Faculty Publications
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Item Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production(Elsevier B.V., 2024) Subramani, A.K.; Ramachandra, R.; Thote, S.; Govindaraj, V.; Vanzara, P.; Raval, R.; Raval, K.Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 μmole min−1 and a Km of 4.6 mg mL−1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg−1 and 625 U mg−1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase. © 2024 Elsevier B.V.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 Marine chitinase AfChi: green defense management against Colletotrichum gloeosporioides and anthracnose(Springer Science and Business Media Deutschland GmbH, 2024) Rajesh, R.; Raval, K.; Raval, R.Anthracnose disease, caused by the Colletotrichum gloeosporioides species, affects vegetables, fruits, pulses, and cereals, leading to significant economic losses worldwide. Although many synthetic fungicides are used to control this pathogen, eco-friendly biological alternatives are gaining popularity. This study focuses on isolating and purifying chitinase (Af Chi)from a marine bacterium and testing its antifungal efficacy against C. gloeosporioides spore germination by targeting the chitin in the fungal cell wall. The chitinase was purified from a marine bacterium A. faecalis from the Arabian Sea and had a molecular mass of 45 kDa and a specific activity of 84.6 U/mg. Af Chi worked best at 50 °C and pH 7.0 in Tris HCl buffer. Na+ ion was the highest cofactor, highlighting the halophilic nature of this chitinase. K+, Ca2+, Cu2+, Mg2+, Mn2+, and EDTA also increased activity, while Fe3+, Zn2+, Co2+, and Pb2+ decreased it. The Km and Vmax values were 1.87 µg/mL and 17.45 U/mL, respectively. Purified Af Chi at 10 mg/mL completely inhibited spore germination within 8 h and reduced the size of the spores. © The Author(s) 2024.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