Abstract
The exogenous lipolytic activities of Kocuria sp. have been recognized earlier but the genus further contains many more unexplored strains. In this study, the extracellular lipase activity of Kocuria flava Y4 (GenBank accession no. MT773277), isolated from Dioscorea villosa during our previous study, was regulated by different physicochemical parameters, such as pH, temperature, shaking speed, and incubation time. For efficient immobilization of the extracellular lipase, 4% sodium alginate, 50 mL of 25 nM CaCl2.2H2O solution, and 15 min. Hardening time of gel beads in calcium chloride was used. For the first time, K. flava Y4 lipase was purified using ammonium sulphate precipitation followed by dialysis and DEAE-Sepharose anion exchange chromatography with Sepharose-6B gel filtration chromatography, yielding ∼15-fold purified lipase with a final yield of 96 U/mL. The SDS-PAGE of purified lipase displayed a single strong band, indicating a monomeric protein of 45 kDa. At a temperature of 35°C and pH 8, the purified lipase showed maximum hydrolytic activity. Using p-nitrophenyl acetate (p-NPA) as the hydrolysis substrate, the values of Km and Vmax derived from the Lineweaver–Burk plot were 4.625 mM and 125 mol/min−1mg−1, respectively.
Highlights
International Journal of Analytical Chemistry quality and they are a good alternative to reduce the cost of lipase production [15]
MT773277), which was locally isolated during our previous study, was used. is strain was maintained on nutrient agar slants at 35°C and stored at 4°C. e nutrient broth medium was inoculated with initial optical density (OD) 0.141 of K. flava Y4 and the overnight grown culture was centrifuged at 10,000 rpm for 15 min to separate the pellet and supernatant
Lipase activity of K. flava Y4 was found to be 41 ± 0.64 U/mL, which was assessed by the p-nitrophenyl acetate (p-NPA) assay
Summary
International Journal of Analytical Chemistry quality and they are a good alternative to reduce the cost of lipase production [15]. Bacterial lipases are of great demand because of their potential industrial applications. E bacterial lipase is used as a key enzyme in various industrial applications due to its environmental safety, nontoxic nature, and generation of no hazardous residues [12]. Lipases in food and dairy industries are widely applicable for milk fat hydrolysis, ripening and flavour enhancement in cheese product, and lipolysis of cream and butter fat [7, 17]. Considering the inherent complex nature of the protein structure, lipase is immobilized using entrapment strategy. For the production of saline-alkaline lipase enzyme immobilized with magnetic Fe3O4 nanoparticles for commercial applications was reported [18]. E goal of this study was purification, optimization, and immobilization of lipase obtained from K. flava Y4, a plant probiotic, which was newly isolated during our previous study. The spatial geometry of K. flava lipase active site residues for optimal activity has been investigated
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