Abstract
Psychrotropic Bacillus sphaericus producing solvent stable cold-active lipase upon growth at low temperature was isolated from Gangotri glacier. Optimal parameters for lipase production were investigated and the strain was able to produce lipase even at 15 °C. An incubation period of 48 h and pH 8 was found to be conducive for cold-active lipase production. The addition of trybutyrin as substrate and lactose as additional carbon source increased lipase production. The enzyme was purified up to 17.74-fold by ammonium sulphate precipitation followed by DEAE cellulose column chromatography. The optimum temperature and pH for lipase activity were found to be 15 °C and 8.0, respectively. The lipase was found to be stable in the temperature range 20–30 °C and the pH range 6.0–9.0. The protein retained more than 83 % of its initial activity after exposure to organic solvents. The lipase exhibited significant stability in presence of acetone and DMSO retaining >90 % activity. The enzyme activity was inhibited by 10 mM CuSO4 and EDTA but showed no loss in activity after incubation with other metals or inhibitors examined in this study.
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Alquati C, De Gioia L, Santarossa G, Alberghina L, Fantucci P, Lotti M (2002) The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling. Eur J Biochem 269:3321–3328
Baghel VS, Tripathi RD, Ramteke PW, Gopal K, Dwivedi S, Jain RK, Rai UN, Singh SN (2005) Psychrotrophic proteolytic bacteria from cold environment of Gangotri glacier, western Himalaya, India. Enzyme Microb Technol 36:654–659
Berglund P, Hutt K (2000) Biocatalytic synthesis of enantiopure compounds using lipases. In: Patel RN (ed) Stereoselective biocatalysis. Marcel Dekker, New York, pp 188–194
Calik PO, Zdamar TH (2001) Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analysis for serine and neutral proteases and a-amylase. Biochem Eng J 8:61–81
Choo DW, Kurihara T, Suzuki T, Soda K, Esaki N (1998) A cold adapted lipase of an alaskan psychtotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization. Appl Environ Microbiol 64:486–491
Feller G, Narinx E, Arpigny JL, Aittaleb M, Baise E, Geniot S, Gerday C (1996) Enzymes from psychrophilic organisms. FEMS Microbiol Rev 18:189–202
Ferrer M, Soliveri J, Plou FJ, Lopez-Corter N, Reyes-Duarte D, Christensen M, Copa-Patino JL, Ballesteros A (2005) Synthesis of sugar esters in solvent mixtures by lipase from Thermomyces lanuginosus and Candida antarctica B and their antimicrobial properties. Enzyme Microb Technol 36:391–398
Gupta A, Joseph B, Mani A, Thomas G (2008) Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World J Microbiol Biotechnol 24:237–243
Hun CJ, Rahman RNZA, Salleh AB, Basri M (2003) A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase. Biochem Eng J 15:147–151
Ionita A, Moscovici M, Popa C, Vamanu A, Popa O, Dinu L (1997) Screening of yeast and fungal strains for lipolytic potential and determination of some biochemical properties of microbial lipases. J Mol Catal B: Enzym 3:147–151
Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13:390–397
Joseph B, Ramteke PW, Kumar PA (2006) Studies on the enhanced production of extracellular lipase by Staphylococcus epidermidis. J Gen Appl Microbiol 52:315–320
Joseph B, Ramteke PW, Thomas G (2008) Cold-active microbial lipases: Some hot issues and recent developments. Biotech Adv 26:457–470
Koops BC, Verheij HM, Slotboom AJ, Egmond MR (1999) Effect of chemical modification on the activity of lipases in organic solvents. Enzyme Microbiol Technol 25:622–631
Lowry OH, Rosebrough NJ, Farr AL, Randal RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Marshall CJ (1997) Cold-adapted enzymes. Trends Biotechnol 15:359–364
Nawani N, Dosanjh NS, Kaur J (1998) A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies. Biotech Lett 20(10):997–1000
Noble ME, Cleasby A, Johnson LN, Egmond MR, Frenken LG (1993) The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate. FEBS Lett 331:123–128
Ogino H, Nakagawa S, Shinya K, Muto T, Fujimura N, Yasudo M, Ishikawa H (2000) Purification and characterization of organic solvent tolerant lipase from organic solvent tolerant Pseudomonas aeruginosa LST-03. J Biosci Bioeng 89:451–457
Rahman RNZRA, Baharum SN, Basri M, Salleh AB (2005) High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5. Anal Biochem 341:267–274
Schmidt-Dannert C, Sztajer H, Stocklein W, Menge U, Schmid RD (1994) Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochim Biophys Acta 1214:43–53
Sharon C, Furugoh S, Yamakido T, Ogawa HI, Kato Y (1998) Purification and characterization of lipases from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis. J Ind Microbiol Biotech 20:304–307
Sikkema J, Bont DJAM, Poolman B (1994) Intercalations of cyclic hydrocarbons with biological membranes. J Biol Chem 269:8022–8028
Singh J, Yadav RR (2000) Tree-rings indications of recent glacier fluctuations in Gangotri, western Himalaya India. Curr Sci 79:1598–1601
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
Soni K, Madamwar D (2000) Ester synthesis by lipase immobilized on silica and microemulsion based organogels (MBGs). Process Biochem 36:607–611
Sugihara A, Tani T, Tominaga Y (1991) Purification and characterization of a novel thermostable lipase from Bacillus sp. J Biochem 109:211–216
Thomas SB, Thomas BF (1973) Psychrotrophic bacteria in refrigerated bulk-collected raw milk. Part II. Dairy Ind 38:61–70
Voet D, Voet JG, Pratt CW (1999) Fundamentals of biochemistry. Wiley, New York
Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–670
Acknowledgement
The authors are grateful to Department of Biotechnology Govt. of India for the grant (BT/PR2639/PID/24/112/2001) received to carry out this study.
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Joseph, B., Ramteke, P.W. Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526: partial purification and characterization. Ann Microbiol 63, 363–370 (2013). https://doi.org/10.1007/s13213-012-0483-y
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DOI: https://doi.org/10.1007/s13213-012-0483-y