Pseudomonas fragi CRDA 037 was used as source of intracellular esterase, which remained attached to the cell membrane, and characterized. Several mechanical methods of disruption were used including glass beads (MSK), sonication, French press and combinations of these methods. The cellular debris were also treated with detergents such as CHAPS and Triton X-100 in the presence of chelating agent ethylenediaminetetra acetic acid (EDTA). The esterase activity remained in the cellular debris which was therefore use as a source of enzyme for kinetic studies. In the case of glass beads homogenization, the activity was found to decrease as a function of time of disruption. The results of chemical treatment showed that the esterase was characterized in terms of detergent and EDA action as well as substrate specificity. Triton X-100 and EDTA had no effect on the esterase activity and did not denature the enzyme. The substrate specificity with cells and cellular debris were carried out. The valeric acid was the best in term of esterase activity among fatty acids used in the study. (Abstract shortened by UMI.)

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.

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

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Induction of extracellular lipases from Pseudomonas fragi CRDA 037 and Geotrichum candidum was used to increase lipase production in a shorter period of time. Induction was performed using edibles oils such as olive oil, canola oil, fish oil and butter fat. Fermentation trials of the microorganisms in the selected media were done in order to optimize the production of lipases. Optimal lipase activity was obtained in the presence of butter fat (1%, v/v) for P. fragi which was cultivated at 15°C during 48 h, and fish oil (0.75%, v/v) for G. candidum incubated at 27°C for 56 h. The induced lipase extracts of P. fragi and G. candidum obtained after these fermentation trials were purified by 3.7 and 5.9-fold respectively using ultrafiltration, while the non-induced fractions were purified by 2.3 and 5.1-fold, respectively. Activities were evaluated using p-nitrophenyl esters such as p-nitrophenyl laurate and p-nitrophenyl palmitate. (Abstract shortened by UMI.)

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