Neerja Thakur

Mining of Microbial Genomes for the Novel Sources of Nitrilases

Next-generation DNA sequencing (NGS) has made it feasible to sequence large number of microbial genomes and advancements in computational biology have opened enormous opportunities to mine genome sequence data for novel genes and enzymes or their sources. In the present communication in silico mining of microbial genomes has been carried out to find novel sources of nitrilases. The sequences selected were analyzed for homology and considered for designing motifs. The manually designed motifs based on amino acid sequences of nitrilases were used to screen 2000 microbial genomes (translated to proteomes). This resulted in identification of one hundred thirty-eight putative/hypothetical sequences which could potentially code for nitrilase activity. In vitro validation of nine predicted sources of nitrilases was done for nitrile/cyanide hydrolyzing activity. Out of nine predicted nitrilases, Gluconacetobacter diazotrophicus, Sphingopyxis alaskensis, Saccharomonospora viridis, and Shimwellia blattae were specific for aliphatic nitriles, whereas nitrilases from Geodermatophilus obscurus, Nocardiopsis dassonvillei, Runella slithyformis, and Streptomyces albus possessed activity for aromatic nitriles. Flavobacterium indicum was specific towards potassium cyanide (KCN) which revealed the presence of nitrilase homolog, that is, cyanide dihydratase with no activity for either aliphatic, aromatic, or aryl nitriles. The present study reports the novel sources of nitrilases and cyanide dihydratase which were not reported hitherto by in silico or in vitro studies.

Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051xa0U/mg dcw (dry cell weight) to 0.209xa0U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80xa0°C were 5 and 1xa0h, respectively. Activity of this enzyme was strongly inhibited by Hg2+, Ag+ and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9

AbstractMutants of Gordonia terrae were ngenerated using chemical mutagens for better activity, stability and higher substrate/product tolerance of its nitrilase enzyme. Mutant E9 showed two-time increase in activity and tolerated p-hydroxybenzonitrile (p-HBN) up to 50xa0mM. Response surface methodology and inducer mediation approach further enhanced the production of enzyme to 2.5-fold. The bench scale production of p-hydroxybenzoic acid (p-HBA) was carried out in a fed-batch reaction (500-mL scale) using whole-cell nitrilase of mutant E9 in 0.1xa0M potassium phosphate buffer (pH 8.0) at 40xa0°C. Total six feedings each at an interval of 45xa0min resulted in accumulation of 360xa0mM (21.6xa0g) of p-HBA with a purity of 99xa0%. The catalytic and volumetric productivity of bioprocess using mutant G. terrae was improved to 1.8xa0gxa0h−1xa0gDCW−1 and 43.2xa0gxa0L−1, respectively, from 0.78xa0gxa0h−1xa0gDCW−1 and 28.8xa0gxa0L−1 using resting cells of wild strain. Km and Vmax of purified nitrilase from mutant E9 were 55xa0Uxa0mg−1 and 1.8xa0mM for p-HBN with a higher turnover number of 36xa0s−1xa0×xa010−3.Graphical Abstract

β-Galactosidase from Lactobacillus brevis PLA28: Purification, Characterization and Synthesis of Galacto-oligosaccharides

Objective: Purification and characterization of β-galactosidase from Lactobacillus brevis PLA28 were carried out and transgalactosylation activity was also studied for the synthesis of galacto-oligosaccharides (GOS). Methods: β-Galactosidase was purified by using ammonium sulphate precipitation method and hydrophobic interaction chromatography. Reaction conditions were optimized for the assay of this β-galactosidase. GOS synthesis was carried out under the optimized reaction conditions in batch mode and the product formed was detected by thin layered chromatography (TLC). Results: β-Galactosidase was purified to 6.6 fold with a yield of 6% and specific activity of 4 U/mg protein. Molecular weight of the purified β-galactosidase was found to be 45 and 60 kDa on SDS PAGE and 105 kDa on native PAGE. The temperature and pH optima of purified enzyme were 30°C and pH 6.5 respectively. The enzyme was found to be stable at 30°C for 6 h. Vmax and KM of the purified β-galactosidase were calculated to be 6.6 U/mg protein and 8.33 mM respectively. GOS synthesis was observed at pH 6.5, 30°C in 6-8 h of incubation by purified enzyme. Conclusion: Lactobacillus brevis PLA28 β-galactosidase has exhibited capability for transgalactosylation reaction with lactose conversion to synthesize GOS.

Nitrile Metabolizing Enzymes in Biocatalysis and Biotransformation

Nitrile metabolizing enzymes, i.e., aldoxime dehydratase, hydroxynitrile lyase, nitrilase, nitrile hydratase, and amidase, are the key catalysts in carbon nitrogen triple bond anabolism and catabolism. Over the past several years, these enzymes have drawn considerable attention as prominent biocatalysts in academia and industries because of their wide applications. Research on various aspects of these biocatalysts, i.e., sources, screening, function, purification, molecular cloning, structure, and mechanisms, has been conducted, and bioprocesses at various scales have been designed for the synthesis of myriads of useful compounds. This review is focused on the potential of nitrile metabolizing enzymes in the production of commercially important fine chemicals such as nitriles, carboxylic acids, and amides. A number of opportunities and challenges of nitrile metabolizing enzymes in bioprocess development for the production of bulk and fine chemicals are discussed.

Aliphatic amidase of Rhodococcus rhodochrous PA-34: Purification, characterization and application in synthesis of acrylic acid.

An intracellular aliphatic amide degrading inducible amidase produced by Rhodococcus rhodochrous PA-34 was characterized and acrylic acid synthesis from acrylamide was carried out using whole cell amidase. A bioprocess was developed at 50 ml fed batch reaction using 400 mM acrylamide feeding at an interval of 30 min resulted in the production of 4 g acrylic acid with volumetric and catalytic productivity of 80 g/l and 19 g/g/h respectively. The amidase of this organism had molecular weight of 40 kDa and was purified to 8.5 fold with 8% yield. This enzyme was active within the temperature range of 30 to 60 °C, with optimum temperature 45 °C and pH 7.5. The Vmax, Km, and kcat of purified amidase were calculated as 250 U/mg protein, 4.5 mM, and 166 sec-1 for acrylamide. The enzyme showed tolerance to metal chelating agent (EDTA) and was strongly inhibited by heavy metal ions Hg2+, Ag2+, Cu2+ and Co2+. R. rhodochrous PA-34 amidase preferentially hydrolyzed small aliphatic toxic amide such as acrylamide. Thus, the amidase of R. rhodochrous PA-34 is promising biocatalyst for the synthesis of industrially important acids and biodegradation of toxic amides.

Organic solvent tolerant metallo protease of novel isolate Serratia marcescens PPB-26: production and characterization

Proteases are a class of enzymes that catalyze hydrolysis of peptide bonds of proteins. In this study, 221 proteolytic bacterial isolates were obtained by enrichment culture method from soils of various regions of Himachal Pradesh, India. From these a hyper producer of protease was screened and identified by morphological and physiological testing and by 16S rDNA sequence as Serratia marcescens PPB-26. Statistical optimization of physiochemical parameters enhanced the protease production by 75xa0%. Protease of S. marcescens PPB-26 was classified as a metalloprotease. It showed optimal activity at 30xa0°C, pH 7.5 (0.15xa0M Tris–HCl buffer) and with 0.8xa0% substrate concentration. It had Kmxa0=xa00.3xa0%, Vmaxxa0=xa034.5xa0μmolxa0min−1 mg−1 protein and a half life of 2xa0days at 30xa0°C. The enzyme was stable in most metal ions but showed increased activity with Fe2+ and Cu2+ while strong inhibition with Co2+ and Zn2+. Further investigation showed that the enzyme could not only retain its activity in various organic solvents but also showed increased activity with methanol and ethanol. The reported metalloprotease is thus a potential candidate for carrying out industrial peptide synthesis.