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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 Screening of chitin deacetylase producing microbes from marine source using a novel receptor on agar plate(Elsevier B.V., 2019) Pawaskar, G.-M.; Pangannaya, S.; Raval, K.; Trivedi, D.; Raval, R.Chitosan is a deacetylated form of naturally occurring polymer; chitin. On an industrial scale, the deacetylation of chitin to chitosan is performed using harsh chemicals like sodium hydroxide. This not only adds to the environmental pollution but the product is also random in terms of its deacetylation. This shortcoming can be addressed by using enzymes like chitin deacetylase (CDA). The screening of these organisms would require a reliable, fast and sensitive screening method. The deacetylation of chitin into chitosan, releases acetate as the byproduct of the reaction. A receptor which specifically binds to the acetate ion was synthesized chemically. The receptor upon binding with the acetate ion emitted a fluorescence which could be viewed using the gel documentation unit. The receptor was optimized for the screening of CDA producing microbes with the positive fungal control as Penicillium sp. and bacterial control as Bacillus megaterium. A parallel study with the 4-Nitroacetanilide, the reported screening indicator for CDA was performed. The results obtained with the receptor in the present study were concordant with the 4-Nitroacetanilide. Upon standardization, the protocol was extended for the screening of CDA producing microbes from the marine crustacean dumped soil and water samples. The CDA activity of these microbes was further confirmed using spectrophotometric MBTH assay. This is the first report using this receptor for the screening of CDA producers. The method is not only sensitive but also reproducible and can be extended for a high throughput screening of CDA producers. © 2019 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 Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22(Springer Science and Business Media Deutschland GmbH, 2021) Pawaskar, G.M.; Raval, K.; Rohit, P.; Shenoy, R.P.; Raval, R.Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni–NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris–HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters Km, Vmax, and Kcat were 3.06E?05 µg mL?1, 3.06E + 01 µM mg?1 min?1 and 3.27E + 04 s?1, respectively, with EGC as the substrate and 7.14E?07 µg mL?1, 7.14E + 01 µM mg?1 min?1 and 1.40E + 06 s?1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis–Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application. © 2021, The Author(s).Item Enhanced degradation of azo dye using mixed cultures of white-rot fungi in a modified rotating packed disc bioreactor and reuse of treated water(Elsevier Ltd, 2023) Kalnake, R.P.; Raval, R.; Murthy, D.V.R.; Vanzara, P.B.; Raval, K.Reactive azo dyes are toxic and carcinogenic. In this study, mixed cultures of white-rot fungi (WRF) are used to treat synthetic reactive black 5 (RB-5) wastewater in a modified rotating packed disc bioreactor (RPDB). The continuous degradation studies were carried out for 25 days under the influence of the recycle stream in which 3665 L of synthetic effluent was treated. The dye wastewater was completely decolorized with more than 93 % chemical oxygen demand (COD) reduction using the mixed fungal culture. During the continuous operation, the COD of influent reduced more than 85 % for successive 25 days of continuous operation at hydraulic retention time of 10.8 h. The dry biomass loading was about 0.14 g/g GAC at the end of the continuous process. The rate of COD removal followed first order kinetics with a rate constant of 0.026 per hour. The treated water was reused to produce melanin from microbial culture. © 2023 Elsevier LtdItem 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 Identification and characterization of chitinase producing marine microorganism: Unleashing the potential of chitooligosaccharides for bioethanol synthesis(Elsevier B.V., 2024) Atheena, P.V.; Rajesh, K.M.; Raval, K.; Subbalaxmi, S.; Raval, R.The dwindling supply of the petroleum product and its carbon footprint has initiated search for a sustainable fuel and alternate feed-stocks. One such underexplored feedstock is chitin, a waste derived from sea food processing. The limitation of insolubility and crystallinity inherent in chitin is addressed with the chitin hydrolysates. In the present study, a chitinases producing marine isolate was isolated from the sediments of Arabian Sea from a depth of 20 m. In order to increase the expression of the chitinases, sequential optimisation using one factor at a time and Taguchi experimental designs were employed which resulted in a yield of 13.46 U/mL which was 2.62 fold higher than the initial bioprocess condition values. In a two-step refinery protocol, Candida albicans was evolved towards chitooligosaccharides using chemically synthesized hydrolysates. In a fed –batch fermentation design the Candida yielded a 12.8 % conversion of these commercial chitin oligosaccharides into bioethanol in a run time of 48 h. This is the first report demonstrating the potential of Candida to utilise chitin oligosaccharides for the production of bioethanol. © 2024 The Author(s)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