Browsing by Author "Raval, Keyur"

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Bioprocess development and optimization of melanin from Pseudomonas stutzeri
(National Institute of Technology Karnataka, Surathkal, 2018) Thaira, Harsha; Raval, Keyur
Melanins are water insoluble polyphenol compounds that are responsible for black, brown and grey pigmentation in eukaryotes and prokaryotes. The presence of indole ring and many functional groups, such as, =O, -OH, -NH, and –COOH makes melanin an ideal choice for many medical, cosmetic and environmental applications. The current market price of melanin is about USD 500 because of unavailability of sustainable manufacturing process. Microorganisms are reported to produce melanin; however, sustainability of a microbial bioprocess depends on raw material requirements and water footprint. A non-pathogenic microorganism capable of growing in seawater and require raw materials, which do not compete for human consumption is ideal for any bioprocess development. Therefore, a marine bacterium known as Pseudomonas stutzeri, which readily grows in seawater and does not require any tyrosine addition was selected for this work on melanin production. The best operating conditions were identified for melanin production. Inoculum age of 12 hours and 10% inoculum gave the best melanin production. Different carbon, nitrogen sources and trace elements were screened and glucose and coconut cake meal were selected as the best carbon and nitrogen sources, respectively. Sulphates of copper, magnesium and iron enhanced melanin production. All these media components were optimized using central composite design and melanin production was increased to 3.4 folds to about 520 mg/L. However, melanin demonstrated biomass growth inhibition as well as feedback inhibition on to the enzyme tyrosinase. An alumina adsorbent based novel adsorptive bioprocess was developed to reduce the feedback inhibition by melanin, which resulted in 8.8 fold enhancement in the production of melanin by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349.03 mg/L). Further, an adsorption based downstream process was developed using alumina, at acidic pH (< 3) which resulted in 98% recovery of melanin from the fermentation broth. Particle size analysis of the biosynthesized melanin indicated that they are nanoparticles with a size of 32 ± 0.98 nm and preliminary investigation indicated that melanin nanoparticles adsorbed different heavy metals at very low metal concentrations.
Cloning, Expression, Purification and Product Characterization of a Novel Chitinase from Bacillus Aryabhattai
(National Institute of Technology Karnataka, Surathkal, 2024) Arun Kumar, S.; Raval, Keyur
Chitin, an abundant polysaccharide in the world, is a primary byproduct of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase transforms chitin into chitin oligomers, which holds significant industrial potential. However, chitin's crystalline and insoluble nature makes the conversion process challenging. This study isolated a novel marine bacterium, Bacillus aryabhattai, from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin and produce chitin oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains, which helps chitinase act on crystalline chitin. Statistical optimization of media and process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulphate as significant media components and 1% colloidal chitin. The optimum process parameters such as pH 7, temperature 40℃, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific chitinase activity to ±115.2 U⸳ mg-1, ±63.02 U⸳ mg-1 and ±146.1 U⸳ mg-1 against chitin powder, flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. Also, TLC and LC-MS analysis confirmed that chitin oligomers (monomer to hexamer) were produced from insoluble chitin powder and flakes by spent media of Bacillus aryabhattai. A recombinant chitinase from the marine bacteria Bacillus aryabhattai (BaChiA) was developed in the future. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. Chitinase characterization against colloidal chitin revealed optimum parameters such as Tris buffer at pH 8, temperature 55℃, with 400 mM sodium chloride. Enzyme kinetics studies for colloidal chitin substrates revealed Vmax of 112.3 μmole⸳ min-1 and Km of 2.5 mg⸳ mL-1. The specific chitinase activity against chitin powder and flakes reached 875 U⸳ mg-1 and 625 U⸳ mg-1, respectively. The chitinase showed antifungal activity against Candida albicans, Fusarium solani, and Penicillium chrysogenum growth with the zone of inhibition of 14 mm and 25 mm diameter for wildtype and recombinant BaChiA, respectively. Thin Layer Chromatography (TLC) and LC-MS confirmed the production of chitin trimer, tetramer, pentamer, and hexamer from chitin powder and flakes using recombinant chitinase.
Studies on Removal of Heavy Metals from Aqueous Solution Using Melanin Coated Matrix
(National Institute of Technology Karnataka, Surathkal, 2019) Vishnu, M.; Raval, Keyur; B, Raj Mohan.
The biopolymer/biopigment Melanin is known for its free radical scavenging property. Melanin obtained from the marine bacteria Pseudomonas stutzeri is used for the removal of Hg (II), As (III), As (V), Pb (II), Cr (VI) and Cu (II) from aqueous medium. The melanin produced by the bacterium is confirmed to be nanoparticle (32 ± 0.98 nm) using TEM and particle size analysis. Different characterisations such as SEM, TGA, DSC, FTIR, BET surface analysis, zeta potential analysis, XRD were conducted to understand the physio-chemical properties of melanin. The kinetic, thermodynamic and equilibrium studies were conducted to achieve the removal of 85%, 87% 92% and 95% Hg (II), Cr (VI), Pb (II) and Cu (II) respectively from water having 10 mg/L adsorbate concentration and 0.2 g/L of adsorbent loading. Trivalent and pentavalent arsenic showed ineffective binding to melanin which was resolved by functionalising melanin using iron and copper. On functionalisation, melanin could remove more than 99 % of As (III) and As (V) from aqueous medium having arsenic concentration of 10 mg/L and loading of 0.8g/L and 2g/L of Fe-melanin and Cu-melanin respectively. For effective heavy metal adsorption and effortless removal of adsorbent from the aqueous medium, melanin nanoparticles were coated on to different matrices and batch removal studies were performed. Adsorption studies using melanin immobilised N, N-diethylacrylamide hydrogel, melanin coated PVDF membrane, melanin impregnated activated carbon were conducted and found that efficient removal of heavy metals was achieved by melanin impregnated activated carbon. Continuous adsorption studies using melanin impregnated activated carbon as a fixed bed column was performed by varying the parameters such as influent flow rate, heavy metal concentration and adsorbent loading in the column. The flow rate of 0.5 mL/min, the heavy metal concentration of 1 mg/L and adsorbent loading of 100 mg were the optimised parameters for efficient heavy metal removal. Thomas model fitted well with the experimental data compared to the Adam-Bohart’s model. Efficient desorption of Hg (II), Pb (II) and Cu (II) were obtained using 3N HCl and Cr (VI) using 1N citric acid. Melanin was re-functionalised after treatment with 5N HCl, and effective reuse of melanin for removal of all heavy metals was achieved until four cycles of study.
Studies on the expression of recombinant Chimeric Chitin Deacetylase
(National Institute Of Technology Karnataka Surathkal, 2023) Bhat, Priyanka; Raval, Keyur
Transdermal Delivery of Donepezil Niosomes For Alzheimer's Disease: Synthesis, Ex Vivo Permeation And Brain Targeting Studies.
(National Institute of Technology Karnataka, Surathkal, 2022) .Archana; Raval, Keyur; Srinikethan, G.
The present investigation aims to encapsulate donepezil (DNP) in niosomes to avert the side effects and deliver the drug to the brain bypassing the skin barrier. 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, hydration volume, and Solulan C24. The optimization process resulted in the formation of stable niosomes of 180.1 ± 1.83 nm entrapping 82.15% ± 1.54% of the drug. The cryo-SEM image and in vitro drug release profile revealed that the optimized niosome is pH-sensitive, and the niosomes undergo membrane destabilization at acidic skin pH. The MN-assisted studies with MN1200 showed a 26-fold increase in transdermal permeation of DNP against the passive method in porcine skin at a steady flux rate of 9.89 ± 0.923 μg/cm2/h. Therefore, it may be inferred that delivering the intact DNP niosomes using MNs across the skin is feasible by a relatively painless and non- invasive method, thereby improving patient compliance. The specificity of a niosome for brain cells was improved by functionalizing the niosomes with NPG, a glucose analog molecule. The molecular docking computational studies identified the interacting amino acids between the NPG ligand and active site, 4PYP of the GLUT-1 transporter protein. In addition, the cell viability studies by AO-EB staining and cell uptake studies by the iron perls staining method presented visual evidence for non-toxicity and internalization of the niosomes. The in vivo studies on AD-induced SD rats demonstrated excellent localization and sustained release of DNP from NPG-fn-niosome by three folds. The results confirmed the actual effectiveness of NPG functionalization and site-directed delivery. However, further preclinical studies are recommended to confirm the efficacy of the engineered drug delivery system.