Virender Kumar

Statistical Enhancement of Cyanide Degradation Using Microbial Consortium

Remediation of cyanide contaminated water bodies using microorganisms is a popular alternative over chemical and physical methods of cyanide detoxification. The objective of the present study is to develop a microbial consortium using three bacteria, i.e., Enterobacter sp. RL2a, Serratia marcescencs RL2b and Achromobacter sp. RL2c for effective degradation of simulated cyanide wastewater. In vitro cyanide degradation was optimum with 2% inoculum volume of cells; pH 6.0, 30°C temperature at 20 mM substrate concentration leading to complete cyanide removal in 36 h. Response surface methodology (RSM) approach was used for optimization of reaction conditions for cyanide degradation using 5 mg ml-1 resting cells of microbial consortium. Plackett-burman design depicted that three variables viz. time, resting cells of strain RL2b and pH exhibit positive effect on cyanide degradation. The analysis of the quadratic regression model suggested that the model was very significant as correlation coefficient (0.847) closer to 1 denotes better correlation between the observed and predicted responses. The model was validated by performing the experiment under optimum conditions, which resulted in 63% cyanide degradation in 1 h reaction and complete degradation of 20 mM cyanide in 6 h. By performing factorial design, there was 1.3 fold (33%) increases in cyanide degradation.

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.

Enzymes of aldoxime–nitrile pathway for organic synthesis

Aldoxime–nitrile pathway is one of the important routes of carbon and nitrogen metabolism in many life forms and a key interface for plant–microbe interactions. This pathway starts with transformation of amino acids to aldoximes, which are converted to nitriles and the later are ultimately hydrolyzed to acids and ammonia. Understanding and engineering of the enzymes involved in this pathway viz. cytochrome P450/CYP79, aldoxime dehydratase, nitrilase, nitrile hydratase, amidase and hydroxynitrile lyase, presents unprecedented opportunities in biocatalysis and green chemistry. Co-expressing these enzymes in prokaryotic and eukaryotic microbial hosts and tailoring their properties i.e. activity, specificity, stability and enantioselectivity may lead to develop sustainable bioprocesses for the synthesis of industrially important nitriles, amides and acids.

Alkaline active cyanide dihydratase of Flavobacterium indicum MTCC 6936: Growth optimization, purification, characterization and in silico analysis

The present work explores a rare cyanide dihydratase of Flavobacterium indicum MTCC 6936 for its potential of cyanide degradation. The enzyme is purified to 12 fold with a yield of 76%. SDS and native-PAGE analysis revealed that enzyme was monomer of 40 kDa size. The enzyme works well in mesophilic range at wide array of pH. The thermostability profile of cyanide dihydratase revealed that the enzyme is quite stable at 30 °C and 35 °C with half-life of 6 h 30 min and 5 h respectively. Km and Vmax for cyanide dihydratase of F. indicum was measured to be 4.76 mM and 45 U mg-1 with kcat calculated to be 27.3 s-1 and specificity constant (kcat/Km) to be around 5.67 mM-1 s-1. MALDI-TOF analysis of purified protein revealed that the amino acid sequence has 50% and 43% sequence identity with putative amino acid sequence of F. indicum and earlier reported cyanide dihydratase of Bacillus pumilus respectively. Homology modeling studies of cyanide dihydratase of F. indicum predicted the catalytic triad of the enzyme indicating Cys at 164, Glu at 46 and Lys at 130th position. The purified enzyme has potential applications in bioremediation and analytical sector.

Purification, Characterization and In-silico Analysis of Nitrilase from Gordonia terrae

An inducible and aromatic nitrilase from Gordonia terrae was purified with a yield of 19%. The enzyme had turnover number of 63 s⁻¹ x 10⁻¹, Km 1.4 mM and Vmax 95 Umg⁻¹ protein for benzonitrile. The nitrilase of G. terrae was active at basic pH (7-10), moderate temperature (20-45 °C) and has a half-life of 4 h at 35 °C. MALDI analysis and amino acid sequence deduced from cloned nucleotide fragment showed 97% homology with putative amidohydrolase of Gordonia sputi NBRC 100414 and G. namibiensis. The enzyme showed regioselectivity towards hydroxybenzonitriles, as different position of hydroxyl group i.e. meta-, para- and orthosubstitutions on benzonitrile effect enzyme activity. The in-silico interactions of these substrates with the predicted 3D model of this enzyme also showed differential interaction between hydroxyl group of substrates and the polar amino acids surrounding enzymes active site. This leads to different proximity and orientation of substrates vis-a-vis their interaction with catalytic residues.