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.
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.
Yen
Chih-Chung
CC
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan.
Malmis
Conmar C
CC
Lee
Guan-Chiun
GC
Lee
Li-Chiun
LC
Shaw
Jei-Fu
JF
D019943:Amino Acid Substitution; D055162:Biocatalysis; D002175:Candida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008049:Lipase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism*
D004952:Esters; D005656:Fungal Proteins; D014280:Triglycerides; D008049:Lipase