Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

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• 作者：S.S. Kanwar ; I.A. Ghazi ; S.S. Chimni ; G.K. Joshi ; G.V. Rao ; R.K. Kaushal ; R. Gupta and V. Punj
• 关键词：Bacillus coagulans MTCC-6375 ; Lipase purification ; Biochemical characterization ; Effect of metal-ions
• 刊名：Protein Expression and Purification
• 出版年：2006
• 出版时间：April 2006
• 年：2006
• 卷：46
• 期：2
• 页码：421-428
• 全文大小：293 K

文摘

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. Mg²⁺, Cu²⁺, Ca²⁺, Hg²⁺, Al³⁺, and Fe³⁺ at 1 mM 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⁺ (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.