Barbara Bisakowski

Characterization of a lipoxygenase extract from Geotrichum candidum

Abstract Geotrichum candidum was cultivated and harvested after 8 days of incubation, a period that corresponded to the maximal dry weight of mycelium as well as to optimal lipoxygenase (LOX) activity. A partially purified LOX enzymic extract was obtained by ammonium sulfate precipitation. The LOX extract exhibited two major activities, at pH 8.00 and 3.75 using linoleic acid as substrate. At pH 3.75, the enzymic extract displayed the highest specific activity using linolenic and linoleic acids as substrates whereas, at pH 8.00, maximal LOX activity was obtained using arachidonic and linoleic acids. At pH 3.75, fatty acid acylglycerols were enzymically oxidized with a similar efficiency to that obtained with free fatty acids. K m and V max values as well as the effect of a wide range of chemical substances on LOX activity were also investigated. High-performance liquid chromatography and gas chromatography/mass spectrometry analyses showed that LOX activity in the microbial enzymic extract converted linoleic acid to the corresponding 9- and 13-hydroperoxides.

Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media

Abstract The enzymatic activity of mushroom tyrosinase was investigated using catechin as substrate in selected organic solvent media. The results showed that optimal tyrosinase activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in the organic solvent media of heptane, toluene, dichloromethane, and dichloroethane, respectively, and at a temperature between 25°C and 27.5°C. In addition, the kinetic studies showed that the K m values were 5.38, 1.03, 2.52 and 4.03 mM, for the tyrosinase-catechin biocatalysis in the reaction media of heptane, toluene, dichloromethane, and dichloroethane, respectively, while the corresponding V max values were 1.22×10 −3 , 0.33×10 −3 , 1.47×10 −3 and 1.20×10 −3 δA per μg protein per second, respectively. The use of acetone as co-solvent for the tyrosinase-catechin biocatalysis showed that acetone concentrations ranging from 5% to 30% (v/v) in the heptane reaction medium produced a decrease of 4.3% to 96.7% in tyrosinase activity. The results also indicated that the presence of 12.5% acetone in the reaction medium of dichloromethane, and 22.0% in those of toluene and dichloroethane produced a maximal increase of 42.6%, 92.1% and 71.8%, respectively, in tyrosinase activity. However, the overall findings indicated that additional increases in acetone concentration resulted in an inhibition of tyrosinase activity.

Partial characterization of a lipoxygenase from fusarium proliferatum

Summary Biomass of the fungus Fusarium proliferatiim was produced and used to obtain a crude extract (FI) of lipoxygenase. The enzyme was further purified by ammonium sulfate precipitation at 40% of saturation (FII). The enzymatic extract (FII) showed its optimum activity at pH 6.0. The apparent K m and V max values for the lipoxygenase (FII) were calculated to be 5.15 × 10−5 M and 1.61 μmol hydroperoxide/mg protein/min, respectively. Enzyme activity remained relatively stable at potassium cyanide concentrations as high as 60 mM. The presence of 5 mM ethylenediaminetetraacetate activated the enzyme by 50%, whereas the use of 1.2 mM hydroquinone resulted in a 2‐fold increase in lipoxygenase activity. The partially purified enzyme (FII) showed a three‐fold enhancement of activity towards linoleic acid compared to linolenic acid as well as mono‐, di‐ and trilinolein.

Biocatalysis of lipoxygenase in selected organic solvent media

Abstract The biocatalysis of purified soybean lipoxygenase (LOX) (EC 1.13.11.12), using linoleic acid as a substrate model, was investigated in selected organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene. The results indicated that there was a 2.6-fold increase in LOX activity in the monophasic iso-octane medium compared to that obtained in the aqueous medium. The results also showed that there was an increase of 2.2- and 1.8-fold in LOX activity in the monophasic reaction media of octane and hexane, respectively. However, an inhibitory effect on enzyme activity was observed when the monophasic reaction media of toluene, chloroform and dichloromethane were used. In addition, the results showed that the optimum concentration of octane and iso-octane in the biphasic medium containing the organic solvent and Tris–HCl buffer solution, was determined to be 3.5% and 4%, respectively, for LOX activity. Moreover, the biocatalysis of LOX in a ternary micellar system, containing either 3.5% octane or 4% iso-octane, Tris–HCl buffer solution and an emulsifier, resulted in an overall increase in enzyme activity. The Km and Vmax values, substrate specificity, optimum protein concentration, optimum reaction temperature as well as the enzymatically catalyzed end-products were investigated for LOX biocatalysis in both ternary micellar systems.

Biocatalysis of immobilized chlorophyllase in a ternary micellar system

The immobilization of chlorophyllase was optimized by physical adsorption on various inorganic supports, including alumina, celite, Dowex-1-chloride, glass beads and silica gel. The enzyme was also immobilized in different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant. The highest immobilization efficiency (84.56%) and specific activity (0.34 mumol hydrolyzed chlorophyll mg protein-1 per min) were obtained when chlorophyllase was suspended in Tris-HCl buffer solution and adsorbed onto silica gel. The effect of different ratios of chlorophyllase to the support and the optimum incubation time for the immobilization of chlorophyllase were determined to be 1-4 and 60 min, respectively. The experimental results showed that the optimum pH and temperature for the immobilized chlorophyllase were 8.0 and 35 degrees C, respectively. The use of optimized amounts of selected membrane lipids increased the specific activity of the immobilized chlorophyllase by approximately 50%. The enzyme kinetic studies indicated that the immobilized chlorophyllase showed a higher affinity towards chlorophyll than pheophytin as substrate.

Partial purified lipoxygenase from Fusarium oxysporum : characterization and kinetic studies

Abstract Crude lipoxygenase was obtained from a biomass culture of Fusarium oxysporum and partially purified by precipitation with ammonium sulphate at 20–80% of saturation. The enzyme exhibited two pH optima, a major activity at pH 10 and a minor one at pH 8. The apparent Km values at pH 8·0 and 10·0 were calculated to be 3·28 × 10−5 m and 3·55 × 10−5 m , respectively. At pH 8·0, lipoxygenase activity was inhibited by 31% at 20 m m potassium cyanide (KCN), exhibiting a non-competitive inhibitory effect. However, at pH 10·0, KCN had relatively little effect on enzyme activity. The addition of EDTA to the reaction medium activated the enzyme activity by 50·3% and 16·6% at pH 8·0 and 10·0, respectively. Substrate specificity of the enzyme was greatest towards linoleic acid, followed by linolenic acid, at both pH 8·0 and 10·0. In addition, the enzyme demonstrated higher activity towards the glycerol fatty acids at pH 10·0 compared to that at pH 8·0. Native polyacrylamide gel electrophoretograms of partially purified enzyme indicated the presence of one major and five minor bands of molecular weights of 67000–140 000 Da.

Biocatalysis of silica gel-immobilized chlorophyllase in a ternary micellar system

Abstract A partially purified enzymic extract from Phaeodactylum tricornutum was immobilized on silica gel and the specific activity of chlorophyllase in its free and immobilized states were compared in a ternary micellar system. The storage stability of the free and immobilized chlorophyllase extracts, maintained at temperatures ranging from 4 to 35°C for a period of 0–20 h, was temperature-dependent. The results also showed that the specific activity of the free and immobilized chlorophyllase extracts was highest at 30°C for long-term incubation, using chlorophyll and pheophytin as substrates and that a three-fold increase in the specific activity of the immobilized chlorophyllase was observed in comparison to that obtained with the free counterpart. The findings indicated that when free and immobilized chlorophyllase extracts were recovered and reused with both substrates, the immobilized chlorophyllase extract could be recycled for longer periods of time, while the free enzyme extract showed no activity after the first cycle.