Expression and Biochemical Characterization of Cold-Adapted Lipases from Antarctic Bacillus pumilus Strains

Abstract

Two lipase genes (bpl1 and bpl3) from Antarctic Bacillus pumilus strains were expressed in Bacillus subtilis. Both recombinant lipases BPL1 and BPL2 were secreted to the culture medium and their activities reached 3.5 U/ml and 5.0 U/ml, respectively. Their molecular masses apparent using SDS-PAGE were 23 kDa for BPL1 and 19 kDa for BPL3. Both lipases were purified to homogeneity using ammonium sulfate precipitation and HiTrap SP FF column and Superose 12 column chromatographies. The final specific activities were estimated to be 328 U/mg for BPL1 and 310 U/mg for BPL3. Both lipases displayed an optimum temperature of 35°C, similar to other mesophilic enzymes. However, they maintained as much as 70% and 80% of the maximum activities at 10°C. Accordingly, their calculated activation energy at a temperature range of 10-35°C was 5.32 kcal/mol for BPL1 and 4.26 kcal/mol for BPL3, typical of cold-adapted enzymes. The optimum pH of BPL1 and BPL3 was 8.5 and 8.0, respectively, and they were quite stable at pH 7.0-11.0, showing their strong alkaline tolerance. Both lipases had a preference toward medium chain length (C6-C10) fatty acid substrates. These results indicate the potential for the two Antarctic B. pumilus lipases as catalysts in bioorganic synthesis, food, and detergent industries.

Characterization of Proteus vulgaris K80 Lipase Immobilized on Amine-Terminated Magnetic Microparticles

“…The hydrolysis rates of various synthetic substrates, including pNP-acetate (C 2 ), pNP-butyrate (C 4 ), pNP-caproate (C 6 ), pNPcaprylate (C 8 ), pNP-caprate (C 10 ), and pNP-laurate (C 12 ), were measured using an established spectrophotometric method. A different assay method was used for pNP-laurate (C 12 ), pNPmyristate (C 14 ), pNP-palmitate (C 16 ), and pNP-stearate (C 18 ) [11]. Lipase solutions were added to 0.88 ml of reaction buffer containing 50 mM Tris-HCl (pH 8.0), 0.1% gum arabic, and 0.2% deoxycholate.…”

“…It functions under aqueous conditions to hydrolyze the ester bonds in triacylglycerol and to liberate fatty acids and glycerol [3]. Lipase is applied widely for biodiesel production, biosensor construction, polymer synthesis, and functional lipid production [5,11,13,16].…”

Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties

“…Low values of E A are typical of coldactive lipases from different organisms such as Psychrobacter sp. (Parra et al 2008), Antarctic Bacillus pumilus strains (Litantra et al 2013) and Photocacterium strain (Kim et al 2012), which displayed E A of 23, 18 and 11.3 kJ mol −1 , in the conditions of the respective studies. In comparison, CaLIP5, the previously identified cold-active lipase belonging to the CaLA superfamily, showed higher activation energy for hydrolysis (36 kJ mol −1 ), in a temperature range of 5-25°C above which its enzymatic rate decreased (Lan et al 2011).…”

“…Cold-active lipases are thus of particular interest for the lipophilization and production of thermolabile compounds at high rates and for the economic benefits through energy saving: functioning in/during cold environments/season, minimizing undesirable higher temperature occurring-chemical reactions, avoiding the requirement of unnecessary heating steps and being rapidly and easily inactivated when required (Gerday et al 2000;Joseph et al 2008). These lipases are usually produced by psychrophilic microorganisms isolated from cold environments such as the Antarctic (Parra et al 2008;Wang et al 2012;Litantra et al 2013), deep-sea sediments (Jeon et al 2009) or other cold regions of the world (Zheng et al 2011;Tanaka et al 2012). In order to survive and proliferate in such extreme conditions, these microorganisms must synthesize naturally evolved enzymes exhibiting high activity at low temperature.…”

New Insight into Old <b><i>Bacillus</i></b> Lipase: Solvent Stable Mesophilic Lipase Demonstrating Enzyme Activity towards Cold

“…The lipase retained high activity at a broad range of temperatures from 2 to 35 ° C, indicating that it has a cold adaptation property. Litantra et al [2013] reported expression and biochemical characterization of cold-adapted lipases from antarctic B. pumilus strains, where they showed that enzyme was retaining its 70% activity at 10 ° C, with optimum enzyme activity at 35 ° C. In a similar study from Pseudomonas sp., Choo et al [1998] found that the lipase was stable in the range of 5-35 ° C, but was highly unstable above 40 ° C. Cold-adapted lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates [Angelaccio et al, 2012]. The optimum temperature and high enzyme activity at low temperature are favorable properties for the production of relatively frail compounds.…”