A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization |
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Authors: | Neerupma Nawani Jyoti Khurana Jagdeep Kaur |
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Institution: | (1) Department of Biotechnology, Punjab University, Chandigarh, 160014, India |
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Abstract: | 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 ∘C and retained 100% activity after incubation at 60 ∘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 ∘C and 23 min incubation at 80 ∘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, βME (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 μM/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. |
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Keywords: | lipase thermostable enzyme purification physico-chemical properties immunointerference |
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