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A Bacillus sp. RK-3 isolated from soil initially produced 3.28 IU/mL of 1, 3 regiospecific lipase in medium containing 1.0% olive oil. After process optimization, 10.56 IU/mL of lipase was produced in medium containing sunflower oil 1.5 %, tryptone 2 %, Ca 2+ 20 mM using 3 % inoculum in 250 mL Erlenmeyer flask containing 50 mL of the medium at pH 7.0, 250 rpm and 30 °C for 36 h. Scale up in 10 L bioreactor with 7.5 L of the optimized medium yielded 16.41 IU/mL in 30 h resulting in net 6.0 fold increase in enzyme units as against initial units of 3.28 IU/mL obtained under unoptimized conditions. The productivity in 10 L bioreactor is 0.547 IU/mL/h as against initial of 0.091 IU/mL/h. The lipase exhibited 95.12 % stability in hexane, followed by THF (75.83 %) and petroleum ether (73.85 %) after 24 h of incubation. Cocoa butter substitute (CBS) synthesis was attempted in a reaction containing 1.2 IU/mg of lipase using palm oil and methyl stearate in hexane. The reaction product being formed was analyzed qualitatively using Thin Layer Chromatography (TLC) and quantified by gas chromatography (GC) which showed 83.17 % conversion efficiency for CBS in 24 h.

Production of 1, 3 regiospecific lipase from Bacillus sp. RK-3: Its potential to synthesize Cocoa Butter Substitute