Faculty Publications
Permanent URI for this communityhttps://idr.nitk.ac.in/handle/123456789/18736
Publications by NITK Faculty
Browse
4 results
Search Results
Item Bioethanol: A New Synergy between Marine Chitinases from Bacillus haynesii and Ethanol Production by Mucor circinelloides(MDPI, 2023) Govindaraj, V.; Subramani, A.K.; Gopalakrishnan, R.; Kim, S.-K.; Raval, R.; Raval, K.The fourth generation of bioethanol production is on a lookout for non-lignocellulosic biomass waste. One such candidate is chitin, the second most abundant biopolymer on earth. However, the crystalline nature of chitin hinders its application potential for bioethanol production. This limitation can be circumvented by hydrolysing this polymer into oligomers using chitinases. We used this hypothesis and isolated a Bacillus haynesii, a marine bacterium that utilizes colloidal chitin as a substrate and produces chitin oligosaccharides. Further, we utilized Mucor circinelloides to produce bioethanol using the chitin oligosaccharides in the shake flask. We investigated the effect of inoculum age, filling volume, different substrates, and substrate concentration on bioethanol production using Mucor circinelloides from Bacillus haynesii-produced chitin oligosaccharides. Bacillus haynesii demonstrated a maximum chitinase activity of 3.08 U/mL with specific activity of 96 U/mg at the 90th h. Chitin oligosaccharides produced by Bacillus haynesii were confirmed using mass spectrometry. Bioethanol concentration was determined using dichromate oxidation assay as well as gas chromatography. The research resulted in 7.4 g/L of ethanol from 30 g/L of chitin oligosaccharides, with a maximum ethanol yield of 0.25 g of ethanol/g substrate at the 55th h with 48 h inoculum in 80 mL of fermentation medium. Results suggest that chitin oligosaccharides from Bacillus haynesii are an effective and renewable substrate for bioethanol production. © 2023 by the authors.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 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.