Amit Pratush

Molecular diversity and functional variability of environmental isolates of Bacillus species

In the present study, out of 264 phosphate (P) solubilizing Bacillus strains isolated from apple rhizosphere, only twelve isolates were found to be efficient (showed most of the plant growth promoting activity) which were further characterized at molecular level using 16S rDNA partial gene sequencing. Out of 12 isolates, MZPSB 207 was found to be most efficient P-solubilizing (864.71xa0μg/ml) isolate which also showed indole acetic acid production (51.83xa0μg/ml), siderophore production, ammonia production, antagonistic property (against Rhizoctonia solani and Fusarium oxysporum), hydrolytic enzymes productions (protease, chitinase and cellulase), 1-aminocyclopropane-1-carboxylate (ACC) deaminase production (7.7xa0μm αKB mg-1xa0h-1). The in-vitro seed germination assay showed that Bacillus (twelve isolates) inoculated seeds showed more seed germination and seedling vigor rate as compared to uninoculated control treatment.For the genetic diversity studies of efficient 12 strains, the polyphasic approach using 16S-rDNA, Repetitive element sequence (rep) based PCR (ERIC-PCR and BOX-PCR) were used. Based on 16S rDNA partial gene sequencing the isolated Bacillus genus was divide into four groups. First group (five isolates), second group (two isolates), third group (three isolates) and fourth group (two isolates) which showed close genetic relatedness to the B. subtilis, B. pumulis, B. megaterium and B. amyloliquefaciens, respectively. The rep PCR fingerprinting showed variability between and within the species. The large variability was showed by ERIC-PCR whereas some variability was showed by BOX-PCR. The results clearly showed that 16S rRNA gene sequencing is unable to discriminate the isolates at strain level. But rep-PCR fingerprinting is excellent tool to characterize and discriminate the strains at the genomic level.

Generation of mutant of Rhodococcus rhodochrous PA-34 through chemical mutagenesis for hyperproduction of nitrile hydratase

Rhodococcus rhodochrous PA-34 has been reported to produce nitrile hydratase enzyme that converts 3-cyanopyridine to nicotinamide. A mutant of R. rhodochrous PA-34 was generated through chemical mutagenesis using N-methyl-N-nitro-N-nitrosoguanidine (MNNG) that exhibited 2 times higher nitrile hydratase activity as compared to wild strain. The reaction conditions using resting cells of this mutant strain for the conversion of nicotinamide were optimized. Under the optimized reaction conditions the mutant strain exhibited maximum nitrile hydratase activity [7.8 U/mgdcm (milligram dry cell mass)] at 55 degrees C in 0.3 M potassium phosphate buffer (pH 5.5).

Cloning, Sequencing, and Expression of Nitrile Hydratase Gene of Mutant 4D Strain of Rhodococcus rhodochrous PA 34 in E. coli

The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9xa0U/mgxa0dcw) as compared to the wild (4.1xa0U/mgxa0dcw) whereas it is less than the mutant strain (8.4xa0U/mgxa0dcw). Addition of cobalt ion in Luria–Bertani medium is needed up to a very small concentration (0.4xa0mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6xa0h of incubation and was purified with a yield of 56xa0% with specific activity of 37.1xa0U/mg protein.

Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34

Nitrile hydratase (NHase; E.C. 4.2.1.84) has been purified and characterized using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography from the mutant 4D of Rhodococcus rhodochrous PA-34. The SDS-PAGE and MALDI-TOF analysis of the purified enzyme revealed that it is dimmer consisting of α- and β-subunits with a molecular mass of 25 and 30xa0kDa, respectively. The Km and Vmax values were 102xa0mM and 350.8xa0μmol/min/mg using 3-cyanopyridine as substrate. The purified NHase was stable in higher concentration of potassium ions and in acidic pH 5.5 as compared to NHase of the wild R. rhodochrous PA-34. The analysis of the N-terminal amino acid sequence of this enzyme revealed that this enzyme has 90xa0% homology with the high molecular weight nitrile hydratase of R. rhodochrous J1.