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Pseudomonas fragi CRDA 037 was used to produce both intracellular and extracellular lipases. The crude lipase fractions were fractionated using ammonium sulfate to obtain partially purified intracellular and extracellular lipases; the fraction precipitated at 20-60% of saturation of the intracellular proteins was retained as the source of the endolipase, whereas the culture medium precipitated at 20-40% of saturation, was used as the source of exolipase. Native gel electrophoresis (PAGE) suggested that the partially purified extracellular lipase has a molecular weight of 25,500 Da. Three major electrophoretic bands were present in the intracellular lipase fraction at 70,000, 49,000, and 35,500 Da. The partially purified lipases were characterized with respect to their optimum pH and temperature for lipase activity, and well as for their kinetics, specificities, and reactions toward inhibitors. The optimum pH for the activity of the endolipase was found to be 9.0 whereas that of the exolipase was 8.75. With respect to optimum temperature, 30$ sp circ$C was determined to be the best for the endolipase while 35$ sp circ$C was the optimal value for the exolipase. Enzyme specificity was carried out using triacetin, tributyrin, trimyristin, and triolein as substrates. The results for the exolipase indicated that the lowest K$ sb{m}$ was obtained with trimyristin and the highest K$ sb{m}$ was obtained with triolein whereas for the endolipase, the highest K$ sb{m}$ was obtained with tributyrin and the lowest was with trimyristin. Experiments carried out with inhibitors indicated that both lipases are serine lipases, sulfydryl enzymes, and that tryptophan is essential for maintaining the conformation of the proteins. The most potent inhibitor was ferrous chloride, and sodium deoxycholate was a weak inhibitor for both lipases, however, it was an activator at low concentrations for the exolipase.

No description available.

The partially purified extracellular lipase from Pseudomonas fragi CRDA 037, obtained by ammonium sulfate precipitation, was subjected to further purification by successive ion-exchange and size-exclusion chromatographies using the Fast Protein Liquid Chromatography system. The purification of the partially purified lipase resulted in two enzymatic fractions, FIVa ' and FIVb', with a purification-fold of 169 and 195, respectively. Native electrophoretic analyses revealed the presence of three bands for fraction FIVa', with estimated molecular weights (MW) of 16.2, 25.8 and 38.5 kDa and two bands for FIVb' , with estimated MW of 15.2 and 25.8 kDa. The two purified fractions, FIVa' and FIVb', showed an optimum pH of 9.5 and 10.0, respectively, and an optimum temperature of 80°C. The Km values for FIVa' and FIVb' were 3.85 and 5.49 mM and the V max values were 2.78 and 2.09 U/mg, respectively, using triacetin as a substrate. The purified lipase fractions retained more than 90% of their activity when stored at room temperature for 36 h. The lipase activity of the purified lipase fractions was completely inhibited by 10 mM of FeCl 2, FeCl3 and Ellman's reagent. However, 10 mM of CaCl2 and EDTA activated the two purified lipase fractions by 20 to 50%. Both fractions exhibited high specificity towards short- and long-chain fatty acid esters of triacylglycerols. Fraction FIVa' showed higher specificity towards triacetin, tristearin and tripalmitin, whereas fraction FIVb' exhibited higher activity with triacetin, trimyristin and triolein. In addition, the two purified lipase fractions were able to catalyze, to almost the same extent, the hydrolysis of butter and olive, canola and fish oils. The gas-liquid chromatography analysis of free fatty acids, obtained by the hydrolysis of the four edible oils, revealed that fraction FIVa' was more specific for the hydrolysis of fatty acid esters chain lengths of C12 to C18 whereas fraction FIVb' showed a non-specific hydrolyzin

Lipase was partially purified from the viscera of grey mullet ( Mugil cephalus) by ammonium sulfate fractionation, simultaneous desalting, and concentration via ultrafiltration and then affinity chromatography on EAH-Sepharose 4B. The partially purified extract was characterized using p-nitrophenyl palmitate (rho-NPP) as substrate. Grey mullet lipase was active within the pH range of 7-10, with an optimum pH of 8.0, and was stable from pH 4-10. The enzyme was active within the temperature range of 20°C and 60°C, and exhibited an optimum for the hydrolysis of rho-NPP at 50°C. The enzyme was stable between 10-50°C, beyond which it lost activity progressively. At 50°C there was ca. 50% residual activity after 60 min incubation. However at 60°C, there was 22%, 20% and 0% remaining activity after 10, 30 and 60 min incubation respectively. Based on the temperature activity data, the activation energy for the hydrolysis of rho-NPP was calculated as 1.94 kcal/mol (8.15 kJ/mol). / The rho-nitrophenyl esters of medium to long chain fatty acid (C10-C16) served as good substrates with the order of ease of hydrolysis as; rho-NP-palmitate > rho-NP-myristate > rho-NP-caprate > rho-NP-caproate > rho-NP-butyrate > rho-NP-acetate. The Km' and Vmax for the hydrolysis of rho-NPP were 0.22 mM and 20 mumol min-1 mg-1 , respectively. The hydrolytic activity of the lipase was enhanced by Mg2+, Mn2+, NaN3, and EDTA, but strongly inhibited by Hg2+, and Cu2+. PMSF (1 mM), Ca2+ (1 mM and 10 mM) had no effect on grey mullet lipase activity. Lower concentrations (25-10% v/v) of water-miscible organic solvents (dimethyl sulfoxide, dimethyl formamide, iso-propanol, and methanol) had negligible effect on the activity of the lipase while higher concentrations (>50% v/v) completely inhibited the enzyme. The grey mullet lipase was remarkably stable in water-immiscible organic solvents (benzene, toluene, hexane, heptane, and isooctane). The water-immiscible solvents also activated the enzyme with hexane giving the most activation. Lower concentrations of trihydroxylated bile salts (sodium taurocholate, and sodium cholate) were more potent activators than the dihydroxylated bile salt (sodium deoxycholate). Sodium dodecyl sulfate at 1 mM, and Tween 80RTM at 1% had 6% and 12% stimulatory effect on the activity of the enzyme respectively, while 1% and 0.5% Triton RTM X-100 caused 67% and 40% inhibition, respectively.

No description available.

No description available.

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No description available.

Using tricaprylin (TC) and trilinolenin (TLN) as substrates, biosynthesis of medium-long-medium (MLM) type structured lipids (SLs), by Lipozyme IM from Rhizomucor meihei and Novozym 435 from Candida antarctica , was investigated to determine their capacity as biocatalysts for the biosynthesis of SLs. At 30°C, Lipozyme IM showed higher bioconversion yield (24.7%) and initial enzyme activity (6.3 mumol CLnC/g enzyme/min) as compared to that of 24.0% and 1.6 mumol CLnC/g enzyme/min, respectively, for the Novozym 435 at 50°C. As a result, Lipozyme IM was subsequently used for further investigations. The SLs were recovered and characterized by silver-ion exchange high-performance liquid chromatography and gas-liquid chromatography. The structural analyses indicated that the major products of the enzymatic reaction were 1,3-dicapryl-2-linolenyl glycerol (CLnC) and 1(3)-capryl-2,3(1)-dilinolenyl glycerol (CLnLn). In order to optimize the bioconversion yield of CLnC, selected parameters, including initial water activity and solvent type, lipase concentration (5 to 20 mg solid enzyme), substrate molar ratios (TC:TLN of 1:4 to 8:1) and molecular sieve (5 to 20 mg/mL, Type 3A), were investigated. The experimental results showed that using hexane at initial aw 0.06, 10 mg solid enzyme/mL and substrate molar ratio of TC to TLN of 6:1 resulted in the highest bioconversion yield of 73.2% of CLnC. However, the addition of molecular sieve to the reaction medium resulted in a 14.0% decrease in the bioconversion yield of CLnC. Using the optimized conditions, the effects of TLN concentration and other selective limiting parameters, including the denaturation of enzyme, aw and the formation of glycerol layer, on the mass productivity (PM), enzymatic productivity (PE) and volumetric productivity (PV) of the interesterification reaction were investigated. Using 80 mM TLN, the maximum PM of 15.5 mg CLnC/g substrates/h was obtained; however, using 200 mM TLN, the maximum PE and PV were 0.07 mg/enzyme unit/h and 6.1 g CLnC/L/h, respectively. The addition of 3 mg Silica gel to the reaction medium resulted in 52.0, 37.3 and 37.3% increase in PM, PE and PV, respectively.