An alkali tolerant α-l-rhamnosidase from Fusarium moniliforme MTCC-2088 used in de-rhamnosylation of natural glycosides.

Analkali tolerant α-l-rhamnosidase has been purified to homogeneity from the culture filtrate of a new fungal strain, Fusarium moniliforme MTCC-2088, using concentration by ultrafiltration and cation exchange chromatography on CM cellulose column. The molecular mass of the purified enzyme has been found to be 36.0 kDa using SDS-PAGE analysis. The Km value using p-nitrophenyl-α-l-rhamnopyranoside as the variable substrate in 0.2 M sodium phosphate buffer pH10.5 at50 °C was 0.50 mM. The catalytic rate constant was15.6 s-1giving the values of kcat/Km is 3.12 × 104M-1 s-1. The pH and temperature optima of the enzyme were 10.5 and 50 °C, respectively. The purified enzyme had better stability at 10 °C in basic pH medium. The enzyme derhamnosylated natural glycosides like naringin to prunin, rutin to isoquercitrin and hesperidin to hesperetin glucoside. The purified α-l-rhamnosidase has potential for enhancement of wine aroma.

Alpha-L-rhamnosidase from Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH.

An alpha-L-rhamnosidase secreting fungal strain has been isolated and identified as Aspergillus clavato-nanicus MTCC-9611. The enzyme was purified to homogeneity from the culture filtrate of the fungus using concentration by ultrafiltration membrane and ion-exchange chromatography on CM-cellulose. The native PAGE analysis confirmed the homogeneity of the purified enzyme. The SDS-PAGE analysis of the purified enzyme revealed a single protein band corresponding to the molecular weight 82 kDa. The alpha-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 had optimum at pH 10.0 and 50 degrees C. The K(m) values of the enzyme were 0.65 mM and 0.95 mM using p-nitrophenyl alpha-L-rhamnopyranoside and naringin as a substrates respectively. The enzyme transforms naringin to prunin at pH 10.0 and further hydrolysis of prunin to naringenin does not occur under these reaction conditions that makes alpha-L-rhamnosidase activity of Aspergillus clavatonanicus MTCC-9611 promising enzyme to get prunin for pharmaceutical purposes.

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice.

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.

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.

Oxidation of polyaromatic hydrocarbons in systems containing water miscible organic solvents by the lignin peroxidase of Gleophyllum striatum MTCC-1117.

Lignin peroxidase has been purified to homogeneity using a process of concentration by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose from the liquid culture filtrate of the brown rot fungi Gleophyllum striatum MTCC-1117. The molecular mass of the purified enzyme is 43 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The K(m) values for the enzyme using veratryl alcohol, hydrogen peroxide and n-propanol were 66 microM, 82 microM and 476 microM, respectively. The pH and temperature optima of the enzyme were 2.8 and 25 degrees C, respectively. The enzyme is completely inhibited by 20% of the water miscible organic solvents acetone dioxane, diethylether, acetonitrile and dimethylformamide. The lignin peroxidase oxidizes polycyclic aromatic hydrocarbons pyrene, acenaphthene, anthracene, dibenothiophene and 9-methyl anthracene.

Purification and characterisation of lignin peroxidase from Pycnoporus sanguineus MTCC-137.

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K(m)(, kcat) and k(cat)/K(m) values of the enzyme using veratryl alcohol and H2O2 as the substrate were 61 microM, 2.13 s(-1), 3.5 x 10(4) M(-1) s(-1) and 71 microM, 2.13 s(-1), 3.0 x 10(4) M(-1) s(-1) respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25 degrees C.

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.

In silico analysis of pectin lyase and pectinase sequences.

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like beta-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVNSNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.

Purification, characterization, and coal depolymerizing activity of lignin peroxidase from Gloeophyllum sepiarium MTCC-1170.

Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K(m) values were 54 and 76 microM for veratryl alcohol and H2O2, respectively. The pH and temperature optima were 2.5 and 25 degrees C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H2O2. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H2O2, and the purified lignin peroxidase. The influence of NaCl and NaN3 and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.

Purification and characterization of pectin lyase secreted by Penicillium citrinum.

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K(m) and k(cat) values were found to be 1 mg/ml and 76 sec(-1), respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0-12.0 range when exposed for 24 h. The optimum temperature was 50 degrees C, and the pectin lyase was found to be completely stable up to 40 degrees C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.

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.

Enzymatic characteristics of ligninperoxidases from Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus using n-propanol as substrate.

The activities of ligninperoxidases from Penicillium citrinum MTCC 3565, Fusarium oxysporum MTCC 3379 and Aspergillus terreus MTCC 3374 have been assayed and the enzymatic characteristics like Km, pH and temperature optima using n-propanol as the substrate have been reported. The results suggest that n-propanol can substitute veratryl alcohol as substrate for assaying ligninperoxidase activities from different fungal strains, without affecting the enzymatic characteristics. The above strains were selected, as they were known to secrete ligninperoxidase in the liquid culture medium.

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.