Production and purification of an alkaline lipase from Bacillus sp. for enantioselective resolution of (±)-Ketoprofen butyl ester.

The present study was conducted to purify lipase from indigenous Bacillus subtilis strain Kakrayal_1 (BSK-L) for enantioselective resolution of racemic-ketoprofen. The production of lipase (BSK-L) was optimized using Plackett-Burman and central composite design of response surface methodology (RSM). The optimized media containing olive oil (3.5%), MnSO4 (8 mM), CaCl2 (5 mM), peptone (20 g/l), pH (8), agitation (180 rpm) and temperature (37 °C) resulted in maximum lipase production of 7500 U/g of cell biomass. The lipase was purified using sequential method to an overall purification fold of 13% with 20% recovery, 882 U/mg specific activity and a molecular weight of 45 kDa. Optimal pH and temperature of purified lipase were found to be 8 and 37 °C, respectively. Furthermore, BSK-L displayed good stability with various organic solvents, surfactants and metal ions. K m and V max values of lipase were observed to be 2.2 mM and 6.67  mmoles of product formed/min/mg, respectively. The racemic ketoprofen butyl ester was hydrolyzed using lipase with 49% conversion efficiency and 69% enantiomeric excess (ee) which was superior to the commercially procured lipase (Candida antarctica lipase). Thus, this enzyme could be considered as a promising candidate for the pharmaceutical industry.

Enantioselective resolution of 2-arylpropionic acid derivatives employing immobilization of lipase from Bacillus subtilis strain Kakrayal_1 (BSK-L).

This work studied the enantioselective resolution of 2-arylpropionic acid derivatives employing immobilization of lipase produced by Bacillus subtilis strain Kakrayal_1 (BSK-L). The efficient immobilization of lipase on modified silica gel was confirmed by Fourier transform infrared spectroscopy. Tethering of lipase facilitated the enhancement of physiochemical properties and stability of enzyme. Covalently immobilized enzyme retained 85% of residual activity even on reuse after 10th reaction cycle. Validation of immobilized lipase for enantioselective resolution of 2-arylpropionic acid derivatives led to 47.8% conversion efficiency with 87% enantiomeric excess (ee) for ketoprofen, and 27.3% conversion efficiency with 75% ee for flurbiprofen. The enantioselective resolution using immobilized lipase (BSK-L) was superior to free and commercially procured lipase, which suggest a potential application of immobilized lipase in the pharmaceutical/chemical industry.

Evaluation of alkali and thermotolerant lipase from an indigenous isolated Bacillus strain for detergent formulation

Background: Lipases are used in detergent industries to minimise the use of phosphate-based chemicals in detergent formulations. The use of lipase in household laundry reduces environmental pollution and enhances the ability of detergent to remove tough oil or grease stains. Results: A lipase-producing indigenous Bacillus subtilis strain [accession no. KT985358] was isolated from the foothills of Trikuta mountain in Jammu and Kashmir, India. The lipase (BSK-L) produced by this strain expressed alkali and thermotolerance. Lipase has an optimal activity at pH 8.0 and temperature 37°C, whereas it is stable at pH 6.0­9.0 and showed active lipolytic activity at temperatures 30 to 60°C. Furthermore, lipase activity was found to be stimulated in the presence of the metal ions Mn2+, K+, Zn2+, Fe2+ and Ca2+. This lipase was resistant to surfactants, oxidising agents and commercial detergents, suggesting it as a potential candidate for detergent formulation. BSK-L displayed noticeable capability to remove oil stains when used in different washing solutions containing buffer, lipase and commercial detergent. The maximum olive oil removal percentage obtained was 68% when the optimum detergent concentration (Fena) was 0.3%. The oil removal percentage from olive oil-soiled cotton fabric increased with 40 U/mL of lipase. Conclusions: This BSK-L enzyme has the potential for removing oil stains by developing a pre-soaked solution for detergent formulation and was compatible with surfactants, oxidising agents and commercial detergents.

Heparin depolymerization by immobilized heparinase: A review.

Heparin is a member of the glycosaminoglycan (GAG) family composed of glucosamine and uronic acid units containing O-sulfo, N-acetyl and N-sulfo groups, which are alternating in the chain and linked by 1→4 manner. It is a naturally occurring anticoagulant that prevents the formation of clots and their growth within blood. Certain low molecular weight heparins (LMWHs) are considered as better therapeutic agents than natural heparin because of the reduced side effects and smaller risk of bleeding. LMWHs can be produced from heparin by chemical or enzymatic depolymerizations. Heparinases catalyze the cleavage of glycosidic linkage between amino sugars and uronic acids in heparin. There are three kinds of heparinases which are frequently used for depolymerization of heparin. Despite wide range of applications of heparinases in health care, their use still has been hampered due to poor stability and high cost. To overcome this problem heparinases are recommended for immobilization to reduce the cost of product and enhance stability. Heparinases have been successfully immobilized using various methods and supports, mostly for deheparinization of blood through extracorporeal devices. The focus of this review is to present the current status of heparinase immobilization including various supports and methods used, stability and applications.