Purification and Characterization of an Alkali-Thermostable Lipase from Thermophilic Anoxybacillus flavithermus HBB 134 = Purification and Characterization of an Alkali-Thermostable Lipase from Thermophilic Anoxybacillus flavithermus HBB 134

다국어 초록 (Multilingual 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. Hg(2+), Fe(3+), Pb(2+), Al(3+), and Zn(2+) 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.

참고문헌 (Reference)

1 Fojan P, "What distinguishes an esterase from a lipase: a novel structural approach" 82 : 1033-1041, 2000

2 Pinzón-Martínez DL, "Thermophilic bacteria from Mexican thermal environments:isolation and potential applications" 31 : 957-966, 2010

3 Rua ML, "Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behaviour and its effect on activity" 56 : 89-102, 1997

4 Lešèiæ I, "Substrate specificity and effects of watermiscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus" 29 : 548-553, 2001

5 Nawani N, "Studies on lipolytic isoenzymes from a thermophilic Bacillus sp.: production, purification and biochemical characterization" 40 : 881-887, 2007

6 Oh BC, "Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning" 179 : 385-392, 1999

7 Castro-Ochoa LD, "Screening, purification and characterization of thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11" 37 : 648-654, 2005

8 Bakir ZB, "Screening for industrially important enzymes from thermophilic bacteria; selection of lipaseproducing microorganisms and optimization of culture conditions" 3 : 43-48, 2015

9 Snellman EA, "Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1" 269 : 5771-5779, 2002

10 Lesuisse E, "Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme" 216 : 155-160, 1993

11 Gaur R, "Purification and characterization of lipase from solvent tolerant Pseudomonas aeruginosa PseA" 43 : 1040-1046, 2008

12 Lotrakul P, "Purification and characterization of lipase from Aeromonas sobria LP004" 54 : 113-120, 1997

13 Ji Q, "Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production" 66 : 264-269, 2010

14 Hiol A, "Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit" 26 : 421-430, 2000

15 Saxena RK, "Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus" 39 : 239-247, 2003

16 Mhetras NC, "Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207" 100 : 1486-1490, 2009

17 Iýzumi T, "Purification and characterization of a thermostable lipase from newly isolated Pseudomonas sp. KWI-56" 54 : 1253-1258, 1990

18 Lee KW, "Purification and catalytic properties of novel enantioselective lipase from Acinetobacter sp. ES-1 for hydrolysis of (S)-ketoprofen ethyl ester" 38 : 443-448, 2006

19 Sharma AK, "Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp. AG-8" 41 : 363-366, 2001

20 Sharma R, "Production, purification, characterization and applications of lipases" 19 : 627-662, 2001

21 Dharmsthiti S, "Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027" 179 : 241-246, 1999

22 Kumar S, "Production, purification and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3" 41 : 38-44, 2005

23 Olusesan AT, "Phenotypic and molecular identification of a novel thermophilic Anoxybacillus species: a lipase-producing bacterium isolated from a Malaysian hotspring" 25 : 1981-1988, 2009

24 Nthangeni MB, "Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases" 28 : 705-712, 2001

25 Tan T, "Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3" 39 : 1495-1502, 2004

26 Emtenani S, "Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: expression and biochemical characterization" 48 : 1679-1685, 2013

27 Chi L, "Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain" 59 : 119-134, 2013

28 Saxena RK, "Microbial lipases: potential biocatalysts for the future industry" 77 : 101-115, 1999

29 Andualema B, "Microbial lipases and their industrial applications: review" 11 : 100-118, 2012

30 Bornscheuer UT, "Microbial carboxyl esterases:classification, properties and application in biocatalysis" 26 : 73-81, 2002

31 Sigurgisladottir S, "Lipase activity of thermophilic bacteria from Icelandic hot springs" 15 : 361-366, 1993

32 Hasan F, "Industrial applications of microbial lipases" 39 : 235-251, 2006

33 Zuridah H, "Identification of lipase producing thermophilic bacteria from Malaysian hot springs" 21 : 3569-3573, 2011

34 Lima VMG, "Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium" 31 : 53-61, 2004

35 Ahmet Colak, "Determination and Characterization of Thermostable Esterolytic Activityfrom a Novel Thermophilic Bacterium Anoxybacillus gonensis A4" 한국생화학분자생물학회 40 (40): 588-594, 2007

36 Ay F, "Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF" 80 : 74-79, 2011

37 Zhang A, "Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963" 56 : 78-84, 2009

38 Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4" 227 : 680-685, 1970

39 Colak A, "Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2" 96 : 625-631, 2005

40 Bradford MM, "Arapid and s ensitiv e method f or the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-254, 1976

41 Chakraborty K, "An extra-cellular alkaline metallolipase from Bacillus licheniformis MTCC 6824:purification and biochemical characterization" 109 : 727-736, 2008

42 Nawani N, "A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization" 290 : 17-22, 2006

43 Nawani N, "A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies" 20 : 997-1000, 1998

44 Sulong MR, "A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OSTlipase gene" 49 : 190-195, 2006

45 Kulkarni N, "A novel alkaline, thermostable, protease free lipase from Pseudomonas sp" 21 : 897-899, 1999