Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: Comparison of glycosylation, activity, and stability

Shye Jye Tang, Jei Fu Shaw, Kuang Hui Sun, Guang Huan Sun, Terng Yuan Chang, Ching Kai Lin, Yuh Chih Lo, Guang Chiun Lee

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59 Citations (Scopus)

Abstract

Although Candida rugosa utilizes a nonuniversal serine codon (CUG) for leucine, it is possible to express lipase genes (LIP) in heterologous systems. After replacing the 19 CUG codons in LIP4 with serine codons by site-directed mutagenesis, a recombinant LIP4 was functionally overexpressed in Pichia pastoris in this study. This recombinant glycosylated lipase was secreted into the culture medium with a high purity of 100 mg/liter in a culture broth. Purified recombinant LIP4 had a molecular mass of 60 kDa, showing a range similar to that of lipase in a commercial preparation. Since LIP4 has only a glycosylation site at position Asn-351, this position may also be the major glycosylation site in C. rugosa lipases. Although the thermal stability of recombinant LIP4 significantly increased from 52 to 58°C after glycosylation, there were no significant differences in the catalytic properties of recombinant glycosylated lipase from P. pastoris and the unglycosylated one from Escherichia coil. These two recombinant LIP4s showed higher esterase activities toward long-chain ester (C16and C18) and exhibited higher lipase activities toward unsaturated and long-chain lipids. In addition, LIP4 does not show interfacial activation as compared with LIP1 toward lipid substrates of tributyrin and triolein. These observations demonstrated that LIP4 shows distinguished catalytic activities with LIP1 in spite of their high sequence homology.

Original languageEnglish
Pages (from-to)93-98
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume387
Issue number1
DOIs
Publication statusPublished - 2001 Mar 1

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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