title: Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris.
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.
authors.affiliation: School of Biology andPharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China. jiangke.yang@gmail.com
authors.forename: Jiangke
authors.initials: J
authors.lastname: Yang
authors.forename: Xiangxiang
authors.initials: X
authors.lastname: Yan
authors.forename: Ribo
authors.initials: R
authors.lastname: Huang
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D003062:Codon; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D003062:Codon; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D008049:Lipase