Advances in cloning, structural and bioremediation aspects of nitrile hydratases.

Nitrile hydratase (NHase) is a prominent enzyme in many microorganisms for its nitrile metabolism. The potentiality in the bioconversion of nitriles to its high-value amides has been extensively used in industries for the production of acrylamide and nicotinamide which are essential chemicals. Enzymologists are still considering NHases for its potential biotechnological applications including biotransformations and bioremediations. But most of the nitrile hydratases have limitations like the low expression, low thermostability and enantioselectivity. Though considerable data has been generated in the area of gene configuration, crystal structure, kinetic mechanism and photoreactivity of NHases, there is a need for constant improvement to develop a robust biocatalyst for bioremediation of toxic nitriles. With these considerations, in the present review, we report advances with the main focus to structure, catalytic mechanism, cloning strategy, gene expression, bioinformatic tools, metagenomics, thermostability and current bioremediation applications of NHases.

Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95.

A putrescine monooxygenase from Shewanella putrefaciens 95 (SpPMO) is the initial enzyme catalyzing the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for the synthesis of a siderophore putrebactin was identified. This PMO clustered together with known characterized NMOs from Shewanella baltica, Bordetella pertussis, Erwinia amylovora, Streptomyces sp. Gordonia rubripertincta, Pseudomonas aeruginosa and outgrouped from Escherichia coli, Nocardia farcinica, and Rhodococcus erythropolis. The deduced SpPMO protein showed 53% and 36% sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively. In this investigation, we have cloned the complete 1518bp coding sequence of pubA from Shewanella putrefaciens 95 encoding the corresponding protein SpPMO. It comprises 505 amino acid residues in length and has approximately a molecular weight of 54 kDa. Chaperone-assisted heterologous expression of SpPMO in pET151Topo expression vector under the control of bacteriophage T7 promoter permitted a stringent IPTG dependent expression. It has been successfully cloned, overexpressed and purified as a soluble His6 -tagged enzyme using E. coli as a cloning and expression host. The expression of recombinant SpPMO was confirmed by Western blotting using anti-His6 antibody. The purified protein showed FAD and NADPH dependent N-hydroxylation activity. This study has paved a way to understand the hydroxylation step of putrebactin synthesis which can be further investigated by studying its kinetic mechanism and physiological role.