Amit Kumar Dubey

In Silico Characterization of Pectate Lyase Protein Sequences from Different Source Organisms

A total of 121 protein sequences of pectate lyases were subjected to homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis. The phylogenetic tree constructed revealed different clusters based on different source organisms representing bacterial, fungal, plant, and nematode pectate lyases. The multiple accessions of bacterial, fungal, nematode, and plant pectate lyase protein sequences were placed closely revealing a sequence level similarity. The multiple sequence alignment of these pectate lyase protein sequences from different source organisms showed conserved regions at different stretches with maximum homology from amino acid residues 439–467, 715–816, and 829–910 which could be used for designing degenerate primers or probes specific for pectate lyases. The motif analysis revealed a conserved Pec_Lyase_C domain uniformly observed in all pectate lyases irrespective of variable sources suggesting its possible role in structural and enzymatic functions.

Purification and biochemical characterization of an alkaline pectin lyase from Fusarium decemcellulare MTCC 2079 suitable for Crotolaria juncea fiber retting

An extracellular pectin lyase secreted by Fusarium decemcellulare MTCC 2079 under solid state fermentation condition has been purified to electrophoretic homogeniety by using ammonium sulfate fractionation, carboxymethyl cellulose and gel filtration (Sephadex G‐100) column chromatographies. The purified enzyme showed single protein band corresponding to molecular mass 45 ± 01 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 9.0 and showed maximum stability in the pH range of 9.0–12.0. The optimum temperature of the purified enzyme was 50 °C and it showed maximum stability upto 40 °C. The energy of activation for the thermal denaturation (Ea) was 59.06 kJ mol−1 K−1. The Km and kcat values using citrus pectin as the substrate were 0.125 mg ml−1 and 72.9 s−1 in 100 mM sodium carbonate buffer pH 9.0 at 50 °C. The biophysical studies on pectin lyase showed that its secondary structure belongs to α + β class of protein with comparatively less of β‐sheets. Purified pectin lyase showed efficient retting of Crotolaria juncea fibers.

Characterization of a neutral pectin lyase produced by Oidiodendron echinulatum MTCC 1356 in solid state fermentation.

A neutral pectin lyase produced by a new fungal strain Oidiodendron echinulatum MTCC 1356 under solid state fermentation using wheat bran as agro waste has been studied. The enzyme was purified by ammonium sulphate precipitation (30–60%), DEAE anion exchange and Sephadex G‐100 column chromatographies. The SDS‐PAGE and native PAGE revealed two bands of sizes 42 and 47 kDa. The enzyme was purified 37 fold with specific activity of 4.5 U/mg and 2.25% yield. The Km and Vmax values determined using citrus pectin were 1.2 mg/ml and 0.36 IU/min respectively. The pH and temperature optima were pH 7.0 and 50 °C, respectively. The pH stability was around 5.0 for 24 h at 20 °C. The purified enzyme retained maximum activity for 30 min upto 50 °C. The activation energy for thermal denaturation of the purified enzyme was found to be 60.0 kJ/Mol. The effects of various metal ions and protein inhibitors on enzyme activity have revealed total inhibition of the enzyme activity in the presence of Ag+and Cu+and KMnO4 at 1 mM. The neutral pectin lyase showed retting of Crotalaria juncea fibre in the presence of EDTA. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938

An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the Km and kcat values of the enzyme were obtained as 1.7 mg/ml and 66 s−1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.

Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre

An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The Km and kcat values of the PG were 2.7 mg/mL and 2.23 × 103 s−1, respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50°C and the enzyme does not loose activity below 30°C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag+, Hg2+ and KMnO4, while it was stimulated to some extent by Co2+. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.

Modulation of arsenic-induced oxidative stress and protein metabolism by diphenyleneiodonium, 24-epibrassinolide and proline in Glycine max L.

Abstract Arsenic (As)-toxicity is a major constraint for crop production. The present study was intended to examine the comparative ameliorative effects of diphenyleneiodonium (DPI), 24-epibrassinolide (EBL) and proline (Pro) on As-stress in Glycine max L. Seeds of Glycine max L. were subjected to As (100 µM) singly, and together with DPI (10 µM), EBL (0.5 µM) or Pro (10 mM), for five days, and were then analyzed. Experimental results showed that As treatment caused a substantial fall in growth traits like germination percentage, radicle length and dry mass, which was accompanied by As accumulation. Additionally, As application also revealed reduced viability, total protein content and activities of antioxidative enzymes (superoxide dismutase, catalase and ascorbate peroxidase), while it increased the levels of total sugar, proline and oxidative stress markers such as electrolyte leakage, reactive oxygen species, lipid oxidized products, protein carbonyls and hydroperoxides, Amadori and Maillard reaction products, malondialdehyde-/4-hydroxy-2-nonenal-protein adducts, protease and proteasome. Isozymes of antioxidative enzymes were also observed to be altered considerably under As-stress. Impressively, DPI, EBL and Pro played their role as protective agents, hence caused enhanced growth and reduced As accumulation. These protective chemicals also improved the viability, accruals of total protein, total sugar and endogenous proline, and activities of antioxidants, while they reduced the levels of oxidative stress markers. Our findings demonstrated the involvement of DPI, EBL and Pro in As-stress tolerance in Glycine max L. Further, Pro appears to be superior to DPI and EBL, in alleviating As-induced responses in Glycine max L.

Modulation in arsenic-induced lipid catabolism in Glycine max using proline, 24-epibrassinolide and diphenylene iodonium

Abstract Proline, 24-epibrassinolide and diphenylene iodonium are few of the novel antioxidant molecules, involved in growth regulation and abiotic stress tolerance of plants. However, these are scarcely explored in relation to their role in arsenic stress tolerance. Therefore, present study was designed to investigate the involvement of proline, 24-epibrassinolide and diphenylene iodonium in conferring tolerance to Glycine max L. against arsenic toxicity. The results showed that arsenic caused decrease in growth attributes like germination percentage, radicle length and dry mass, which were accompanied by the accumulation of arsenic. The application of arsenic steeply reduced total lipid content while increased the levels of oxidative stress markers such as superoxide anion, hydroxyl radical, hydrogen peroxide, free fatty acid, conjugated diene, lipid hydroperoxide, malondialdehyde and 4-hydroxy-2-nonenal, and the activities of lipase and lipoxygenase. Impressively, proline, 24-epibrassinolide and diphenylene iodonium played their roles as protective agents, and caused enhanced growth and reduced arsenic accumulation. These protective molecules enhanced the total lipid content while reduced the levels of oxidative stress markers and activities of lipase and lipoxygenase. The results indicated that proline, 24-epibrassinolide and diphenylene iodonium served as potential inhibitors of As-induced oxidative stress in Glycine max L.

Molecular Biology of Microbial Pectate Lyase: A Review

Pectate lyase represents an important member of pectinase group of enzymes responsible for the pathogenesis and softening of plant tissues. It also has role in fruit juice clarification and in retting of natural fibers. The biochemical characterization of pectate lyases from diverse microbial sources and plants along with an insight to the protein structure has been dealt earlier but there is a lack of exclusive review on the molecular biology of pectate lyases. This review tries to fill the gap by highlighting the various aspects of molecular biology of microbial pectate lyases especially the cloning and expression of pectate lyase genes from diverse sources attempted so far. The topics covered in this review are a brief description about enzymes associated with degradation of pectin, its classification, applications, updated information about the biochemical characterization of microbial pectate lyases and cloning and expression of microbial pectate lyase genes.

Genome-Wide Assessment of Polygalacturonases-Like (PGL) Genes of Medicago truncatula , Sorghum bicolor , Vitis vinifera and Oryza sativa Using Comparative Genomics Approach

AbstractThe polygalacturonases (PG) is one of the important members of pectin-degrading glycoside hydrolases of the family GH28. In plants, PG represents multigene families associated with diverse processes. In the present study, an attempt has been made to investigate the diversity of PG genes among monocots and dicots with respect to phylogeny, gene duplication and subcellular localization to get an insight into the evolutionary and functional attributes. The genome-wide assessment of Medicago truncatula, Vitis vinifera Sorghum bicolor, and Oryza sativa L. ssp. japonica genomes revealed 53, 49, 38 and 35 PG-like (PGL) genes, respectively. The predominance of glyco_hydro_28 domain, hydrophilic nature and genes with multiple introns were uniformly observed. The subcellular localization showed the presence of signal sequences targeting the secretory pathways. The phylogenetic tree constructed marked uniformity with three distinct clusters for each plant irrespective of the variability in the genome sizes. The site-specific selection pressure analysis based on Ka/Ks values showed predominance of purifying selection pressures among different groups identified in these plants. The functional divergence analysis revealed significant site-specific selective constraints. Results of site-specific selective pressure analysis throw light on the functional diversity of PGs in various plant processes and hence its constitutive nature. These findings are further strengthened by functional divergence analysis which reveals functionally diverse groups in all the four species representing monocots and dicots. The outcome of the present work could be utilized for deciphering the novel functions of PGs in plants.

Molecular Biology, Genomics and Bioinformatics Insights into Fungal Pectin Lyase: An overview

Pectinase represents an industrially important group of enzymes, comprising pectin lyases (PNL), pectate lyases (PL), polygalacturonases (PG), and pectin methylesterases (PME) as a well-studied member with diverse applications. Among pectinases, pectin lyase occupies unique positions based on its reaction mechanisms. Pectin lyase is associated with degradation of pectin polymer directly by β-elimination, and 4, 5-unsaturated oligogalacturonide is the product formed. In case of other pectinases, sequential degradation of pectin molecule occurs. Further, its relevance in fruit juice clarification is mainly because of two reasons. First, they are known to degrade pectin without altering the ester group responsible for the specific aroma of the juice and second, the highly toxic methanol is not generated by the process of degradation of pectin-by-pectin lyases. The applications of pectin lyases in retting processes have recently been elucidated. There exist substantial reports of fungal pectin lyases especially from Aspergillus and Penicillium species, with sole emphasis on production optimization, purification, and biochemical characterizations. As far as molecular biology of pectin lyases is concerned, few studies have been performed especially on Aspergillus species and molecular cloning and expression of relevant pectin lyases genes have been reported. Efforts have been made to decipher the factors influencing pectinases gene regulation, though not extensively. Molecular cloning of pectin lyase gene from diverse sources, its manipulation by using directed evolution techniques, and searching for novel sources by utilizing metagenomic-based approach are some of the recent developments in pectin lyase research, which is a subject matter for the present review.