Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme

Shu Wei Chang, Guan Chiun Lee, Jei Fu Shaw

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZαC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5′ end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.

Original languageEnglish
Pages (from-to)5831-5838
Number of pages8
JournalJournal of Agricultural and Food Chemistry
Volume54
Issue number16
DOIs
Publication statusPublished - 2006 Aug 9

Fingerprint

Candida rugosa
Pichia pastoris
Pichia
Candida
Lipase
codons
Codon
Genes
Enzymes
enzymes
Polymerase Chain Reaction
serine
Serine
Protein Isoforms
genes
Mutagenesis
Cloning
site-directed mutagenesis
lips
Lip

Keywords

  • Candida rugosa lipase
  • Codon optimization
  • Isoforms
  • N-terminal peptide
  • Pichia pastoris

ASJC Scopus subject areas

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

Cite this

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title = "Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme",
abstract = "Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZαC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5′ end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.",
keywords = "Candida rugosa lipase, Codon optimization, Isoforms, N-terminal peptide, Pichia pastoris",
author = "Chang, {Shu Wei} and Lee, {Guan Chiun} and Shaw, {Jei Fu}",
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AU - Chang, Shu Wei

AU - Lee, Guan Chiun

AU - Shaw, Jei Fu

PY - 2006/8/9

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N2 - Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZαC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5′ end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.

AB - Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZαC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5′ end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.

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