Optimizing The Medium Of Candida Rugosa Lipase Production And Cloning Of Candida Rugosa Lipase 1

Candida rugosa (formerly Candida cylindarcea) lipase is widely used inchemical pharmacy and food industry. It is one of the most popular commerciallipases in the world. Several isozymes encoded by the lip gene family, namelylip1 to lip5, possess distinct thermal stability and substrate specificity. They areall of great potential values in many areas.Several lipase genes that revealed a high homology (between 60% and 70%)have been cloned from C. rugosa, namely lip1 to lip5. So they share similarproperties and it is very hard to separate them from each other. The currentcommercial lipases are quite impure samples with variable amount of proteins,lactose, different concentrations of lipases and, of course, different isoenzymaticprofiles depending on the sample. The erratic biocatalytical results led thescientists to consider whether the production of lipases, and furthermore theisoenzyme biosynthesis and secretion, could be modulated depending on thefermentation conditions.In the flask experiments, we explore the factors that might influence thelipase production. Enlightened by the published papers, we picked up 8 factorsand carried out the plackett burman experiment. Results revealed that peptoneand maltose were crucial to lipase production. And the sequent steepest ascentexperiment confined the range of the proportion of peptone and maltose. Later,response surface methodology helped us to determine the optimal mediumcomposition: peptone 24g/L;maltose 14.2 g/L;glucose 1 g/L,K2HPO4 10 g/L,yeast extract 1g/L. The lipase production of this medium reaches 570 U/mL,which will be of great help for further industry production.Among the five isoenzymes, Lip 1 possessed the greatest catalytic activityand it is the major component in the commercial samples. Due to the similarproperties of these isoenzymes, cloning and expression of the individual lipgenes is thought to be the most suitable approach for the production andcharacterization of pure C. rugosa lipase isoform with optimized properties forbiocatalytic application. The PCR products with Candida rugosa genome as thetemplate would be impure with all possible isoenzymes genes. However,wefound these five isoenzyme genes can be digested by NcoI into differentfragments. In this way, we obtained the Candida rugosa lip 1 gene and thesequence has been confirmed by sequencing.The production of native CRL is poor, which results in the relatively highproduction cost. Therefore, we want to enhance the production by heterologousexpression. Unfortunately, Candida rugosa utilizes a non-universal code system,that is, the triplet CTG, a universal code for Leu, is read as Ser. CTG encodes 19Ser residues in Candida rugosa lip1, including the catalytic site. So, theheterologous expression of this gene may result in the inactive production of thisprotein. We altered the 19 Ser encoded by CTG by overlap extension PCR. Uptill now, sequence analysis indicated that 17 of them had been altered.In this study, we found a basis to produce the native Candida rugosalipase and made preparations for further heterologous expression. This work isof practical and theoretical value.