Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation

Three distinct forms (Lip A, Lip B and Lip C) of extra-cellular lipases (EC - 3.1.1.3), produced by Candida rugosa in solid state fermentation (SSF) we re purified and characterised. SSF was carried out in glass columns using c oconut oil cake and wheat bran. The enzyme was purified from the aqueous ex tract of fermented matter by ammonium sulphate precipitation, dialysis, ult ra-filtration and gelfiltration using Sephadex-200 to a 43-fold purificatio n and 64.35-mg/ml specific activity SDS-PAGE of purified enzyme revealed th ree distinct bands, indicating the existence of three iso-forms, Lip A, Lip B and Lip C with apparent molecular weight about 64,000 62,000 and 60,000 Da, respectively. All the three iso-forms were optimally active at 35-40 de greesC and pH 7-8 They showed marked differences in their Km values with di fferent saturated and unsaturated triacyl glycerols. Ag++ and Hg++ strongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect and Ca+ and Mg++ enhanced the activity. EDTA also strongly inhibited the enzyme a ctivities of iso-forms. However, activities of all the three lipases were c ompletely inhibited by serine protease inhibitors such as 3,4-dichloroisoco umarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the b est of our knowledge, this is the first report describing the purification and characterisation of C. rugosa lipase iso-forms from solid cultures. The se lipase iso-forms with diverse characteristics produced in solid cultures may find potential application in biomedical field.