The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH7 and a broad temperature optimum in the range 30–50°C. The enzyme retained 80% of residual activity after being heated at 70°C for 10min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12–C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.
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September 2002
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Research Article|
September 01 2002
Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris
Guan-Chiun LEE;
Guan-Chiun LEE
∗Institute of Botany, Academia, Sinica, Nankang, Taipei, Taiwan 11529,
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Li-Chiun LEE;
Li-Chiun LEE
†Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 20224
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Vasyl SAVA;
Vasyl SAVA
1
∗Institute of Botany, Academia, Sinica, Nankang, Taipei, Taiwan 11529,
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Jei-Fu SHAW
Jei-Fu SHAW
2
∗Institute of Botany, Academia, Sinica, Nankang, Taipei, Taiwan 11529,
2To whom correspondence should be addressed (e-mail boplshaw@gate.sinica.edu.tw).
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Publisher: Portland Press Ltd
Received:
March 13 2002
Revision Received:
May 15 2002
Accepted:
May 21 2002
Accepted Manuscript online:
May 21 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (2): 603–611.
Article history
Received:
March 13 2002
Revision Received:
May 15 2002
Accepted:
May 21 2002
Accepted Manuscript online:
May 21 2002
Citation
Guan-Chiun LEE, Li-Chiun LEE, Vasyl SAVA, Jei-Fu SHAW; Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris. Biochem J 1 September 2002; 366 (2): 603–611. doi: https://doi.org/10.1042/bj20020404
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