The colder regions of Earth are inhabited by cold-adapted microorganisms designated as psychrophiles that are known to produce cold-active enzymes, such as peptidases, chaperones, lipases, cellulases, and phosphatases. These types of enzymes are a major part of the market of industrial enzymes. Bacteria isolated from water samples collected from the Chamba region in the Himalayas were screened for peptidase production using skim milk agar plates. Among the peptidase-producing bacteria isolated, 20% of the isolates exhibited fast growth and maximum zones of clearance, and thus, were used for further studies. The 16S rDNA sequence analysis of isolate S1DI 10 identified it as a Bacillus sp. The peptidase was cloned in pET28a vector and expressed in Escherichia coli BL21(DE3) and the His-tagged recombinant protein was purified using Ni-NTA column. The purified peptidase of SIDI 10 was found to be an alkaline, cold-active peptidase with optimal enzyme activity at 10°C and pH 8. An approach of one variable at a time was used to further study the effect of various metal ions, organic solvents and detergents on the peptidase enzyme. The peptidase activity was enhanced in the presence of Fe²⁺ and Mn²⁺ (metal ions), hexane (organic solvent), SDS- sodium dodecyl sulfate (anionic detergent) and Tween 80 (nonionic detergent). Response surface methodology (RSM) was used to determine the cumulative effect of these five variables. A 2⁵ full factorial central composite design was applied for the five independent variables to determine the optimal combinations of these constituents at the maximum peptidase activity.

The foremost concern in forensic entomology is the explicit identification of the species recovered from the crime scene. From the different orders of insects, Diptera is the prime focus in this field, followed by Coleoptera, whose identification can be extremely helpful for corpses in later decomposition stages. In this study, cytochrome oxidase subunit II (COII) was used to check its adequacy as a genetic marker and to create a reference database for eleven species belonging to five families of Coleoptera, namely, Silphidae, Staphylinidae, Histeridae, Dermestidae and Scarabaeidae, from two different states in India to assist in the accurate identification of imperative beetle species in medico-legal entomology. To achieve this, standard protocols of DNA isolation, amplification and sequencing were followed. We concluded that the COII gene can be used as a molecular marker for the identification of forensically relevant species, as observed from the similarities between the phylogenetic relationship constructed by COII and morphological data.

Background and Objective: Demodex mites are tiny parasites that live around hair follicles of mammals. The two main species of Demodex i.e. Demodex folliculorum and Demodex brevis present in humans are found near the hair follicles of eyes. The present study was to understand the presence of Demodex mites in people suffering from blepharitis in Amritsar, Punjab. Material and Methods: Demodex mites samples present in blepharitis patients were isolated from the eyelashes. DNA was isolated from three mites and used for PCR amplification of mitochondrial (mt) 16S rDNA. The amplified PCR product were purified and used for molecular identification. Results: The amplified mt16s rDNA product was sequenced and subjected to BLAST search in the NCBI database for molecular identification. The identified mite belongs to Demodex folliculorum species. The phylogenetic tree constructed by using mt16s rDNA sequence suggests that D. folliculorum is closer to D. canis than to D. brevis. Conclusion: All the three isolates belong to D. folliculorum and the mitochondrial DNA 16S rDNA partial sequence is applicable for phylogenetic relationship analysis.

Background: Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. Results: In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% β-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. Conclusion: MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfp_mut1. The promoter region of the cry gene was amplified to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfp_mut1. The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfp_mut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by fluorescence spectroscopy. The fluorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable fluorescence that increased with time up to 12 hours, but the increase in the fluorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specific activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.