請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/69639
標題: Site-Specific Saturation Mutagenesis on Residues 132 and 450 of Candida rugosa LIP2 Enhances Catalytic Efficiency and Alters Substrate Specificity in Various Chain Lengths of Triglycerides and Esters
作者: Yen, C.C.
Malmis, C.C.
Lee, G.C.
Lee, L.C.
Shaw, J.F.
關鍵字: Candida rugosa LIP2
Pichia pastoris
site-specific saturation
mutagenesis
mutants
substrate specificity
pichia-pastoris
biochemical-characterization
lipase
gene
cylindracea
stability
purification
expression
isoenzyme
sequences
期刊/報告no:: Journal of Agricultural and Food Chemistry, Volume 58, Issue 20, Page(s) 10899-10905.
摘要: The catalytic versatility of recombinant Candida rugosa LIP2 has been known to have potential applications in industry. In this study, site-specific saturation mutagenesis on residues L132 and G450 of recombinant LIP2 has been employed to investigate the impact of both residues on substrate specificity of LIP2. Point mutations on L132 and G450 were done separately using mutagenic degenerate primer sets containing 32 codons to generate two libraries of mutants in Pichia pastoris. Replacements of amino acid on these mutants were identified as L132A, L132I, G450S, and G450A. In lipase activity assay, L132A and L132I mutants showed a shift of preference from short- to medium-chain triglyceride, whereas G450S and G450A mutants retained preferences as compared to wild-type LIP2. Among mutants, G450A has the highest activity on tributyrin. However, hydrolysis of p-nitrophenyl (p-NP) esters with L132A, L132I, and G450S did not show differences of preferences over medium- to long-chain esters except in G450A, which prefers only medium-chain ester as compared to wild-type LIP2. All mutants showed an enhanced catalytic activity and higher optimal temperature and pH stability as compared to wild-type LIP2.
URI: http://hdl.handle.net/11455/69639
ISSN: 0021-8561
文章連結: http://dx.doi.org/10.1021/jf1004034
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