K. D. S. Yadav

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 β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, 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 and characterization of lignin peroxidase from Loweporus lividus MTCC-1178

Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC‐1178 has been purified to homogeneity using Amicon concentration and DEAE cellulose chromatography. The molecular weight of the purified lignin peroxidase using SDS‐PAGE analysis has been found to be 40 kDa. The Km values for veratryl alcohol and H2O2 for the purified enzyme were 58 and 83 μM, respectively. The calculated kcat value of the purified enzyme using veratryl alcohol as the substrate was 2.5 s−1. The pH and temperature optima of lignin peroxidase have been found to be 2.6 and 24°C, respectively.

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 Km and kcat 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°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.

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 Km values were 54 and 76 µM for veratryl alcohol and H2O2, respectively. The pH and temperature optima were 2.5 and 25°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 characterisation of an acidic pectin lyase produced byAspergillus ficuum strain MTCC 7591 suitable for clarification of fruit juices

An acidic pectin lyase (E.C. 4.2.2.10) produced byAspergillus ficuum MTCC 7591 of molecular weight 31.6 kD was purified to apparent homogeneity by ion exchange and gel filtration chromatography. Eighty-six fold purification with 60% yield and a specific activity of 7.8 U/mg protein was obtained. The Km and calculated turnover number (kcat) of the purified enzyme were found to be 0.60 mg/ml and 74 s−1 respectively using citrus pectin as the substrate. The pH and temperature optima were 5.0 and 50°C respectively. Exposed to 24 hours at a particular pH the enzyme was found to be relatively stable in the pH range 2.0–9.0. Exposed to a particular temperature for 1 hour, the enzyme retains full activity up to 40°C. Metal ions and protein inhibitors did not have significant effects on the activity of the enzyme. The enzyme has been found to be very effective in the clarification of sweet lime and orange juices.

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 µM, 82 µM and 476 µM, respectively. The pH and temperature optima of the enzyme were 2.8 and 25°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.

N-Oxidation of arylamines to nitrosobenzenes using chloroperoxidase purified from Musa paradisiaca stem juice

Abstract N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase purified from Musa paradisiaca stem juice has been examined. The enzymatic characteristics of the stem chloroperoxidase using 4-chloroaniline as substrate were determined. The Km values for 4-chloroaniline and H2O2 were 770 μM and 154 μM respectively, while the pH and temperature optima were 4.4 and 30°C respectively. The substrate specificities of the enzyme for the arylamines 3,4-dichloroamine, p-aminobenzoic acid, p-toluidine, p-anisidine, m-anisidine, p-aminophenol, o-aminophenol and m-aminophenol have been characterized. The feasibility of using concentrated M. paradisiaca stem juice for the specific conversion of 4-chloroaniline to 4-chloronitrosobenzene has been demonstrated. This enzyme can be used for the N-oxidation of other arylamines.

Purification and characterization of lignin peroxidase from Pleurotus sajor caju MTCC-141.

Abstract The culture conditions for extracellular secretion of lignin peroxidase by Pleurotus sajor caju MTCC–141 in the liquid culture growth medium amended with lignin containing natural substrates have been studied. Secretion of lignin peroxidase has been found to be maximum in the presence of bagasse. Lignin peroxidase from the liquid culture filtrate has been purified to homogeneity. Two isozymes having relative molecular masses 38 and 40 kDa have been isolated. The enzymatic characteristics like Km, pH, and temperature optima of the major isozyme (40 KDa) has been determined using veratryl alcohol, n-propanol, and H2O2 as the substrate. The Km values for veratryl alcohol, n-propanol, and H2O2 have been found to be 57 μ M, 500 μ M, and 80 μ M, respectively. The pH and temperature optima of lignin peroxidase have been found to be 3 and 30°C, respectively. The inhibition of the enzyme activity by sodium azide has been studied and it has been found to be uncompetitive, with KI value of 4 mM.

α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, Km and kcat values of the enzyme were 0.65mM and 43.65s-1, respectively. The pH and temperature optima of the enzyme were 6.5 and 57°C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin.

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 Km, kcat and kcat/Km values of the enzyme using veratryl alcohol and H2O2 as the substrate were 61 M, 2.13 s−1, 3.5 × 104 M−1s−1 and 71 M, 2.13 s−1, 3.0 × 104 M−1 s−1 respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.