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) w ere purified and characterised. SSF was carried out in glass columns using coconut oil cake and wheat bran. The enzyme was purified from the aqueous e xtract of fermented matter by ammonium sulphate precipitation, dialysis, ul tra-filtration and gel filtration using Sephadex-200 to a 43-fold purificat ion and 64.35-mg/ml specific activity. SDS-PAGE of purified enzyme revealed three 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 35-4 0 degreesC and pH 7-8. They showed marked differences in their KM values wi th different saturated and unsaturated triacyl glycerols. Ag++ and Hg++ str ongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect an d Ca++ and Mg++ enhanced the activity. EDTA also strongly inhibited the enz yme activities of iso-forms. However, activities of all the three lipases w ere completely inhibited by serine protease inhibitors such as 3,4-dichloro isocoumarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the best of our knowledge, this is the first report describing the purifica tion and characterisation of C. rugosa lipase iso-forms from solid cultures . These lipase iso-forms with diverse characteristics produced in solid cul tures may find potential application in biomedical field.