Construction and Activity Screening of Lipase Mutagenesis Library from Candida rugosa

• Main Authors: Ming-Li Su, 蘇明俐
• Other Authors: Shye-Jye Tang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/08728813230506557531

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 100 === Candida rugosa lipases (CRLs) have been used in a wide catalytic reaction in bioindustry. To investigate the relationship of enzyme activities with protein sequence and to screen engineering lipase with higher enzymatic activity, we constructed three libraries containing mutation at CRL alcohol binding sites. Amino acids in the sites were replaced into hydrophobic amino acid or the similar with properties. Libraries were constructed by Quick Change Mutagenesis PCR using DNA template with low lipase activity. The libraris were transformed into Sacchromyces cerevisiae for selection. Results indicated library giving expected mutantions. After transformation, colonies were observed clear zone in tributyrin plates. These results showed that we have set up a reliable and feasible strategy to construct and screen lipase library. In the future we are able to construct new mutants using template with different lipase activity and to obtain engineering lipases with higher lipase activity.