UV-induced yeast lipase producer with a wide substrate specificity - selection, characterization and production of the enzyme

Lipases are capable of changing the quantitative and/or qualitative characteristics of fat-containing raw materials and widely used for various tasks in modern food and agricultural industries. To meet the growing demand for these enzymes, highly effective producers are needed, especially those exhibiting multiple activity to lipids of different structure and origin. The aim of the study was to search for a new yeast strain with high production of lipase with broad substrate specificity, and to optimize its fermentation conditions. This work objectives also included obtaining an enzyme with a high grade of hydrolysis of various oils, and the study of its technological properties. Lipolytic characteristics were studied in 110 yeast isolates obtained from natural sources and the collections of Moscow State University of Food Production and State Research Institute of Genetics and Selection of Industrial Microorganisms. Qualitative assay of lipase activity was carried out using a differential nutrient medium with tributyrin and dye methyl red; quantitative analysis was carried out in accordance with the modified Y. Ota & K. Yamada method at pH 5.5. Of the 23 strains with lipolytic activity (LA), 12 had sufficiently high LA indices from 2.5 to 7.5 U/cm3, of which M10 isolate with maximum activity was selected. For this isolate, morphological, cultural, physiological and biochemical properties were studied and molecular genetic identification was performed. The strain was identified as Candida parapsilosis (99 % of homology) using phylogenetic analysis and deposited in the All-Russian Collection of Industrial Microorganisms under the number Y-4055. After UV mutagenesis, a highly active mutant C. parapsilosis M10-10 was obtained. Using mathematical planning methods, the optimal nutrient medium for its growth and lipase production was determined as, %: mustard oil - 2.6, yeast extract - 1.8, soy flour - 1, glucose - 0.5, Tween 80 - 0.42, CaCO3 - 0.3, KH2PO4 - 0.03, MgSO4 · 7H2O - 0.02. An enzyme with a purification grade of 20× and lipolytic activity of 30630 U/g was obtained after culturing the strain M10-10 at a 30-40 °С temperature and pH of the nutrient medium 5.5-6.5. It was found that by 48 hours of fermentation, lipase reaches the highest activity in the culture medium when inoculum M10-10 is in an amount of 5%. Optimal conditions for the enzyme were determined as 37 °С and pH 5.5. In terms of activity, the resulting product is not inferior to commercial domestic and foreign enzymes, including Novozym 435 (Sigma-Aldrich, USA) with a lipolytic activity of 24020 U/g. The fatty acid specificity of the new lipase was determined by enzymatic treatment of various vegetable oils and gas chromatography of lipid products using a Shimadzu GC 2010 (Shimadzu, Japan). Modification of vegetable oils with M10-10 lipase in an oil/water emulsion significantly reduced 2.4, 4.6, 2.9 and 1.5 times the saturated fatty acids fraction (including palmitic and stearic acids) and increased 1.5, 1.6, 1.1 and 12 times the polyunsaturated fatty acids fraction (including w-3 linolenic and w-6 linoleic acids) for olive, mustard, sunflower and coconut oils, respectively.