Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1780-1788, 2011.
Article
in English
| WPRIM
| ID: wpr-304521
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pichia
/
Rhizopus
/
Substrate Specificity
/
Enzyme Stability
/
Recombinant Proteins
/
Codon
/
Protein Engineering
/
Chemistry
/
Protein Folding
/
Enzyme Precursors
Language:
English
Journal:
Chinese Journal of Biotechnology
Year:
2011
Type:
Article
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