rhizomucor miehei lipase as the catalyst in the resolution of chiral compounds:an overview [J]. Chem Phys Lipids, 1998, 93(1-2):169-184.
[5] Krishna SH, Divakar S, Prapulla SG, et al. Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei [J]. J Biotechnol, 2001, 87:193-201.
[6] Han Z, Han S, Zheng S, et al. Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeastcell surface [J]. Appl Microbiol Biotechnol, 2009, 85:117-126.
[7] 孙金鹏, 钱圣一, 敬科举, 等.南极假丝酵母脂肪酶B在毕赤酵母的表达及学性质研究[J].厦门大学学报:自然科学版, 2011, 50(4):765-771.
[8] 杨梅, 温真, 林丽玉, 等.毕赤酵母蛋白表达系统研究进展[J].生物技术通报, 2011(4):46-51.
[9] 王乐乐, 喻晓蔚, 徐岩, 等.华根霉(Rhizopus chinensis)前导肽脂肪酶基因的克隆及其在Pichia pastoris中的表达[J].高技术通讯, 2009, 19(1):105-110.
[10] 孙金鹏, 钱圣一, 敬科举, 等.南极假丝酵母脂肪酶B在毕赤酵母的表达及学性质研究[J].厦门大学学报:自然科学版, 2011, 50(4):765-771.
[11] 阎金勇, 杨江科, 徐莉, 等.白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达[J].微生物学报, 2008, 48(2):184-190.
[12] Huge-Jensen B, Andreasen F, Christensen T, et al. Rhizomucor mie-hei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae [J]. Lipids:1989, 24:781-785.
[13] 李强.扩展青霉FS1884脂肪酶基因在毕赤酵母GS115中的表达和定向进化研究[D].福州:福建师范大学, 2009.
[14] Quyen DT, Schmidt-Dannert C, Schmid RD, et al. High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase [J]. Protein Expression and Purification, 2003, 28(1):102-110.
[15] Wu XY, J??skel?inen S, Linko WY. Purification and partial characterization of Rhizomucor miehei lipase for ester synthesis [J]. Appl Biochem Biotech, 1996, 59:145-158.
[16] Hoekman SK, Broch A, Robbins C, et al. Review of biodiesel composition, properties, and specifications [J]. Renewable & Sustainable Energy Reviews, 2012, 16(1):143-169.
[17] Li X, He XY, Li ZL, et al. Enzymatic production of biodiesel from Pistacia chinensis bge seed oil using immobilized lipase [J]. Fuel, 2012, 92(1):89-93.
[18] Yu M, Wen S, Tan T, et al. Enhancing production of Yarrowia lipolytica lipase Lip2 in Pichia pastoris [J]. Engineering in Life Sciences, 2010, 10(5):458-464.
[19] Yang J, Liu L. Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris [J]. Journal of Molecular Catalysis B, Enzymatic, 2010, 63(3/4):164-169.
[20] 王刚, 罗宇, 江正兵, 等.新型脂肪酶LipB52催化生物柴油 [J].华东理工大学学报:自然科学版, 2008, 34(2):197-202.