Faculty Publications
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Item Expression studies of Bacillus licheniformis chitin deacetylase in E. coli Rosetta cells(Elsevier B.V., 2017) Raval, R.; Simsa, R.; Raval, K.Chitin, the biopolymer of the N-acetylglucosamine, is the most abundant biopolymer on the planet after cellulose. However owing to its crystalline nature, its deacetylated derivative; chitosan is industrially more potent. This conversion on an enzymatic scale can be made using chitin deacetylase. The metagenomics library constructed from the soil exposed to chitin and chitosan yielded chitin modifying enzymes, one of them being chitin deacetylase (CDA) utilized for the present study. The gene was amplified and expressed using the pET 22b vector in E. coli Rosetta cells. The effect of two additives; chitin and glycerol on the CDA activity were studied. The inclusion of glycerol in the medium improved the biomass by 50% from the initial value of 1.25 g/l to 2.5 g/l. The activity of CDA increased from 90 ?mol/min/ml to 343 ?mol/min/ml. The CDA activity reported in the present paper is the highest observed for any strain. The addition of glycerol to the media not only helped improve the yield of the chitin deacetylase but also imparted value addition to the waste of the biofuel industry. © 2017 Elsevier B.V.Item Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation(Elsevier B.V., 2019) Bhat, P.; Pawaskar, G.-M.; Raval, R.; Cord-Landwehr, S.; Moerschbacher, B.; Raval, K.Chitosan obtained by enzymatic deacetylation of chitin using chitin deacetylase (CDA) holds promise primarily due to the possibility to yield chitosan with non-random patterns of acetylation and more environmentally friendly process compared to chemical deacetylation. In the present study, a sustainable bioprocess is reported for over-expression of a bacterial CDA in E. coli pLysS cells. A Bacillus licheniformis CDA gene is identified in the genome of the bacterium, cloned, and expressed, yielding enzymatically active recombinant protein. For enzyme production, a growth medium is formulated using carbon and nitrogen sources, which do not compete with the human food chain. The maximum enzyme activity of 320 ± 20 U/mL is achieved under optimized conditions. The CDA productivity is improved by about 23 times in shake flask culture by optimizing operating conditions and medium components. The CDA is purified and the enzyme kinetic values i.e. Km, Vmax and Kcat are reported. Also the effect of cofactors, temperature, and pH on the enzyme activity is reported. Further, economic yield is proposed for production of CDA through this bioprocess. © 2019 Elsevier B.V.Item A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation(Taylor and Francis Ltd., 2022) Subramani, A.K.; Raval, R.; Sundareshan, S.; Sivasengh, R.; Raval, K.Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin’s crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium Bacillus aryabhattai is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like Candida albicans and Fusarium oxysporum revealed a zone of inhibition with 14 mm diameter. © 2022 Taylor & Francis Group, LLC.Item Marine Bacillus haynesii chitinase: Purification, characterization and antifungal potential for sustainable chitin bioconversion(Elsevier Ltd, 2024) Govindaraj, V.; Kim, S.-K.; Raval, R.; Raval, K.The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0–9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL−1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL−1 and Vmax of 5.75 mmol min−1. Kcat and catalytic efficiency were measured at 1.91 s−1 and 191 mL mg−1 s−1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent. © 2024Item Process optimisation for improved chitinase production from marine isolate Bacillus haynesii and bioethanol production with Saccharomyces cerevisiae(Springer Nature, 2025) Govindaraj, V.; Anandan, D.K.; Kim, S.-K.; Raval, R.; Raval, K.In the quest for sustainable fuel sources, chitin-based biorefineries are gaining recognition as chitin is the second most abundant bioresource after cellulose. This approach not only provides an effective method for converting shell waste from seafood processing into valuable bioethanol but also helps in waste management. In this study, Bacillus haynesii, a marine isolate, was investigated and this is the first report on optimisation of process parameters for chitinase production from Bacillus haynesii. The One Factor at a Time (OFAT) method was used to optimize process parameters including inoculum age, inoculum size, temperature, pH, and filling volume, with colloidal chitin identified as the best carbon source for the growth of Bacillus haynesii. The Plackett-Burman Design (PBD) was employed to screen media components, followed by optimization using the Taguchi Orthogonal Array method. The media components investigated included glycerol, yeast extract, MnCl2·4H2O, MgSO4·7H2O, NH4Cl, and colloidal chitin. As a result, the optimized media—comprising 7.5 g/L yeast extract, 7.5% (w/v) glycerol, 0.6% (w/v) colloidal chitin, 1.44 g/L MnCl2·4H2O, and 1.2 g/L MgSO4·7H2O—yielded an enzyme activity of 6.85 U/mL with a specific activity of 28.87 U/mg. Furthermore, ethanol production from chitin oligosaccharides by Saccharomyces cerevisiae was quantified using the potassium dichromate oxidation method, achieving a bioethanol concentration of 2.48% v/v from 33.18 g/L of chitin oligosaccharides. These results demonstrate the potential of Bacillus haynesii-derived chitin oligosaccharides as a promising substrate for bioethanol production. © The Author(s) 2025.Item Identification, purification and functional characterization of a thermostable marine chitinase for potential fungal control via chitin degradation mechanism(Elsevier Ltd, 2025) Atheena, P.V.; Raval, K.; Raval, R.The growing prevalence of treatment-resistant Candida species highlights an urgent need for innovative antifungal therapies. The current range of antifungals, limited to polyenes, azoles, and echinocandins, are becoming insufficient due to the rise of resistance, including cross-resistance among fungal strains. Marine environment is an underexplored reservoir of unique enzymes which can be extremophilic. This study presents the cloning and expression of a chitinase gene from the bacterium Bacillus thuringiensis (BtChi), expressed in an E. coli system, yielding a protein with a molecular weight of approximately 71 kDa. Disc diffusion and MIC experiments indicated that 5 ?g/mL chitinase efficiently suppressed the growth of Candida albicans. Initial characterization identified the optimal activity at 40 °C and pH 7.0. The enzyme retained over 75 % activity across a pH range of 4–8 and a temperature range of 30–70 °C after 120 min. Activity was further enhanced by 24 % with 100 mM Na+. Kinetic parameters with colloidal chitin revealed Km and Vmax values to be 0.05 mg/mL and 1.37 U/mL respectively. This study holds the potential of developing a potent natural anti-fungal against the present day chemical counterparts. © 2025 The Authors