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Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate
Ist Teil von
Protein expression and purification, 2006-04, Vol.46 (2), p.421-428
Ort / Verlag
United States: Elsevier Inc
Erscheinungsjahr
2006
Quelle
Elsevier ScienceDirect Journals
Beschreibungen/Notizen
A novel extra-cellular lipase from
Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103
kDa. The lipase was optimally active at 45
°C and retained approximately 50% of its original activity after 20
min of incubation at 55
°C. The enzyme was optimally active at pH 8.5. Mg
2+, Cu
2+, Ca
2+, Hg
2+, Al
3+, and Fe
3+ at 1
mM enhanced hydrolytic activity of the lipase. Interestingly, Hg
2+ ions resulted in a maximal increase in lipase activity but Zn
2+ and Co
2+ ions showed an antagonistic effect on this enzyme. EDTA at 150
mM concentration inhibited the activity of lipase but Hg
2+ or Al
3+ (10
mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15
mM) decreased 98% of original activity of lipase. The lipase was more specific to
p-nitrophenyl esters of 8 (
pNPC) and 16 (
pNPP) carbon chain length esters. The lipase had a
V
max and
K
m of 0.44
mmol
mg
−1
min
−1 and 28
mM for hydrolysis of
pNPP, and 0.7
mmol
mg
−1
min
−1 and 32
mM for hydrolysis of
pNPC, respectively.