Ashok Kumar Verma

Isolation of phytase-producing bacteria from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea)

Phytase-producing bacteria (PPB) is being investigated as plant growth promoting rhizobacteria (PGPR) to improve the phosphorus (P) nutrition and growth of plants grown in soil with high phytate content. Phytate is dominant organic P forms in many soils and must be hydrolyzed to be available for plants. Indian mustard (Brassica juncea) is a plant with economic importance in agriculture and phytoremediation, therefore biotechnological tools to improve growth and environmental stress tolerance are needed. In this study, we isolated and characterized PPB from Himalayan soils and evaluated their effect on growth and P uptake by B. juncea under greenhouse conditions. Sixty five PPB were isolated and based on phytate hydrolysis, three efficient PPB were chosen and identified as Acromobacter sp. PB-01, Tetrathiobacter sp. PB-03 and Bacillus sp. PB-13. Selected PPB showed ability to grow at wide range of pH, temperature and salt concentrations as well as to harbour diverse PGPR activities, such as: solubilization of insoluble Ca-phosphate (193–642xa0μgxa0ml−1), production of phytohormone indole acetic acid (5–39xa0μgxa0ml−1) and siderophore. Tetrathiobacter sp. PB-03 and Bacillus sp. PB-13 showed 50 and 70xa0% inhibition of phytopathogen Rhizoctonia solani, respectively. Greenhouse potting assay also showed that the bacterization of B. juncea seeds with Tetrathiobacter sp. PB-03 and Bacillus sp. PB-13 significantly increased the biomass and P content in 30xa0days old seedlings. This study reveals the potential of PPB as PGPR to improve the growth of B. juncea.

Development of a β-glucosidase hyperproducing mutant by combined chemical and UV mutagenesis

The extracellular β-glucosidase from microorganisms is generally produced in low levels. Therefore, in this study, a β-glucosidase hyperproducing mutant was developed by multiple exposures of ethyl methyl sulfonate (EMS) and ultraviolet (UV) radiation (both individually and jointly) to Bacillus subtilis strain (PS). The developed mutants were screened, selected and characterized. The mutant, PS-UM1 developed after UV exposure alone, indicated a small increase in β-glucosidase production (718xa0U/l) in comparison to the wild-type strain, PS (675xa0U/l). The mutant, PS-CM5 developed after EMS exposure alone, displayed a slightly better production (762xa0U/l) than both the above strains. However, after exposure of the wild-type strain to both UV and EMS mutagens jointly, a better mutant (PS-CM5-UM3) was developed with 1.2-fold increase in production (806xa0U/l). Further, optimization of culture conditions by classical “one-variable-at-a-time” approach was done to determine the optimum, pH, temperature and nitrogen sources. The selected mutant (PS-CM5-UM3) produced up to 1,797xa0U/l enzyme and was found to be stable for ten generations. The β-glucosidase from the selected mutant (PS-CM5-UM3) was concentrated and purified using ammonium sulfate, dialysis and size-exclusion chromatography. The enzyme displayed maximal activity at 60xa0°C and it was found to be fairly stable at temperatures up to 70xa0°C for 30xa0min. Its molecular weight was determined to be around 60xa0kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Secretory expression, characterization and docking study of glucose-tolerant β-glucosidase from B. subtilis.

The thermostable, glucose tolerant β-glucosidase gene (bgl) of Glycoside hydrolase family 1, isolated from Bacillus subtilis, was cloned and overexpressed in Escherichia coli. The bgl has open reading frame of 1,407 bp, encoding 469 amino acids with predicted molecular weight of 53 kDa. The recombinant protein (BGL) was purified 10.76 fold to homogeneity with specific activity of 54.04U/mg and recovery of 38.67%. The purified BGL was optimally active at pH 6.0 and temperature 60°C. The enzyme retained more than 85% of maximum activity after 1h preincubation at 60°C. The kinetic analysis indicated that BGL has highest catalytic efficiency (Kcat/Km) against p-nitrophenyl-β-d-xylopyranoside (654.58 mM(-1)s(-1)) followed by p-nitrophenyl-β-d-glucopyranoside (292.53 mM(-1)s(-1)) and p-nitrophenyl-β-d-galactopyranoside (61.17 mM(-1)s(-1)). The Ki value for glucose and δ-gluconolactone was determined to be 1.9 mM and 0.018 mM, respectively. The BGL exhibited high tolerance against detergents and organic solvents. The homology modeling revealed that protein has 19 α-helices and 4 β-sheets and adopted (α/β)8 TIM barrel structure. Substrate docking and LigPlot analysis depicted the amino acids of active site involved in hydrogen bonding and hydrophobic interactions with substrates. The efficient BGL secretion with exploration of structural and functional relationship offer vistas for large scale production and various industrial applications.

Cloning, Sequencing, and In Silico Analysis of β-Propeller Phytase Bacillus licheniformis Strain PB-13.

β-Propeller phytases (BPPhy) are widely distributed in nature and play a major role in phytate-phosphorus cycling. In the present study, a BPPhy gene from Bacillus licheniformis strain was expressed in E. coli with a phytase activity of 1.15u2009U/mL and specific activity of 0.92u2009U/mg proteins. The expressed enzyme represented a full length ORF “PhyPB13” of 381 amino acid residues and differs by 3 residues from the closest similar existing BPPhy sequences. The PhyPB13 sequence was characterized in silico using various bioinformatic tools to better understand structural, functional, and evolutionary aspects of BPPhy class by multiple sequence alignment and homology search, phylogenetic tree construction, variation in biochemical features, and distribution of motifs and superfamilies. In all sequences, conserved sites were observed toward their N-terminus and C-terminus. Cysteine was not present in the sequence. Overall, three major clusters were observed in phylogenetic tree with variation in biophysical characteristics. A total of 10 motifs were reported with motif “1” observed in all 44 protein sequences and might be used for diversity and expression analysis of BPPhy enzymes. This study revealed important sequence features of BPPhy and pave a way for determining catalytic mechanism and selection of phytase with desirable characteristics.

Targeting of Metabolic Pathways for Genetic Engineering to Combat Abiotic Stress Tolerance in Crop Plants

Abiotic stress is a serious threat to sustainable agriculture. Plant adaptation to suboptimal environmental conditions is controlled by cascades of molecular networks involved in stress perception, signal transduction, activation of new biochemical pathways, and repression of others. Protective metabolic adaptations alter physiological homeostatic of the whole plant. Use of modern molecular biology tools for elucidating abiotic stress tolerance relies on expression of specific stress-related gene and gene encoding enzymes present in biosynthetic pathways of functional and structural metabolites. Paramount among the mechanisms are reactive oxygen species scavenging, maintenance of ion uptake and water balance, and accumulation of compatible solutes such as betaines, proline, and alcohol sugars. Instead of single gene manipulation approach, targeting the regulatory machinery involving transcription factors has emerged as new potent tool for developing stress-tolerant transgenic crops. Under this chapter we highlight recent advances to our knowledge that emphasize the development of transgenic crops with improved stress tolerance by targeting different genes of various metabolic pathways.

Quality Evaluation of Jaggery Chocolate Under Various Storage Conditions

Characteristics of developed jaggery chocolate under different storage conditions were investigated. Jaggery chocolates were prepared by standard method and packaged in different packaging materials subsequently subjecting the samples to different storage conditions, viz., incubator (40–45°C), refrigerator (8–10°C) and ambient conditions (25–35°C). Moisture content, hardness, optical density and sensory properties of the stored jaggery chocolates were determined at an interval of 15xa0days during the 60xa0days storage period. During different storage conditions, moisture content, optical density and hardness of chocolates varied from 6.09 to 8.42%, 0.12 to 0.529 and 4.29 kgf to 11.58 kgf respectively. Results showed an initial increase in moisture content followed by decrease with increasing storage period. However, optical density, hardness and appearance increased gradually with the increase in storage period in all storage conditions. Quality of prepared chocolates was evaluated for its sensory characteristics on 1–10 scale. The colour, taste, texture, flavour and overall acceptability were found to be in range of 6–8.5.