Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris.
- Author:
Jiangke YANG
1
;
Xiangxiang YAN
;
Ribo HUANG
;
Bo ZHANG
Author Information
1. School of Biology andPharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China. jiangke.yang@gmail.com
- Publication Type:Journal Article
- MeSH:
Codon;
Enzyme Precursors;
biosynthesis;
chemistry;
genetics;
Enzyme Stability;
Lipase;
biosynthesis;
genetics;
metabolism;
Pichia;
enzymology;
genetics;
Protein Engineering;
methods;
Protein Folding;
Recombinant Proteins;
biosynthesis;
genetics;
Rhizopus;
enzymology;
genetics;
Substrate Specificity
- From:
Chinese Journal of Biotechnology
2011;27(12):1780-1788
- CountryChina
- Language:English
-
Abstract:
Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
