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1.  In Silico Analysis of β-Galactosidases Primary and Secondary Structure in relation to Temperature Adaptation 
Journal of Amino Acids  2014;2014:475839.
β-D-Galactosidases (EC hydrolyze the terminal nonreducing β-D-galactose residues in β-D-galactosides and are ubiquitously present in all life forms including extremophiles. Eighteen microbial β-galactosidase protein sequences, six each from psychrophilic, mesophilic, and thermophilic microbes, were analyzed. Primary structure reveals alanine, glycine, serine, and arginine to be higher in psychrophilic β-galactosidases whereas valine, glutamine, glutamic acid, phenylalanine, threonine, and tyrosine are found to be statistically preferred by thermophilic β-galactosidases. Cold active β-galactosidase has a strong preference towards tiny and small amino acids, whereas high temperature inhabitants had higher content of basic and aromatic amino acids. Thermophilic β-galactosidases have higher percentage of α-helix region responsible for temperature tolerance while cold loving β-galactosidases had higher percentage of sheet and coil region. Secondary structure analysis revealed that charged and aromatic amino acids were significant for sheet region of thermophiles. Alanine was found to be significant and high in the helix region of psychrophiles and valine counters in thermophilic β-galactosidase. Coil region of cold active β-galactosidase has higher content of tiny amino acids which explains their high catalytic efficiency over their counterparts from thermal habitat. The present study has revealed the preference or prevalence of certain amino acids in primary and secondary structure of psychrophilic, mesophilic, and thermophilic β-galactosidase.
PMCID: PMC3982409  PMID: 24790757
2.  Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of Geobacillus pallidus BTP-5x MTCC 9225 
Indian Journal of Microbiology  2011;52(1):76-82.
The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90–95% (mol mol−1) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.
PMCID: PMC3298591  PMID: 23449317
Geobacillus pallidus BTP-5x MTCC 9225; Acetohydroxamic acid; Thermophilic amidase; Acyl transferase activity; Hydroxamic acid
3.  Comparative analysis of amino acid sequences from mesophiles and thermophiles in respective of carbon–nitrogen hydrolase family 
3 Biotech  2013;3(6):491-507.
A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of protein from the nitrilase/cyanide hydratase family. This family contains nitrilases that break carbon–nitrogen bonds and appear to be involved in the reduction of organic nitrogen compounds and ammonia production. They all have distinct substrate specificity and include nitrilase, cyanide hydratases, aliphatic amidases, beta-alanine synthase, and a few other proteins with unknown molecular function. These sequences were analyzed for different physical and chemical properties and to relate these observed differences to the thermostability properties, phylogenetic tree construction and the evolutionary relationship among them. In this work, in silico analysis of amino acid sequences of mesophilic (15) and thermophilic (archaea, 15 and bacteria, 15) proteins has been done. The physiochemical properties of these three groups of nitrilase/cyanide hydratase family also differ in number of amino acids, molecular weight, pI values, positively charged ions, i.e. Arg + Lys, aliphatic index and grand average of hydropathacity (GRAVY). The amino acid Ala (1.37-fold) was found to be higher in mesophilic bacteria as compared to thermophilic bacteria but Lys and Phe were found to be significantly high (1.43 and 1.39-fold, respectively) in case of thermophilic bacteria. The amino acids Ala, Cys, Gln, His and Thr were found to be significantly higher (1.41, 1.6, 1.77, 1.44 and 1.29-fold, respectively) in mesophilic bacteria as compared to thermophilic archaea, where Glu, Leu and Val were found significantly high (1.22, 1.19 and 1.26-fold, respectively).
PMCID: PMC3824785
Carbon–nitrogen bonds; Nitrilase/cyanide hydratase family; Nitrilase; Cyanide hydratase; Thermostability; Phylogenetic tree
4.  Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34 
3 Biotech  2012;3(2):165-171.
Nitrile hydratase (NHase; E.C. 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 30 kDa, respectively. The Km and Vmax values were 102 mM and 350.8 μmol/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 90 % homology with the high molecular weight nitrile hydratase of R. rhodochrous J1.
PMCID: PMC3597139
Characterization; Mutant; Purification; Rhodococcus rhodochrous PA-34
5.  Nocardia globerula NHB-2 nitrilase catalysed biotransformation of 4-cyanopyridine to isonicotinic acid 
AMB Express  2012;2:25.
Isonicotinic acid (INA) is an important pyridine derivative used in the manufacture of isoniazid (antituberculosatic drug) and other pharmaceutically important drugs. Nitrilase catalysed processes for the synthesis of pharmaceutically important acids from their corresponding nitriles are promising alternative over the cumbersome, hazardous, and energy demanding chemical processes. Nitrilase of Nocardia globerula NHB-2 (NitNHB2) is expressed in presence of isobutyronitrile in the growth medium (1.0% glucose, 0.5% peptone, 0.3% beef extract, and 0.1 % yeast extract, pH 7.5). NitNHB2 hydrolyses 4-cyanopyridine (4-CP) to INA without accumulation of isonicotinamide, which is common in the reaction catalysed via fungal nitrilases. The NitNHB2 suffers from substrate inhibition effect and hydrolysing activity up to 250 mM 4-CP was recorded. Complete conversion of 200 mM 4-CP to INA was achieved in 40 min using resting cell concentration corresponding to 10 U mL-1 nitrilase activity in the reaction. Substrate inhibition effect in the fed batch reaction (200 mM substrate feed/40min) led to formation of only 729 mM INA. In a fed batch reaction (100 mM 4-CP/20min), substrate inhibition effect was encountered after 7th feed and a total of 958 mM INA was produced in 400 min. The fed batch reaction scaled up to 1 L and 100% hydrolysis of 700 mM of 4-CP to INA at 35°C achieved in 140 min. The rate of INA production was 21.1 g h-1 mgDCW-1. This is the fastest biotransformation process ever reported for INA production with time and space productivity of 36 g L-1 h-1 using a bacterial nitrilase.
PMCID: PMC3403844  PMID: 22537922
4-Cyanopyridine; Isonicotinic acid; Isobutyronitrile; Bacterial nitrilase; Biotransformation; Substrate inhibition; Fed batch

Results 1-5 (5)