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 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 Adsorptive removal of trivalent and pentavalent arsenic from aqueous solutions using iron and copper impregnated melanin extracted from the marine bacterium Pseudomonas stutzeri(Elsevier Ltd, 2020) Manirethan, V.; Raval, K.; Mohan Balakrishnan, R.M.The metalloid arsenic is one of the most conspicuous groundwater contaminants in the Indian subcontinent and its removal from aqueous medium is the main focus of this study. The study aims at functionalising melanin using iron and copper for the efficient removal of arsenic and rendering water fit for consumption. Melanin obtained from the marine bacteria Pseudomonas stutzeri was functionalised by iron impregnation (Fe-melanin) and copper impregnation (Cu-melanin). Morphological studies using FESEM portrayed the impregnated iron and copper granules on the surface of melanin, while XRD analysis confirmed the presence of Fe2O3 and CuO on melanin. Adsorption studies on As (V) and As (III) were conducted using Fe-melanin and Cu-melanin for different operating variables like pH, temperature and contact time. More than 99% per cent of As (III) and As (V) from water was removed at a pH range between 4 and 6 within 50 min in the case of Fe-melanin and 80 min for Cu-melanin. Adsorption equilibrium studies showed better fit with Langmuir adsorption isotherm and had good agreement with Redlich-Peterson's three-parameter model. The maximum adsorption capacities of Fe-melanin and Cu-melanin obtained from Langmuir adsorption model are 50.12 and 20.39 mg/g, respectively, for As (V) and similarly 39.98 and 19.52 mg/g, respectively, for As (III). Arsenic-binding to the functionalised melanin was confirmed using FT-IR and the XPS analysis. Reuse of the adsorbent was effectively done by desorbing the iron and copper together with the bound As (III) and As (V) and further re-impregnation of iron and copper in melanin. Re-functionalised melanin showed 99% adsorption efficiency up to four cycles of adsorption/desorption. A novel iron and copper impregnated melanin was synthesized to remove As (III) and As (V) from groundwater and the adsorption process was optimized. © 2019 Elsevier LtdItem Tailoring solulan C24 based niosomes for transdermal delivery of donepezil: In vitro characterization, evaluation of pH sensitivity, and microneedle-assisted Ex vivo permeation studies(Editions de Sante editions.de.sante@wanadoo.fr, 2020) Nayak, A.S.; Chodisetti, S.; Gadag, S.; Nayak, U.Y.; Srinikethan, S.; Raval, K.The present investigation aims at encapsulating donepezil (DNP) in a niosomes to avert the side effects and to deliver the intact carrier across the skin barrier by modulating its physicochemical properties. The finding conclusively demonstrated that entrapment efficiency and the alteration in the niosome size are associated with the change in the span 60: cholesterol ratio, sonication, and hydration volume. The addition of 5 mM of solulan C24 to the optimized formulation (NSV5SolC24) formed stable niosomes with a mean particle size of 180.1 ± 1.83 nm and entrapment efficiency of 82.15% ± 1.54%. The cryo-SEM image and in vitro drug release profile revealed that the NSV5SolC24 is pH-sensitive. FTIR spectral analysis of NSV5SolC24 suggested that the ether and ester group in the NSV5SolC24 complex undergoes SN2 cleavage and hydrolysis at lower pH, thus enhancing DNP release. The microneedle (MN)-assisted studies with MN1200 showed a 29-fold increase in transdermal permeation of intact NSV5SolC24 against the passive method in porcine skin. The intact NSV5SolC24 carrying DNP was translocated across the skin barrier successfully at a steady flux rate of 9.89 ± 0.923 ?g/cm2/h. Nevertheless, further in vivo studies are recommended to elucidate the pH sensitivity and clinical efficacy of the prepared drug delivery system. © 2020 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 Experimentally Induced Hyperglycemia in Prepubertal Phase Impairs Oocyte Quality and Functionality in Adult Mice(Endocrine Society, 2022) Predheepan, D.; Daddangadi, A.; Uppangala, S.; Koulmane Laxminarayana, S.L.K.; Raval, K.; Kalthur, G.; Kovaĉiĉ, B.; Kumar Adiga, S.Reproductive abnormalities in women with a history of childhood diabetes are believed to be partially attributed to hyperglycemia. Prolonged hyperglycemia can negatively affect ovarian function and fertility during reproductive life. To address this in an experimental setting, the present study used streptozotocin-induced hyperglycemic prepubertal mouse model. The impact of prolonged hyperglycemic exposure during prepubertal life on ovarian function, oocyte quality, and functional competence was assessed in adult mice. The ovarian reserve was not significantly altered; however, the in vitro maturation potential (P<0.001), mitochondrial integrity (P<0.01), and meiotic spindle assembly (P<0.05-0.001) in oocytes were significantly affected in hyperglycemic animals in comparison to control groups. The results from the study suggest that prepubertal hyperglycemia can have adverse effects on the oocyte functional competence and spindle integrity during the reproductive phase of life. Because these changes can have a significant impact on the genetic integrity and developmental potential of the embryos and fetus, the observation warrants further research both in experimental and clinical settings. © 2022 The Author(s). Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved.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