Overexpression of lipases from Candida rugosa for industrial applications

Abstract

Lipases are the most studied enzymes and the most used in industry. In this work, we were interested in four lipases of industrial interest. There are belonging to the lipase family of Candida rugosa (wild type, Lip1, Lip3 and Lip4). We first tested a new expression system, a specific strain of Yarrowia lipolytica, for expression of C. rugosa. This strain JMY1212 enables integration to be targeted to a special locus of the Y. lipolytica genome. We demonstrated that it is the first expression system in which it is possible to compare statistically variant activities directly from the supernatant of the culture. Three lipases of C. rugosa were cloned successfully in this strain and their activities and specificities with respect to fatty acid chain lengths were studied. Lip1 and Lip3 have specificity for the fatty-acids of medium chain (C8-C12) whereas Lip4 prefers C18: 1. Moreover, for the first time, purification, from a mixture of ethyl esters issued from fish oil, polyunsaturated fatty acids (PUFAs); cis-5, 8, 11, 14, 17-eicosapentaenoic acid (EPA) and cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA), molecules with health benefits, was realised with the three C. rugosa lipases, separately. Whatever the enzyme the recovery of DHA is superior to 90 % (97, 100 and 93 % for Lip1, Lip3 and Lip4 respectively). The maximal DHA purity ~60 % was obtained with Lip3 and Lip4, with an initial ethyl ester mixture containing 25% DHA. A remarkable difference between these enzymes lies in the fact that Lip4 is able to better hydrolyse the EPA esters (60% against 13% and 16% respectively for Lip1 and Lip3). Lip4 is also able to hydrolyse DHA (7% against 3 and 0 % for Lip1 and Lip3 respectively). The second part of this work was devoted to the improvement of the enantioselectivity of the two enzymes studied with respect to the resolution of a racemic mixture of pharmaceutical industry, the R, S esters of 2-bromo aryl acetic acid. The 3 lipases of C. rugosa proved to be remarkable from the point of view of enantioselectivity. In spite of their high homology, their specificity is different. Lip1 and Lip3 are completely specific for the S enantiomer (E>300), whereas Lip4 is R specific (E=15). The molecular docking of the S and R enantiomers in the active site of Lip1 and Lip4 lipases enables the observed differences in specificity to be better understood and targets for site-directed mutagenesis to be proposed. We demonstrated that the nature of the amino acid present in position 296 is crucial for the discrimination of these enzymes. The third part of reaction was biodiesel production from immobilized C. rugosa lipase. Optimum conditions were as follow; substrate molar ratio was Palm oil to methanol; 1:4, Lipase was immobilized on macroporous resin namely, NKA-9. Optimum temperature was 30 oC for transesterification. The result obtained 75 % of methyl ester production.