Browsing by Author "Arun Kumar, S."

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    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.
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    Machinability of Hardened Alloy Steel using Cryogenic Machining
    (Elsevier Ltd, 2018) Arun Kumar, S.; Yoganath, V.G.; Krishna, P.
    Machining of hardened alloy steels demand special cutting tools such as PCBN, ceramic. However, these cutting tools are uneconomical and also demand machine tool structures, which have high stiffness and vibration dampening properties. In the current trend towards Green manufacturing it is desired to produce more with less. Green manufacturing also emphasizes on an eco-friendly process. Hence, it is postulated to improve the machinability of these materials by alternate, economical means. One such alternative is cryogenic machining. In the current research work, it is envisaged to study the machinability of hardened alloy steel using commercially available cutting tools (coated carbide) under the influence of cryogenic as the coolant. Machinability factors under influence of cryogenic machining such as tool life, surface roughness and power consumption are studied. Results show that cryogenic as an alternative to coolant during machining of hardened materials increases the process efficiency by reducing energy consumption and also showed significant improvement in tool life. The process thus demonstrates the capability of replacing the special cutting tools that are required for hard turning applications. The portability of the setup for commercial use is also considered. © 2017 Elsevier Ltd.