Sangeeta Yadav

In Silico Characterization of Alkaline Proteases from Different Species of Aspergillus

A total of 49 protein sequences of alkaline proteases retrieved from GenBank representing different species of Aspergillus have been characterized for various physiochemical properties, homology search, multiple sequence alignment, motif, and super family search and phylogenetic tree construction. The sequence level homology was obtained among different groups of alkaline protease enzymes, viz alkaline serine protease, oryzin, calpain-like protease, serine protease, subtilisin-like alkaline proteases. Multiple sequence alignment of alkaline protease protein sequence of different Aspergillus species revealed a stretch of conserved region for amino acid residues from 69 to 110 and 130–204. The phylogenetic tree constructed indicated several Aspergillus species-specific clusters for alkaline proteases namely Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus. The distributions of ten commonly observed motifs were analyzed among these proteases. Motif 1 with a signature amino acid sequence of 50 amino acids, i.e., ASFSNYGKVVDIFAPGQDILSCWIGSTTATNTISGTSMATPHIVGLSCYL, was uniformly observed in proteases protein sequences indicating its involvement with the structure and enzymatic function. Motif analysis of acidic proteases of Aspergillus and bacterial alkaline proteases has revealed different signature amino acid sequences. The superfamily search for these proteases revealed the presence of subtilases, serine-carboxyl proteinase, calpain large subunit, and thermolysin-like superfamilies with 45 representing the subtilases superfamily.

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 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.

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 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.

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.

Overview and Principles of Bioengineering: The Drivers of Omics Technologies

Abstract The science of “omics” reflects the innovations in diverse technologies leading to studies of life processes in totality. With the initial emergence of genomics, proteomics, and metabolomics associated with the comprehensive studies of genes, proteins, and metabolites of an organism, respectively, there has been a substantial growth in other branches of omics such as lipidomics, cytomics, phenomics, and transcriptomics. The omics-driven science has a potential application in crop-improvement program, human health sector, environmental sector, and industrial sector. The recent developments in genome sequencing have further expanded the scope of the omics technologies for elucidating the complexity of life processes and understanding the gene expression and regulation in diverse systems. The concept of bioengineering is more relevant to the human health and is often referred to as biomedical engineering. In general, it covers tools and technologies associated with the manipulation of life processes for the benefit of the humankind. In biological system, the central dogma of molecular biology, i.e., replication, transcription, and translation, is the main center of attraction for manipulation using appropriate tools of omics. This chapter highlights the basics of science of omics along with relevant tools and diverse applications.

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.

Purification and characterization of a highly alkaline pectin lyase from Fusarium lateritum MTCC 8794

Abstract A highly alkaline pectin lyase (PNL) produced by Fusarium lateritum MTCC 8794 using solid-state fermentation was purified and biochemically characterized. The enzyme was purified to homogeneity by comparatively simple method involving ammonium sulfate precipitation and cation exchange chromatography resulting in a final purification fold of 5.5 with specific activity of 1.9 U/mg and yield of 3.42%. The SDS-PAGE of the purified enzyme revealed a single protein band of approximately 16 kDa. The pH optimum was found to be 10.0, while the enzyme was stable in the pH range 6.0–10.0. The optimum temperature of the purified PNL was 40◦C, the enzyme being stable upto 50◦C for 30 min. The Km value calculated by Michaelis-Menten curve was found to be 0.79 mg/mL, while Vmax and kcat of the purified enzyme were found to be 0.57 international unit and 41.6 s−1, respectively. The enzyme showed inhibition by most of the divalent cations at 1 mM concentration.