Debjani Dutta

Extracellular alkaline protease from Bacillus licheniformis NCIM‐2042: Improving enzyme activity assay and characterization

Optimization of enzyme assay conditions for alkaline protease from Bacillus licheniformis NCIM‐2042 was carried out by a statistical approach. Four key determinants such as pH, temperature, buffer concentration, and incubation time were optimized by response surface methodology using rotatable central composite design. Maximum enzyme activity was found to be at pH 9.0, temperature 75°C in phosphate buffer (50 mM) when incubated for 10 min. Protease was stable over a broad range of pH 6.0–12.0 and it was stable at 50°C for 1 h. The protease was completely inhibited by PMSF (5 mM), suggesting that the enzyme is a serine alkaline protease. This enzyme had good stability in the presence of H2O2, SDS, Triton X‐100, and retained more than 88% of its initial activity after preincubation for 30 min at room temperature in the presence of 25% v/v DMSO, methanol, ethanol, ACN, and 2‐propanol.

Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3

Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth) were optimized by rotatable central composite design (RCCD) using response surface methodology (RSM). The effects of all five chemical components (considered as independent variables), namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable) were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone—5.71, soya peptone—4.9, dextrose—1.45, NaCl—6.08, and dipotassium phosphate—0.47 in g/L). To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine.

Characterization and Antioxidant Potential of a Carotenoid from a Newly Isolated Yeast

A newly isolated yeast strain was identified and explored for characteristic growth and pigment production in batch cultures. Based on nucleotide homology and phylogenetic analysis, the strain was identified as Sporidiobolus pararoseus DAGIII (Accession ID-KF724150). Pigment production was carried out using 1% (v/v) of an inoculum at 25ºC and 120 rpm after incubation for 5 days. By HPLC, Elemental, and FTIR analysis, the produced pigment was identified as β-carotene. The antiradical activity was measured and the half maximal inhibitory concentration (IC50) value was 449.11 μg/mL. Optimization of β-carotene production was achieved using a Plackett-Burman design and response surface methodology. A maximum concentration of 15.2614 mg/L of β-carotene was obtained for cultures in a medium containing 21.77 gm/L of dextrose, 20 gm/L of peptone, and 10 gm/L of yeast extract with incubation at 26ºC, an initial pH of 5.3, a shaker speed of 120 rpm, and a percentage inoculum=1.5%.

Encapsulation and release of a bacterial carotenoid from hydrogel matrix: Characterization, kinetics and antioxidant study

Various natural polymers with hydrophilic properties have been used to form hydrogels for the encapsulation and delivery of nutrients and drugs in food and pharmaceutical industries. Among them, chitosan (ChiHG)‐ and alginate (AlgHG)‐ based hydrogels have been extensively explored for delivery of several nutraceuticals in recent years. Release of natural canthaxanthin (CX) obtained from Dietzia maris NITD (accession number: HM151403) has been investigated with emphasis on biomedical applications. Significant changes (P < 0.05) in degree of swelling (%) and moisture content (% dry basis) were found after encapsulation of bacterial canthaxanthin (BCX), but the gel content remained unchanged. BCX encapsulation efficiency was calculated to be 55.92% and 60.45% in ChiHG and AlgHG, respectively. A noticeable change in heat of fusion (ΔHm) d melting point (Tm) was recorded in ChiHG and AlgHG after BCX encapsulation. Swelling and BCX release from gel matrix was performed under two different pH (1.2 and 7.4). The results showed that swelling of hydrogel and BCX release was facilitated at higher pH (7.4) than acidic pH (1.2). With regard to the release kinetics data, it was found that BCX is released from both ChiHG and AlgHG in a non − Fickian diffusion transport method. In addition, antioxidant activity of BCX encapsulated hydrogels was found significantly higher (P < 0.001) in terms of DPPH, ABTS, nitrite, hydroxyl radical scavenging and reducing power assay. These results indicated that BCX can be successfully encapsulated into a polymeric hydrogel to obtain a dynamic biomaterial that may be used in drug delivery applications in future.

Effective Decolorization of Malachite Green Using Kocuria marina DAGII and its Toxicological Study

Malachite Green (MG) is a highly toxic synthetic dye which is a major component of textile industry. It alone accounts for the two-third of the total production of the wastewater. Biological decolorization of dye is highly effective and a promising method due to its low cost and environmental friendly approach. The present study involves efficient detoxification of Malachite Green by the bacterium Kocuria marina DAG II through batch process. It was observed that more than 99 % dye removal was achieved within 8 hours at 28°C with an initial MG concentration of 13mg/L, initial pH of 7.2 and an inoculum size of 1.2% (v/v). Microbial toxicity test was conducted against Bradyrhizobium sp. using Agar well diffusion method. It was inferred that the treated effluent had no zone of inhibition but on contrary the untreated MG solution exhibited 35 ±5 mm inhibition zone in agar plates. Additionally the toxicity of the treated dye solution was checked on the seeds of Vigna radiata (Indian mung bean), a major cultivated Indian pulse crop. Though the phytotoxicity results revealed 100% germination in distilled water (positive control), untreated and treated dye solution, however the root and the shoot length were negligible in case of untreated dye solution. In contrast, the root and shoot length for treated MG solution was similar to the distilled water. Thus, the toxicological studies indicated that the treated dye solution was appreciably less toxic compared to untreated MG solution.

Growth profiling, kinetics and substrate utilization of low-cost dairy waste for production of β-cryptoxanthin by Kocuria marina DAGII

The dairy industry produces enormous amount of cheese whey containing the major milk nutrients, but this remains unutilized all over the globe. The present study investigates the production of β-cryptoxanthin (β-CRX) by Kocuria marina DAGII using cheese whey as substrate. Response surface methodology (RSM) and an artificial neural network (ANN) approach were implemented to obtain the maximum β-CRX yield. Significant factors, i.e. yeast extract, peptone, cheese whey and initial pH, were the input variables in both the optimizing studies, and β-CRX yield and biomass were taken as output variables. The ANN topology of 4-9-2 was found to be optimum when trained with a feed-forward back-propagation algorithm. Experimental values of β-CRX yield (17.14 mg l−1) and biomass (5.35 g l−1) were compared and ANN predicted values (16.99 mg l−1 and 5.33 g l−1, respectively) were found to be more accurate compared with RSM predicted values (16.95 mg l−1 and 5.23 g l−1, respectively). Detailed kinetic analysis of cellular growth, substrate consumption and product formation revealed that growth inhibition took place at substrate concentrations higher than 12% (v/v) of cheese whey. The Han and Levenspiel model was the best fitted substrate inhibition model that described the cell growth in cheese whey with an R2 and MSE of 0.9982% and 0.00477%, respectively. The potential importance of this study lies in the development, optimization and modelling of a suitable cheese whey supplemented medium for increased β-CRX production.