Site-Specific Saturation Mutagenesis

Site-Specific Saturation Mutagenesis - J. Agric. Food Chem....

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
pubs.acs.org/JAFC Published on Web 09/27/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 10899–10905 10899 DOI:10.1021/jf1004034 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 C HIH -C HUNG Y EN , †,§, ) C ONMAR C. M ALMIS , †,#, , z G UAN -C HIUN L EE , ^ L I -C HIUN L EE ,* ,† AND J EI -F U S HAW * ,†,#, X Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan, § Institute of Plant Biology, National Taiwan University, Taipei, 10617, Taiwan, # Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan, ^ Department of Life Science, National Taiwan Normal University, Taipei, 11677, Taiwan, X Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan, and z Taiwan International Graduate Program, Academia Sinica, Taipei, 115, Taiwan. Co-first authors (these authors contributed equally to this work). The catalytic versatility of recombinant Candida rugosa LIP2 has been known to have potential appli- cations 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; Pichia pastoris ; site-specific saturation mutagenesis; mutants; substrate specificity INTRODUCTION For over 150 years, lipases have been extracted, purified, and characterized. Lipases belong to a class of biocatalysts with an unsurpassable role in swiftly growing biotechnology and have remarkable ability to carry out novel reactions in both aqueous and nonaqueous media ( 1 ). These enzymes are involved in a wide variety of reactions on fatty acids, alcohols, or esters for foods, flavors, fragrances, and pharmaceuticals ( 2 ). The progress at this stage in producing lipases is largely dependent on the develop- ment of molecular biology tools. Among many lipase-producing types of yeast,
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 7

Site-Specific Saturation Mutagenesis - J. Agric. Food Chem....

This preview shows document pages 1 - 2. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online