A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization.
A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60-65 ( composite function)C and retained 100% activity after incubation at 60 ( composite function)C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ( composite function)C and 23 min incubation at 80 ( composite function)C. Catalytic function of lipase was activated by Mg(++) (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, betaME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ) and V (max) of 0.5 mM and 0.139 microM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.
Nawani
Neerupma
N
Department of Biotechnology, Punjab University, Chandigarh 160014, India.
Khurana
Jyoti
J
Kaur
Jagdeep
J
D001426:Bacterial Proteins; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification*; D004364:Pharmaceutical Preparations; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004364:Pharmaceutical Preparations; D008049:Lipase; C428386:thermostable lipase