Elsevier

New Biotechnology

Volume 28, Issue 6, October 2011, Pages 738-745
New Biotechnology

Research paper
Purification, characterization and thermal inactivation kinetics of a non-regioselective thermostable lipase from a genotypically identified extremophilic Bacillus subtilis NS 8

https://doi.org/10.1016/j.nbt.2011.01.002Get rights and content

Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07–70.40 kJ mol−1, −83.58 to −77.32 kJ mol−1 K−1 and 95.60–98.96 kJ mol−1, respectively. Lipase activity was slightly enhanced when treated with Mg2+ but there was no significant enhancement or inhibition of the activity with Ca2+. However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.

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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