Screening, Cloning And Expression Lipases From Various Sources And Their Enzymatic Properties

Lipase is one of the most important industrial enzymes. In order to meet the increasing needs, lipase research mainly focused on the development of new lipases and redesingning the existing lipases. This study is designed to screen and clone the lipases with potential applications, and realize highly effective expression to prompt their industrial application.Three lipase-producing strains were obtained through the screening procedure. They belong to the Proteus sp., Bacillus pumilus and Psedomonas aeruginosa bacteria respectively. Lipases from these three strains were cloned and managed to build several different recombinant plasmids. Through enzyme activity determination, optimal expression recombinant plasmid of every lipase gene was obtained. Their enzyme properties were also investigated in detail.The lipase gene lipK19from Proteus consisted of864nucleotides, encoding a protein of287amino acids and having92%homology blasted with lipase gene K80, PVL which had been reported. Plasmid pET28a was selected as the expression plasmid. The lipase activity attained to1500U/L. This lipase had an optimal temperature of40℃and an optimal pH of9with a certain tolerance in alkaline pH rang. Ca2+promoted the enzyme activity, while EDTA and surfactants had strong inhibitory action.The lipase had a certain organic tolerance.The lipase gene lipS6from Bacillus pumilus consisted of648nucleotides, encoding a protein of215amino acids and having96%homology blasted with lipase B26which had been reported. Plasmid pET32a was selected as the expression plasmid. The lipase activity was up to2000U/L. This lipase had an optimal temperature of35℃and an optimal pH of9, while the thermostability and the pH tolerance were not very good. Ca2+promoted the enzyme activity. The most suitable acid and alcohol substrates for esterification were tetradecanoic acid and butyl alcohol respectively.The Pseudomonas aeruginosa lipase gene lipA consisted of936nucleotides, encoding311amino acids. Chaperon lipB size was1023bp, encoding340amino acids. Both had99%homology with reported sequence. Selected pACYCDuer-lipA-lipB as the recombinant expression plasmid and lipase activity was up to8500U/L. The lipase had an optimal temperature of30℃and an optimal pH of9with a strong pH tolerance. Ca2+promoted the enzyme activity. The most suitable acid and alcohol substrates for esterification were toctanoic acid and hexanol respectively.