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

Chih Chung Yen, Conmar C. Malmis, Guan Chiun Lee, Li Chiun Lee, Jei Fu Shaw

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Abstract

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.

Original languageEnglish
Pages (from-to)10899-10905
Number of pages7
JournalJournal of Agricultural and Food Chemistry
Volume58
Issue number20
DOIs
Publication statusPublished - 2010 Oct 27

Fingerprint

Candida rugosa
Mutagenesis
Candida
substrate specificity
Substrate Specificity
Site-Directed Mutagenesis
Chain length
catalytic activity
mutagenesis
Esters
Triglycerides
esters
triacylglycerols
mutants
Substrates
Pichia
Lipase
Point Mutation
Codon
Libraries

Keywords

  • Candida rugosa LIP2
  • Mutants; substrate specificity
  • Pichia pastoris
  • Site-specific saturation mutagenesis

ASJC Scopus subject areas

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

Cite this

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title = "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",
abstract = "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.",
keywords = "Candida rugosa LIP2, Mutants; substrate specificity, Pichia pastoris, Site-specific saturation mutagenesis",
author = "Yen, {Chih Chung} and Malmis, {Conmar C.} and Lee, {Guan Chiun} and Lee, {Li Chiun} and Shaw, {Jei Fu}",
year = "2010",
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T1 - 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

AU - Yen, Chih Chung

AU - Malmis, Conmar C.

AU - Lee, Guan Chiun

AU - Lee, Li Chiun

AU - Shaw, Jei Fu

PY - 2010/10/27

Y1 - 2010/10/27

N2 - 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.

AB - 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.

KW - Candida rugosa LIP2

KW - Mutants; substrate specificity

KW - Pichia pastoris

KW - Site-specific saturation mutagenesis

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