Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites

Li Chiun Lee, Yu Ting Chen, Chih Chung Yen, Teresa Ching Yn Chiang, Shye Jye Tang, Guan-Chiun Lee, Jei Fu Shaw

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.

Original languageEnglish
Pages (from-to)5103-5108
Number of pages6
JournalJournal of Agricultural and Food Chemistry
Volume55
Issue number13
DOIs
Publication statusPublished - 2007 Jun 27

Fingerprint

Candida rugosa
Candida
tributyrin
substrate specificity
Substrate Specificity
Lipase
binding sites
engineering
Triglycerides
Candida cylindracea
triacylglycerols
Binding Sites
Pichia pastoris
Substrates
Chain length
esters
long chain triacylglycerols
Esters
medium chain triacylglycerols
point mutation

Keywords

  • Candida rugosa
  • Lipase
  • Pichia pastoris
  • Saturation mutagenesis
  • Substrate specificity

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Food Science
  • Chemistry (miscellaneous)

Cite this

Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites. / Lee, Li Chiun; Chen, Yu Ting; Yen, Chih Chung; Chiang, Teresa Ching Yn; Tang, Shye Jye; Lee, Guan-Chiun; Shaw, Jei Fu.

In: Journal of Agricultural and Food Chemistry, Vol. 55, No. 13, 27.06.2007, p. 5103-5108.

Research output: Contribution to journalArticle

Lee, Li Chiun ; Chen, Yu Ting ; Yen, Chih Chung ; Chiang, Teresa Ching Yn ; Tang, Shye Jye ; Lee, Guan-Chiun ; Shaw, Jei Fu. / Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites. In: Journal of Agricultural and Food Chemistry. 2007 ; Vol. 55, No. 13. pp. 5103-5108.
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abstract = "Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.",
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AU - Tang, Shye Jye

AU - Lee, Guan-Chiun

AU - Shaw, Jei Fu

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AB - Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.

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