Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372.

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+ and Fe3+ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions synergized and Zn2+ and Co2+ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p-nitrophenyl palmitate and showed a Vmax and Km of 0.70 mmol.mg⁻¹.min⁻¹ and 32 mM for hydrolysis of pNPP.

[1]  S. S. Kanwar,et al.  Microbial lipases: at the interface of aqueous and non-aqueous media. A review. , 2008, Acta microbiologica et immunologica Hungarica.

[2]  R. Gupta,et al.  Bacterial lipases: an overview of production, purification and biochemical properties , 2004, Applied Microbiology and Biotechnology.

[3]  Y. Pyun,et al.  Isolation and characterization of a thermophilic lipase from bacillus thermoleovorans ID-1. , 1999, FEMS microbiology letters.

[4]  M. Reetz,et al.  Overexpression, immobilization and biotechnological application of Pseudomonas lipases. , 1998, Chemistry and physics of lipids.

[5]  J. Kaur,et al.  Purification, characterization and thermostability of lipase from a thermophilic Bacillus sp. J33 , 2000, Molecular and Cellular Biochemistry.

[6]  S. S. Kanwar,et al.  Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3. , 2005, Protein expression and purification.

[7]  S. S. Kanwar,et al.  Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372. , 2008, Acta microbiologica et immunologica Hungarica.

[8]  Y. Tsai,et al.  Effect of triton X-100 on alkaline lipase production by Pseudomonas pseudoalcaligenes F-111 , 1995, Biotechnology Letters.

[9]  S. S. Kanwar,et al.  Methods for inhibition of residual lipase activity in colorimetric assay: a comparative study. , 2005, Indian journal of biochemistry & biophysics.

[10]  Y. Pyun,et al.  Purification and characterization of two distinct thermostable lipases from the gram-positive thermophilic bacterium Bacillus thermoleovorans ID-1 , 2001 .

[11]  B. Dijkstra,et al.  Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. , 1999, Annual review of microbiology.

[12]  Magyar Tudományos Akadémia.  Acta microbiologica et immunologica Hungarica , 1994 .

[13]  S. S. Kanwar,et al.  Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate. , 2006, Protein expression and purification.

[14]  S. S. Kanwar,et al.  Properties and application of poly(methacrylic acid-co-dodecyl methacrylate-cl-N,N-methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetate , 2008 .

[15]  Pramod P. Wangikar,et al.  Lipase-Catalyzed Esterification , 2000 .

[16]  S. Erazo,et al.  Cocoa butter equivalent through enzymic interesterification of palm oil midfraction , 2001 .

[17]  F. Molinari,et al.  Isolation of a novel carboxylesterase from Bacillus coagulans with high enantioselectivity toward racemic esters of 1,2-O-isopropylideneglycerol , 1996 .

[18]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[19]  Karl-Erich Jaeger,et al.  Lipases for biotechnology. , 2002, Current opinion in biotechnology.

[20]  Babu Joseph,et al.  Standard Review Cold-active microbial Lipases: a versatile tool for industrial applications , 2007 .

[21]  A. Bhaduri,et al.  Purification and characterization of an alkaline lipase from a newly isolated Pseudomonas mendocina PK-12CS and chemoselective hydrolysis of fatty acid ester. , 2003, Bioorganic & medicinal chemistry.

[22]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[23]  A. Kademi,et al.  Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans , 2000, Applied Microbiology and Biotechnology.

[24]  U. Winkler,et al.  Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens , 1979, Journal of bacteriology.

[25]  Sajja Hari Krishna,et al.  Optimizing lipases and related enzymes for efficient application. , 2002, Trends in biotechnology.

[26]  Abdul Hameed,et al.  Industrial applications of microbial lipases , 2006 .

[27]  H. El-Shafei,et al.  Production, purification and characterization of Bacillus lipase. , 1997, Microbiological research.

[28]  D. Freire,et al.  Penicillium restrictum lipases: A comparative study and characterization of enzymes with different degrees of purity , 1999 .

[29]  Abel Hiol,et al.  Production, purification and characterization of an extracellular lipase from Mucor hiemalis f. hiemalis , 1999 .