Research paperPurification, characterization and thermal inactivation kinetics of a non-regioselective thermostable lipase from a genotypically identified extremophilic Bacillus subtilis NS 8
Introduction
Lipase represents a group of enzymes with the ability to hydrolyze triacyglycerols at lipid–water interface [1]. Lipase acts as the interface and catalyses hydrolysis of fats and mono- and di-glycerides to free fatty acids and glycerols [2]. An important characteristic of lipases is their ability not only to hydrolyze the ester bonds, transesterify triglycerides and resolve racemic mixture but also to synthesize ester bonds in non-aqueous media [48]. Purification of lipase allows for better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters [3]. Many bacillus lipases have been purified to homogeneity using a variety of methods involving ammonium sulfate precipitation, ion exchange chromatography followed by gel filtration [4]. However, the use of ammonium sulfate precipitation has been reported to cause low enzyme yield [5]. Purified microbial lipases have also been characterized in terms of their activity and stability profiles with respect to pH, temperature, and effects of metal ions [6] as well as their molecular weights [7].
It is pertinent to have a deeper study on the thermal behaviour of lipases because they are mostly utilized industrially at elevated temperatures. Thus, thermostable enzymes have been the target of many studies of the development of strategies to enhance stability [8]. To the best of our knowledge, infinitesimal or no information is available on a comprehensive study of thermal behaviour and thermodynamic properties of lipases from extremophiles. In the present study therefore, an extracellular lipase from an extremophilic Bacillus subtilis NS 8 which has been previously isolated from hotspring and identified by phenotypic methods and confirmed by the beneficial genotypic techniques of 16S rRNA sequence analysis was purified and characterized.
Section snippets
Chemicals
Sodium dodecyl sulfate-polyacrylamide gels, silver staining kit, standard molecular weight protein markers, pure 1,2-diolein, 1,3-diolein, olein, oleic acid, sodium acetate potassium phosphate, Tris–HCl and glycine NaOH were purchased from Sigma Chemical Co., USA. DEAE-Toyopearl 650-M and Sephadex G-75 were purchased from Pharmacia, Sweden. All other chemicals used were of analytical grade.
Enzyme production and assay
The pure culture of the Bacillus strain NS 8 which produces an alkaline thermostable lipase was obtained
Purification of lipase and molecular weight determination
The extracts from the fermentation in the bioreactor were concentrated using a Millipore PLGC UF membrane (10 kDa) cut-off and 600 ml of the concentrated crude lipase was used for the purification studies. The results of the procedure for the purification are summarized in Table 1. A three-step purification increased the specific activity of lipase by 500-fold with a yield of 16% (Table 1). The purification fold and yield obtained in this study are higher than those reported in previous studies.
Conclusion
Purification of B. subtilis NS 8 lipase was achieved using three purification steps. It was active at an optimum temperature of 60°C and stable in the pH and temperature range of 7.0–9.0 and 40–70°C. It was highly stable at 60, 70 and 80°C with half-lives of 273.38, 51.04 and 41.58 min, respectively. The D-values were 788.70, 169.59 and 138.15 min at 60, 70 and 80°C, respectively. Lipase activity was slightly enhanced when treated with Mg2+ but there was no significant enhancement or inhibition
Acknowledgements
We appreciate the Malaysian Ministry of Science, Technology and Innovation (MOSTI) for the financial support awarded to Professor Dr. Nazamid Saari under Sciencefund Project No. 05-01-04-SF0397.
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