A laccase of Fomes durissimus MTCC-1173 and its role in the conversion of methylbenzene to benzaldehyde.

A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl aminoethyl cellulose. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined K (m) and k (cat) values of the laccase are 182 µM and 0.35 s(-1), respectively, giving a k (cat)/K (m) value of 1.92 × 10(3) M(-1) s(-1). The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence of mediator molecules, property exhibited by yellow laccases.

Luffa aegyptiaca (Gourd) Fruit Juice as a Source of Peroxidase.

Peroxidases have turned out to be potential biocatalyst for a variety of organic reactions. The research work reported in this communication was done with the objective of finding a convenient rich source of peroxidase which could be used as a biocatalyst for organic synthetic reactions. The studies made have shown that Luffa aegyptiaca (gourd) fruit juice contains peroxidase activity of the order of 180 enzyme unit/mL. The K(m) values of this peroxidase for the substrates guaiacol and hydrogen peroxide were 2.0 and 0.2 mM, respectively. The pH and temperature optima were 6.5 and 60°C, respectively. Like other peroxidases, it followed double displacement type mechanism. Sodium azide inhibited the enzyme competitively with K(i) value of 3.35 mM.

Lignin peroxidases of some indigenous ligninolytic fungi: secretion and enzymatic characteristics.

Secretion and enzymatic characteristics of lignin peroxidases from Gloeophyllum sepiarium MTCC 1170, Cladosporium herbarum MTCC 346, Lenzites betulina MTCC 1183, Daedalea flavida MTCC 145, Hexagonia teruis MTCC 1119 and Coirolopsis floccosa MTCC 1177 ligninolytic fungal strains have been reported. Secretion of lignin peroxidase by these ligninolytic fungal strains have been found to be in the range of 0.86 to 3.0 enzyme unit per ml of the culture medium. The enzymatic characteristics like K(m), pH and temperature optima of all the lignin peroxidases of the above fungal strains have been determined using veratryl alcohol and H(2)O(2) as the variable substrates. The K(m) values using veratryl alcohol as the substrate were found to be 65.0 µM, 58.5 µM, 63.0 µM, 54.5 µM, 54.6 µM and 61.0 µM respectively. The K(m) values using H(2)O(2) as the substrate were found to be 88.0 µM, 86.0 µM, 71.0 µM, 67.0 µM, 80.0 µM and 78.0 µM respectively. The pH optima values for lignin peroxidases of the above ligninolytic fungal strains were found to be 2.5, 2.4, 2.4, 2.25, 2.5 and 2.8 respectively, where as the temperature optima values were 25°C, 24°C, 25°C, 23°C, 24°C and 25°C respectively.

Purification and characterization of laccase secreted by L. lividus.

The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been optimized. The laccase from the culture filtrate of L. lividus MTCC-1178 has been purified to homogeneity. The molecular weight of the purified laccase is 64.8 kDa. The enzymatic characteristics like K(m), pH, and temperature optimum using 2,6-dimethoxyphenol have been determined and found to be 480 microM, 5.0, and 60 degrees C, respectively. The K(m) values for other substrates like catechol, m-cresol, pyrogallol, and syringaldazine have also been determined and found to be 230, 210, 320, and 350 microM, respectively.

Enzymatic characteristics of quercetinases from some indigenous Aspergillus flavus strains.

Five indigenous Aspergillus flavus strains MTCC-2206, 1884, 1883, 1783 and 2456 were screened for the secretion of quercetinase. Fungal strains MTCC-2206, 1884, 1883, and 1783 were found to secrete the quercetinase in the range of 0.24-0.36 enzyme unit/mL of the culture medium, while MTCC-2456 secreted only 0.04 enzyme unit/mL. The enzymatic characteristics of quercetinase were determined. The Km values using quercetin as the substrate were 12.5 microM, 14.0 microM, 12.5 microM and 13.0 microM for the quercetinase produced by MTCC-2206, 1884, 1883 and 1783, respectively. The pH optima for the above enzymes were 6.5, 6.5, 6.0 and 6.0 and temperature optima were 45, 40, 45 and 50 degrees C, respectively. The partial purification from only one strain MTCC-2206 was achieved (nearly 3-fold purification).

Purification and characterization of extracellular laccase secreted by Pleurotus sajor-caju MTCC 141.

The effect of lignin containing natural substrates corn-cob, coir-dust, saw-dust, wheat straw and bagasse particles on the extracellular secretion of laccase in the liquid culture growth medium of Pleurotus sajor-caju MTCC 141 has been studied. The culture conditions for maximum secretion of laccase by Pleurotus sajor-caju MTCC 141 have been optimized. Homogeneous preparation of laccase from the culture filtrate of the fungus has been achieved using ammonium sulphate precipitation, anion exchange chromatography on DEAE and gel filtration chromatography on Sephadex G-100. The purified enzyme preparation gave a single protein band in SDS-PAGE analysis indicating a molecular weight of 90 kD. The enzymatic characteristics Km, k(cat), pH and temperature optima of the purified laccase have been determined using 2, 6-dimethoxyphenol as the substrate and have been found to be 35 micromol/L, 0.30 min(-1), 4.5 and 37 degrees C respectively. The Km values for the other substrate like catechol, m-cresol, pyrogallol and syringaldazine have also been determined which were found to be 216 micromol/L, 380 micromol/L, 370 micromol/L and 260 micromol/L respectively.

Purification of a peroxidase from Solanum melongena fruit juice.

Solanum melongena fruit juice contains peroxidase activity of the order of 0.125 IU/mL. A method for the 11-fold purification of the enzyme was developed. The Km values of the peroxidase for the substrates guaiacol and hydrogen peroxide were 6.5 mM and 0.33 mM, respectively. The pH and temperature optima were 5.5 and 84 degrees C, respectively using guaiacol as the substrate. Sodium azide and phenyl hydrazine inhibited the enzyme competitively.

Secretion of ligninperoxidase by Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus.

Secretion of ligninperoxidase [E.C.1.11.1.7] by Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus in liquid culture growth medium has been demonstrated. Enzymatic characteristics like Km, pH and temperature optima using veratryl alcohol as the organic substrate of ligninperoxidases from above sources have been determined. Km values using veratryl alcohol as substrate for enzymes from P. citrinum, F. oxysporum and A. terreus were 69, 64 and 60 microM respectively. Km values using H2O2 as the variable substrate were 64, 72 and 80 microM. The pH optima were 4.0, 2.3 and 2.0 respectively. The values of temperature optima were 30 degrees, 25 degrees and 22 degrees C for the enzymes from P. citrinum, F. oxysporum and A. terreus respectively.