It has been possible to demonstrate and characterize high phospholipase activities in mycelia of Rhizopus arrhizus and Mucor javanicus by use of a system in which substrates were dissolved in diisopropyl ether. Such activities were associated with bound enzymes and would have been difficult to detect using aqueous assay systems. In both cases, phosphatidylcholine hydrolysis was by phospholipase A1 (EC 3.1.1.32) activity followed by the action of lysophospholipase (EC 3.1.1.5). Phospholipase D (EC 3.1.4.4) activity was also detected. The methods used appear to be of general applicability for the detection and study of insoluble phospholipases.
SummaryContinuous hydrolysis of triglyceride in organic solvent systems using Rhizupus arrhizus mycelia as a source of insolubilized lipase has been studied in packed-bed and stirred-tank reactors. Typically a packed bed reactor containing I g of mycelia fed at I mL/min with a solution of 2.5% ( w h ) olive oil in di-isopropyl ether gave a fatty acid yield of 45% at 30°C. The optimum water concentration was found to be 0.17% ( w h ) except under conditions of high oil feed concentration and high yield where no optimum was established. N o temperature optimum was observed over the range 20-55°C. Calculated activation energies of 13-20 k J h o l . depending on temperature. were lower, while K,,,(app) values of 0. I-0.3M were higher than those for hydrolysis in conventional aqueous emulsion systems. N o evidence of any significant diffusional limitation. which could account for these values. was obtained. The mycelia showed a loss of activity of 0.6-1.0%1h at 30°C. The packed bed proved markedly superior to the stirred tank for this system.
Mucorpusillus was grown in different media for a period of 92 h, and the media were investigated for both milk-clotting and protease activities. It was observed that the ratio of extracellular milk-clotting activity to protease activity was the highest for 3% corn steep liquor containing 1% glucose as the source of carbon. Variation of both milk-clotting and protease activities was studied during the growth of the organism in the medium stated above. Separation of protease was carried out by ion-exchange chromatography at pH 8.0. Fractions collected were assayed for both activities simultaneously. The findings suggested that, instead of only one major acid protease, as reported by previous workers, two major acid proteases were produced. One of them had significant rennin-like activity, and the other lacked it. The former could be assumed to be the enzyme reported and studied by previous workers. The existence oftwo proteases was further confirmed by the appearance of two protease activity bands on polyacrylamide gels after electrophoresis. An attempt was made to separate the rennin-like enzyme from nonspecific protease activity by ammonium sulfate fractionation followed by ionexchange chromatography at pH 6.0. The results indicated that the nonspecific protease activity due to the enzyme that lacked rennin action was substantially removed by the ammonium sulfate fractionation.
Certain long-chain polyacetylenic fatty acids prove t o be more potent competitive inhibitors of lipoxidase than any of the compounds which have been examined previously. Inhibition by nordihydroguaiaretic acid under similar conditions is of the induction period type and is considered not to be competitive.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.