Four trypsin inhibitors, AMTI-I, AMTI-II, AMTI-III, and AMTI-IV, have been isolated and purified to homogeneity from the seeds of Abelmoschus moschatus following ammonium sulphate fractionation, DEAE-cellulose ion exchange chromatography and gel permeation on Sephadex G-100, and their molecular weights were determined to be 22.4, 21.2, 20.8 and 20.2 kDa respectively by SDS-PAGE. While all the four inhibitors were very active against bovine trypsin, two of them (AMTI-III and AMTI-IV) showed moderate activity towards bovine chymotrypsin. AMTI-I and AMTI-II were found to be glycoproteins with neutral sugar content of 2.8 and 4 %, respectively, and all the four inhibitors were devoid of free sulphhydryl groups. The inhibitors were quite stable up to 80 °C for 10 min and were not affected at alkaline as well as acidic conditions tested. Treating them with 8 M urea and 1 % SDS for 24 h at room temperature did not result in any loss of their antitryptic activities. However, they lost considerable antitryptic activity when treated with 6 M guanidine hydrochloride. Activities of the inhibitors were unaffected even after their reduction with DTT suggesting that disulphide bonds are not needed for their inhibitory activities.

Background Microbial community is one of the diversified communities of the marine environment. Studies have shown that microorganisms isolated from the marine environment are metabolically active and have adapted to life in the ocean. The marine microorganisms use various survival strategies to combat heavy metal stress and decolorization of various textile dyes, thus playing an important role in the bioremediation of cadmium and degradation of textile dyes. The present study deals with the isolation and 16S rRNA molecular characterization of M3 and M8 bacterial strains isolated from marine water samples collected from Visakhapatnam harbor. M3 and M8 isolates were also checked for their efficacy in the removal of cadmium and decolorization of various textile dyes from the environment. Results The water sample was subjected to tube dilution method to isolate bacterial strains, and ten different isolates were screened. The biochemical tests were performed for the isolates to prove their validity and 16S rRNA molecular sequencing and phylogenetic analysis for species identification. Out of interest, two bacterial strains, namely, M3 and M8 were subjected to 16S rRNA molecular sequencing and phylogenetic analysis and were identified as Bacillus subtilis and Pseudomonas resinovorans. The two bacterial strains showed promising dye degradation property when checked with nine different textile dyes of wavelength ranging from 400 to 600 nm and removal of cadmium from the growth medium. Conclusion The present study demonstrates the isolates M3 and M8 to be potential strains having dye decolorization and bioremediation of cadmium applications.

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Objective:The objective of the present study was to characterize the monoheaded trypsin inhibitors, Abelmoschus moschatus trypsin inhibitor-I (AMTI-I) and AMTI-II from the seeds of A. moschatus with respect to their specificity, mode of action, and active site residues.Methods: Standard methods were followed in determining inhibitory activities of monoheaded inhibitors. IC 50 values and inhibitory constants (Ki) of AMTI-I and AMTI-II were determined. Studies on complex formation and chemical modification of inhibitors were performed.Results: AMTI-I and AMTI-II were found to be serpins, strongly active against trypsin, moderately active against porcine elastase, Staphylococcus aureus protease, and Aspergillus oryzae protease. AMTI-I and AMTI-II have shown non-competitive type of inhibition toward bovine trypsin with K i values of inhibitors for trypsin found to be 0.25±0.02 nM and 0.22±0.06 nM, respectively. Complex studies revealed the formation of stable 1:1 complex of trypsin with both AMTI-I and AMTI-II. Chemical modification of the functional groups of the inhibitors by selective reagents indicated that arginine residues are essential for their trypsin inhibitory activities. Conclusion:Investigations on the specificity of protease inhibitors are important for understanding their physiological role, control mechanisms involved in the regulation of proteolysis in biological systems and mode of action.

Alkaline proteases are active from neutral to alkaline pH range and have extensive applications in detergent and leather industries. In the present research, bacteria isolated from marine water samples were screened for proteolytic activity. Among the isolates, M2 showed maximum proteolysis with a clear zone when cultured on skim milk agar plates at 37°C for 24 h. Molecular identification using 16S rRNA sequencing and phylogenetic analysis revealed that M2 has sequence identity (99.93%) to Bacillus paramycoides. SEM analysis was carried for determining the morphology of M2 and also for enzyme treated skin. FAME analysis using GCMS was performed for the determination of fatty acids in the strain. The selected isolate was inoculated into protease production medium under submerged fermentation conditions at 37ºC for 48 h with a constant agitation of 120 rpm. Protease activity was determined under varying conditions of pH, incubation temperature, carbon and nitrogen sources, metal ions and NaCl (1- 5%) using casein as substrate. The isolate M2 utilized molasses and peptone as carbon and nitrogen sources for better alkaline protease production at 40°C and pH 10 under optimal conditions. The dehairing experiments with M2 alkaline protease revealed dehairing efficacy of protease over chemical treatment. Hence, extracellular alkaline protease from M2 isolate could find potential application in leather processing industries and can be exploited commercially.