Production, purification and characterization of lipase by the heat-resistant mold, Byssochlamys fulva

The production of lipase by Byssochlamys fulva in a basal liquid medium was investigated. Of the four strains of Byssochlamys fulva investigated, strain H-25 produced the highest lipase activity after 96 hr of growth on a shaker at 30{dollar}spcirc{dollar}C. Olive oil and peptone were the best carbon and nitrogen sources, respectively. The enzyme hydrolyzed corn oil most rapidly than the other natural oils and fats. The enzyme was specific for triglycerides and monoesters of C12 or C14 fatty acids. Of the esters of unsaturated C18 fatty acids tested, methyl linoleate was hydrolyzed most quickly.; One hundred and two lipase over-producing mutants were isolated by mutagenizing a parental strain of Byssochlamys fulva H-25 with UV (ultraviolet light) or/and NTG (N-methyl-{dollar}Nspprime{dollar}-nitro-N-nitrosoguanidine). NTG was proved to be a better mutagenic agent than UV for higher mutant frequency and selection of stable lipase over-producing mutants. When cultured in a basal liquid medium at 30{dollar}spcirc{dollar}C for 96 hr, strain NTG9 obtained by treatment of the parental strain with 0.01% NTG for 10 min was found to yield the highest specific activity (7.58 U/mg protein).; Using the response surface methodology, the optimal conditions for lipase specific activity were 5.7% peptone, initial pH 5.5, and a shaker speed of 210 rpm. The predicated and experimental values were 10.61 U/mg protein and 20.36 U/mg protein, respectively. The highest mycelial dry weight was obtained with 3.5% peptone, pH 6.6, and a shaker speed of 350 rpm. The predicated and experimental values were 7.54 and 7.95 g/l, respectively.; The lipase of Byssochlamys fulva NTG9 was purified 17-fold with a 7.3% yield. The purified enzyme was homogeneous and had a specific activity of 153 U/mg protein. The enzyme was monomeric and its molecular weight was estimated to be 15 kDa by gel filtration with Sephadex G-100, 20 kDa by SDS-PAGE and 14.7 kDa by mass spectrometry. Its isoelectric point was 5.1. The optimum pH and temperature were 8.5 and 30{dollar}spcirc{dollar}C, respectively. The enzyme was stable over a pH range of 6 to 8 at 25{dollar}spcirc{dollar}C for 24 hr, and its activity was stable up to 50{dollar}spcirc{dollar}C for 30 min. The lipase from B. fulva was a 1,3 positional specific lipolytic enzyme, and could be activated with the addition of Ca{dollar}sp{lcub}2+{rcub}{dollar} ion to the reaction mixture.; Synthesis of sugar esters from sugars and fatty acids by lipase from Byssochlamys fulva NTG9 in tertiary butyl alcohol was investigated. Linoleic acid was found to give the highest percentage of esterification (65.5%). Of the sugars tested, fructose yielded the highest percentage of esterification (71.3%).