Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate.
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 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) 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 Vmax and Km 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.
Kanwar
S S
SS
Department of Biotechnology, Himachal Pradesh University, Shimla-171 005, India. kanwarss2000@yahoo.com
Ghazi
I A
IA
Chimni
S S
SS
Joshi
G K
GK
Rao
G V
GV
Kaushal
R K
RK
Gupta
R
R
Punj
V
V
D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D004492:Edetic Acid / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006868:Hydrolysis / Q000187:drug effects; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry
D001426:Bacterial Proteins; D008667:Metalloproteins; D008670:Metals; D004492:Edetic Acid; D008049:Lipase