Genetic and Phenotypic Heterogeneity of the Nocardiopsis alba Strains of Seawater.

This study deals with the genetic and phenotypic heterogeneity of the marine Nocardiopsis alba strains isolated during pre-monsoon, monsoon and post-monsoon seasons. The isolates were characterized for their morphological and biochemical attributes, growth media preferences, antibiotic susceptibility and extracellular enzyme secretion. Nocardiopsis alba strains were assessed against 12 different antibiotics, and the responses were expressed in terms of the multiple antibiotic resistance (MAR) number. The majority of the strains produced multiple extracellular enzymes: proteases, amylases and lipases. Further, the strains were characterized on the basis of 16S rRNA gene sequencing and the majority were identified as Nocardiopsis alba along with few strains of Streptomyces lopnurensis, Nocardiopsis synnemataformans and Nocardiopsis dassonvillei. Neighbor-joining (NJ) phylogenetic tree suggested variation among the genetically similar Nocardiopsis alba species. The study establishes significant heterogeneity with respect to genetic and phenotypic characteristics of the strains of Nocardiopsis alba. Phylogenetic tree and phenogram-based comparison reflect the heterogeneity in terms of different clustering patterns of the strains. Further, the whole genome sequence data available in the literature also confirm the observed heterogeneity. Nocardiopsis alba strains displayed a relatively regressive pattern of dependence on the environmental factors based on the canonical correspondence analysis plot. The study represents cultivation, characterization, phylogenetic analysis and enzymatic potential of the Nocardiopsis alba species of seawater origin.

Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5.

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.

Phylogeny, novel bacterial lineage and enzymatic potential of haloalkaliphilic bacteria from the saline coastal desert of Little Rann of Kutch, Gujarat, India.

This report describes cultivation-dependent diversity, phylogeny and enzymatic potential of the haloalkaliphilic bacteria isolated from the unvegetated desert soil of yet unexplored, saline desert of Little Rann of Kutch (LRK), India. The LRK is a unique ecosystem displaying a combination of Dry Rann and Wet Rann. A total of 25 bacteria were isolated and characterized on the basis of colony morphology, biochemical profile, sugar utilization, secretion of the extracellular enzymes and antibiotic sensitivity. Further, the identification and phylogenetic relatedness of 23 bacteria were established by the analysis of 16S rRNA gene sequences. The phylogenetic analysis indicated that the isolates belong to the phylum Firmicutes, comprising low G + C, Gram-positive bacteria, with different genera: Bacillus (~ 39%), Staphylococcus (~ 30%), Halobacillus (~ 13%), Virgibacillus (~ 13%), Oceanobacillus (~ 4%). Majority of the bacterial isolates produced proteases (30% isolates) followed by cellulases (24% isolates), CMCases (24% isolates) and amylases (20% isolates). Halobacillus, Virgibacillus and Bacillus predominantly produced hydrolases, while many produced multiple enzymes at high salinity and alkaline pH. Highest antibiotic resistance was observed against Ampicillin and Penicillin (32%) followed by Cefaclor (20%); Colistin, Cefoperazone and Cefotaxime (16%); Cefuroxime (12%); Gentamycin and Cefixime (8%); Erythromycin, Cefadroxil, Azithromycin, Co-trimoxazole, Amoxycillin, Norfloxacin, Cefpodoxime, Amikacin and Augmentin (4%). KJ1-10-99 and KJ1-10-93 representing < 97% of 16S rRNA gene sequence similarity belong to a novel lineage within the family Bacillaceae. Comparison of the phenogram and phylogram revealed the contradiction of the phenogram pattern and the phylogenetic placement of the isolates. The isolates belonging to same species have shown considerable phenotypic variation. The study on the cultivable haloalkaliphilic bacteria of an unexplored enigmatic niche reflects ecological and biotechnological significance.

Purification strategies, characteristics and thermodynamic analysis of a highly thermostable alkaline protease from a salt-tolerant alkaliphilic actinomycete, Nocardiopsis alba OK-5.

An alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 was purified to homogeneity by 27 and 13 fold with a yield of 35 and 13% using two-steps and one-step method, respectively. The purification methods involved hydrophobic interaction on phenyl sapharose matrix. The apparent molecular mass was 20 kDa. The temperature optimum shifted from 70 to 80°C in 4M NaCl and 30% Na-glutamate, with significant stability at 60-80°C in Na-glutamate. Deactivation rate constant (K(d)) increased and half life (t(1/2)) decreased with the increasing temperatures from 37 to 80°C. The order of stability was: 30% Na-glutamate>4M NaCl>2M NaCl>0M NaCl. The enzyme was stable even at 80°C in 30% Na-glutamate with K(d) 4.11 and t(1/2) 168.64 min. The activation energies (E), enthalpy (ΔH*) and entropy (ΔS*) for protease deactivation in with Na-glutamate were 31.97 kJ/mole, 29.23 kJ/mole and -211.83 J/mole, respectively. The change in free energy (ΔG*) for protease deactivation at 60°C in 30% Na-glutamate was 101.70 kJ/mole. Protease had the highest activity and stability at pH 10-11. While the enzyme was highly resistant against chemical denaturation, it had varied responses to metal ions. Complete inhibition by PMSF confirmed serine nature of the protease. Na-glutamate, H(2)O(2), ß-mercaptoethanol and different surfactants enhanced the activity.