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Research article
Biocatalysis and Fermentation Technology
Purification and Characterization of an Alkali-Thermostable Lipase from Thermophilic Anoxybacillus flavithermus HBB 134
Department of Biology, Faculty of Science and Arts, Adnan Menderes University, 09010 Aydn, Turkey
Received: December 21, 2015; Accepted: March 17, 2016
J. Microbiol. Biotechnol. 2016; 26(6): 1087-1097
Published June 28, 2016 https://doi.org/10.4014/jmb.1512.12056
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The
molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the
enzyme was at pH 9.0 and 50oC. The enzyme was stable between pH 6.0 and 11.0 at 25°C,
40°C, and 50°C for 24 h. The Km and Vmax of the enzyme for pNPL substrate were determined as
0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme
thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and
diethyl ether. The presence of PMSF, NBS, DTT and β-mercaptoethanol inhibited the enzyme
activity. Hg2+, Fe3+, Pb2+, Al3+, and Zn2+ strongly inhibited the enzyme whereas Li+, Na+, K+, and
NH4
+ slightly activated it. At least 60% of the enzyme activity and stability were retained
against sodium deoxycholate, sodium taurocholate, n-octyl-β-D-glucopyranoside, and CHAPS.
The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The
enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The
lipase of HBB 134 cleaved triolein at the 1- or 3-position.
Keywords
Anoxybacillus, lipase, purification, thermophilic, characterization
References
- Andualema B, Gessesse A. 2012. Microbial lipases and their industrial applications: review. Biotechnology 11: 100-118.
- Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO. 2011. Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF. Protein Expr. Purif. 80: 74-79.
- Bakir ZB, Metin K. 2015. Screening for industrially important enzymes from thermophilic bacteria; selection of lipaseproducing microorganisms and optimization of culture conditions. Eur. J. Biotechnol. Biosci. 3: 43-48.
- Bornscheuer UT. 2002. Microbial carboxyl esterases:classification, properties and application in biocatalysis. FEMS. Microbiol. Rev. 26: 73-81.
- Bradford MM. 1 976. A r apid and s ensitiv e method f or the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
- Castro-Ochoa LD, Rodríguez-Gómez C, Valerio-Alfaro G, Ros RO. 2005. Screening, purification and characterization of thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enzyme Microb. Technol. 37: 648-654.
- Chakraborty K, Raj RP. 2008. An extra-cellular alkaline metallolipase from Bacillus licheniformis MTCC 6824:purification and biochemical characterization. Food. Chem. 109: 727-736.
- Chi L, Hriscu M, Bica A, To a M, Nagy G, Róna G, et al. 2013. Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain. J. Gen. Appl. Microbiol. 59: 119-134.
- Colak A, Sisik D, Saglam N, Guner S, Canakcý S, Belduz AO. 2005. Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2. Bioresour. Technol. 96: 625-631.
- Dharmsthiti S, Luchai S. 1999. Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027. FEMS Microbiol. Lett. 179: 241-246.
- Emtenani S, Asoodeh A, Emtenania S. 2013. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: expression and biochemical characterization. Process Biochem. 48: 1679-1685.
- Faiz Ö, Çolak A, Saglam N, Çanakçi S, Beldüz AO. 2007. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4. J. Biochem. Mol. Biol. 40: 588-594.
- Fojan P, Jonson PH, Petersen MTN, Petersen SB. 2000. What distinguishes an esterase from a lipase: a novel structural approach. Biochimie 82: 1033-1041.
- Gaur R, Gupta A, Khare SK. 2008. Purification and characterization of lipase from solvent tolerant Pseudomonas aeruginosa PseA. Process Biochem. 43: 1040-1046.
- Hasan F, Shah AA, Hameed A. 2006. Industrial applications of microbial lipases. Enzyme Microb. Technol. 39: 235-251.
- Hiol A, Jonzo MD, Rugani N, Druet D, Sarda L, Comeau LC. 2000. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb. Technol. 26: 421-430.
- Iýzumi T, Nakamura K, Fukase T. 1990. Purification and characterization of a thermostable lipase from newly isolated Pseudomonas sp. KWI-56. Agric. Biol. Chem. 54: 1253-1258.
- Ji Q, Xiao S, He B, Liu X. 2010. Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production. J. Mol. Catal. B Enzym. 66: 264-269.
- Kulkarni N, Gadre R. 1999. A novel alkaline, thermostable, protease free lipase from Pseudomonas sp. Biotechnol. Lett. 21:897-899.
- Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R. 2005. Production, purification and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3. Protein Expr. Purif. 41: 38-44.
- Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
- Lee KW, Bae HA, Shin GS, Lee YH. 2006. Purification and catalytic properties of novel enantioselective lipase from Acinetobacter sp. ES-1 for hydrolysis of (S)-ketoprofen ethyl ester. Enzyme Microb. Technol. 38: 443-448.
- Lešèiæ I, Vukeliæ B, Majeriæ-Elenkov M, Saenger W, Abramiæ M. 2001. Substrate specificity and effects of watermiscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus. Enzyme Microb. Technol. 29:548-553.
- Lesuisse E, Schanck K, Colson C. 1993. Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur. J. Biochem. 216: 155-160.
- Lima VMG, Krieger N, Mitchell DA, Baratti JC, de Filippis I, Fontana JD. 2004. Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium. J. Mol. Catal. B Enzym. 31: 53-61.
- Lotrakul P, Dharmsthiti S. 1997. Purification and characterization of lipase from Aeromonas sobria LP004. J. Biotechnol. 54: 113120.
- Mhetras NC, Bastawde KB, Gokhale DV. 2009. Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207. Bioresour. Technol. 100: 1486-1490.
- Nawani N, Dosanjh N, Kaur J. 1998. A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies. Biotechnol. Lett. 20: 997–1000.
- Nawani N, Kaur J. 2007. Studies on lipolytic isoenzymes from a thermophilic Bacillus sp.: production, purification and biochemical characterization. Enzyme Microb. Technol. 40: 881-887.
- Nawani N, Khurana J, Kaur J. 2006. A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization. Mol. Cell. Biochem. 290: 17-22.
- Nthangeni MB, Patterton HG, Tonder AV, Vergeer WP, Litthauer D. 2001. Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases. Enzyme Microb. Technol. 28: 705712.
- Oh BC, Kim HK, Lee JK, Kang SC, Oh TK. 1999. Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning. FEMS Microbiol. Lett. 179: 385-392.
- Olusesan AT, Azura LK, Abubakar F, Hamid NSA, Radu S, Saari N. 2009. Phenotypic and molecular identification of a novel thermophilic Anoxybacillus species: a lipase-producing bacterium isolated from a Malaysian hotspring. World J. Microbiol. Biotechnol. 25: 1981-1988.
- Pinzón-Martínez DL, Rodríguez-Gómez C, Miñana-Galbis D, Carrillo-Chávez JA, Valerio-Alfaro G, Oliart-Ros R. 2010. Thermophilic bacteria from Mexican thermal environments:isolation and potential applications. Environ. Technol. 31:957-966.
- Rua ML, Schmidt-Dannert C, Wahl S, Sprauer A, Schmid RD. 1997. Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behaviour and its effect on activity. J. Biotechnol. 56: 89-102.
- Saxena RK, Ghosh PK, Gupta R, Davidson WS, Bradoo S, Gulati R. 1999. Microbial lipases: potential biocatalysts for the future industry. Curr. Sci. India 77: 101-115.
- Saxena RK, Davidson WS, Sheoran A, Giri B. 2003. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem. 39: 239-247.
- Sharma AK, T war RP, Hoondal GS. 2001. Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp. AG-8. J. Basic Microbiol. 41: 363-366.
- Sharma R, Chisti Y, Banerjee UC. 2001. Production, purification, characterization and applications of lipases. Biotechnol. Adv. 19: 627-662.
- Sigurgisladottir S, Kanarosdottir M, Jonsson A, Kristjansson JK, Mathiasson E. 1993. Lipase activity of thermophilic bacteria from Icelandic hot springs. Biotechnol. Lett. 15: 361-366.
- Snellman EA, Sullivan ER, Colwell RR. 2002. Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1. Eur. J. Biochem. 269: 5771-5779.
- Sulong MR, Raja ABD, Rahman RNZ, Salleh AB, Basri M. 2006. A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OSTlipase gene. Protein Expr. Purif. 49: 190-195.
- Tan T, Zhang M, Xu J, Zhang J. 2004. Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3. Process Biochem. 39: 1495-1502.
- Zhang A, Gao R, Diao N, Xie G, Gao G, Cao S. 2009. Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963. J. Mol. Catal. B Enzym. 56: 78-84.
- Zuridah H, Norazwin N, Siti Aisyah M, Fakhruzzaman MNA, Zeenathul NA. 2011. Identification of lipase producing thermophilic bacteria from Malaysian hot springs. Afr. J. Microbiol. Res. 21: 3569-3573.
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Research article
J. Microbiol. Biotechnol. 2016; 26(6): 1087-1097
Published online June 28, 2016 https://doi.org/10.4014/jmb.1512.12056
Copyright © The Korean Society for Microbiology and Biotechnology.
Purification and Characterization of an Alkali-Thermostable Lipase from Thermophilic Anoxybacillus flavithermus HBB 134
Zehra Burcu Bakir 1* and Kubilay Metin 1
Department of Biology, Faculty of Science and Arts, Adnan Menderes University, 09010 Aydn, Turkey
Received: December 21, 2015; Accepted: March 17, 2016
Abstract
An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The
molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the
enzyme was at pH 9.0 and 50oC. The enzyme was stable between pH 6.0 and 11.0 at 25°C,
40°C, and 50°C for 24 h. The Km and Vmax of the enzyme for pNPL substrate were determined as
0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme
thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and
diethyl ether. The presence of PMSF, NBS, DTT and β-mercaptoethanol inhibited the enzyme
activity. Hg2+, Fe3+, Pb2+, Al3+, and Zn2+ strongly inhibited the enzyme whereas Li+, Na+, K+, and
NH4
+ slightly activated it. At least 60% of the enzyme activity and stability were retained
against sodium deoxycholate, sodium taurocholate, n-octyl-β-D-glucopyranoside, and CHAPS.
The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The
enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The
lipase of HBB 134 cleaved triolein at the 1- or 3-position.
Keywords: Anoxybacillus, lipase, purification, thermophilic, characterization
References
- Andualema B, Gessesse A. 2012. Microbial lipases and their industrial applications: review. Biotechnology 11: 100-118.
- Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO. 2011. Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF. Protein Expr. Purif. 80: 74-79.
- Bakir ZB, Metin K. 2015. Screening for industrially important enzymes from thermophilic bacteria; selection of lipaseproducing microorganisms and optimization of culture conditions. Eur. J. Biotechnol. Biosci. 3: 43-48.
- Bornscheuer UT. 2002. Microbial carboxyl esterases:classification, properties and application in biocatalysis. FEMS. Microbiol. Rev. 26: 73-81.
- Bradford MM. 1 976. A r apid and s ensitiv e method f or the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
- Castro-Ochoa LD, Rodríguez-Gómez C, Valerio-Alfaro G, Ros RO. 2005. Screening, purification and characterization of thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enzyme Microb. Technol. 37: 648-654.
- Chakraborty K, Raj RP. 2008. An extra-cellular alkaline metallolipase from Bacillus licheniformis MTCC 6824:purification and biochemical characterization. Food. Chem. 109: 727-736.
- Chi L, Hriscu M, Bica A, To a M, Nagy G, Róna G, et al. 2013. Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain. J. Gen. Appl. Microbiol. 59: 119-134.
- Colak A, Sisik D, Saglam N, Guner S, Canakcý S, Belduz AO. 2005. Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2. Bioresour. Technol. 96: 625-631.
- Dharmsthiti S, Luchai S. 1999. Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027. FEMS Microbiol. Lett. 179: 241-246.
- Emtenani S, Asoodeh A, Emtenania S. 2013. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: expression and biochemical characterization. Process Biochem. 48: 1679-1685.
- Faiz Ö, Çolak A, Saglam N, Çanakçi S, Beldüz AO. 2007. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4. J. Biochem. Mol. Biol. 40: 588-594.
- Fojan P, Jonson PH, Petersen MTN, Petersen SB. 2000. What distinguishes an esterase from a lipase: a novel structural approach. Biochimie 82: 1033-1041.
- Gaur R, Gupta A, Khare SK. 2008. Purification and characterization of lipase from solvent tolerant Pseudomonas aeruginosa PseA. Process Biochem. 43: 1040-1046.
- Hasan F, Shah AA, Hameed A. 2006. Industrial applications of microbial lipases. Enzyme Microb. Technol. 39: 235-251.
- Hiol A, Jonzo MD, Rugani N, Druet D, Sarda L, Comeau LC. 2000. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb. Technol. 26: 421-430.
- Iýzumi T, Nakamura K, Fukase T. 1990. Purification and characterization of a thermostable lipase from newly isolated Pseudomonas sp. KWI-56. Agric. Biol. Chem. 54: 1253-1258.
- Ji Q, Xiao S, He B, Liu X. 2010. Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production. J. Mol. Catal. B Enzym. 66: 264-269.
- Kulkarni N, Gadre R. 1999. A novel alkaline, thermostable, protease free lipase from Pseudomonas sp. Biotechnol. Lett. 21:897-899.
- Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R. 2005. Production, purification and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3. Protein Expr. Purif. 41: 38-44.
- Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
- Lee KW, Bae HA, Shin GS, Lee YH. 2006. Purification and catalytic properties of novel enantioselective lipase from Acinetobacter sp. ES-1 for hydrolysis of (S)-ketoprofen ethyl ester. Enzyme Microb. Technol. 38: 443-448.
- Lešèiæ I, Vukeliæ B, Majeriæ-Elenkov M, Saenger W, Abramiæ M. 2001. Substrate specificity and effects of watermiscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus. Enzyme Microb. Technol. 29:548-553.
- Lesuisse E, Schanck K, Colson C. 1993. Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur. J. Biochem. 216: 155-160.
- Lima VMG, Krieger N, Mitchell DA, Baratti JC, de Filippis I, Fontana JD. 2004. Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium. J. Mol. Catal. B Enzym. 31: 53-61.
- Lotrakul P, Dharmsthiti S. 1997. Purification and characterization of lipase from Aeromonas sobria LP004. J. Biotechnol. 54: 113120.
- Mhetras NC, Bastawde KB, Gokhale DV. 2009. Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207. Bioresour. Technol. 100: 1486-1490.
- Nawani N, Dosanjh N, Kaur J. 1998. A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies. Biotechnol. Lett. 20: 997–1000.
- Nawani N, Kaur J. 2007. Studies on lipolytic isoenzymes from a thermophilic Bacillus sp.: production, purification and biochemical characterization. Enzyme Microb. Technol. 40: 881-887.
- Nawani N, Khurana J, Kaur J. 2006. A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization. Mol. Cell. Biochem. 290: 17-22.
- Nthangeni MB, Patterton HG, Tonder AV, Vergeer WP, Litthauer D. 2001. Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases. Enzyme Microb. Technol. 28: 705712.
- Oh BC, Kim HK, Lee JK, Kang SC, Oh TK. 1999. Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning. FEMS Microbiol. Lett. 179: 385-392.
- Olusesan AT, Azura LK, Abubakar F, Hamid NSA, Radu S, Saari N. 2009. Phenotypic and molecular identification of a novel thermophilic Anoxybacillus species: a lipase-producing bacterium isolated from a Malaysian hotspring. World J. Microbiol. Biotechnol. 25: 1981-1988.
- Pinzón-Martínez DL, Rodríguez-Gómez C, Miñana-Galbis D, Carrillo-Chávez JA, Valerio-Alfaro G, Oliart-Ros R. 2010. Thermophilic bacteria from Mexican thermal environments:isolation and potential applications. Environ. Technol. 31:957-966.
- Rua ML, Schmidt-Dannert C, Wahl S, Sprauer A, Schmid RD. 1997. Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behaviour and its effect on activity. J. Biotechnol. 56: 89-102.
- Saxena RK, Ghosh PK, Gupta R, Davidson WS, Bradoo S, Gulati R. 1999. Microbial lipases: potential biocatalysts for the future industry. Curr. Sci. India 77: 101-115.
- Saxena RK, Davidson WS, Sheoran A, Giri B. 2003. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem. 39: 239-247.
- Sharma AK, T war RP, Hoondal GS. 2001. Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp. AG-8. J. Basic Microbiol. 41: 363-366.
- Sharma R, Chisti Y, Banerjee UC. 2001. Production, purification, characterization and applications of lipases. Biotechnol. Adv. 19: 627-662.
- Sigurgisladottir S, Kanarosdottir M, Jonsson A, Kristjansson JK, Mathiasson E. 1993. Lipase activity of thermophilic bacteria from Icelandic hot springs. Biotechnol. Lett. 15: 361-366.
- Snellman EA, Sullivan ER, Colwell RR. 2002. Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1. Eur. J. Biochem. 269: 5771-5779.
- Sulong MR, Raja ABD, Rahman RNZ, Salleh AB, Basri M. 2006. A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OSTlipase gene. Protein Expr. Purif. 49: 190-195.
- Tan T, Zhang M, Xu J, Zhang J. 2004. Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3. Process Biochem. 39: 1495-1502.
- Zhang A, Gao R, Diao N, Xie G, Gao G, Cao S. 2009. Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963. J. Mol. Catal. B Enzym. 56: 78-84.
- Zuridah H, Norazwin N, Siti Aisyah M, Fakhruzzaman MNA, Zeenathul NA. 2011. Identification of lipase producing thermophilic bacteria from Malaysian hot springs. Afr. J. Microbiol. Res. 21: 3569-3573.
