Summary
A thermostable lipase gene from Pseudomonas fluorescens SIK W1 was overexpressed in Escherichia coli BL21 using expression vector pTTY2. The amount of lipase produced by E. coli BL21 with pTTY2 was more than 40% of the total cell proteins when induced with isopropyl-β-d-thiogalactopyranoside. The lipase was produced as inclusion bodies in the cytoplasm of E. coli. They were solubilized by 8 m urea and refolded into biologically active form. The refolded lipase showed high thermostability; the time required for 90% inactivation of the enzyme (D-value) was 4 h at 95°C and the increment of temperature to reduce heating times by 90% (z d value) was 76°C.
Similar content being viewed by others
References
Adams DM, Brawley TG (1981) Heat resistant bacterial lipases and ultra-high temperature sterilization of dairy products. J Dairy Sci 64:1951–1957
Andersson RE, Hedlund CB, Jonsson U (1979) Thermal inactivation of a heat-resistant lipase produced by the psychrotrophic bacterium Pseudomonas fluorescens. J Dairy Sci 62:361–367
Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–264
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Kosugi Y, Suzuki H, Funada T (1988) Hydrolysis of beef tallow by lipase from Pseudomonas sp. Biotechnol Bioeng 31:349–356
Kwon DY, Rhee JS (1986) A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J Am Oil Chem Soc 63:89–92
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Liu WH, Beppu T, Arima K (1972) Purification and general properties of the lipase of thermophilic fungus Humicola lanuginosa S-38. Agric Biol Chem 37:157–163
Maniatis T, Fritch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Marston FAO (1986) The purification of eucaryotic polypeptides synthesized in Escherichia coli. Biochem J 240:1–12
Marston FAO (1987) The purification of eucaryotic polypeptides expressed in Escherichia coli. In: Glover DM (ed) DNA cloning, a practical approach, vol III. IRL Press, Oxford, pp 59–88
Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321
Nishio T, Chikano T, Kamimura M (1987) Purification and some properties of lipase produced by Pseudomonas fragi 22.39B. Agric Biol Chem 51:181–186
Schoner RG, Ellis LF, Schoner BE (1985) Isolation and purification of protein granules from Escherichia coli cells overproducting bovine growth hormone. Bio/Technology 3:151–154
Stark MJR (1987) Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene 51:255–267
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned gene. J Mol Biol 189:113–130
Yamane T (1988) Enzyme technology for the lipids industry: an engineering overview. In: Applewhite TH (ed) Proceedings of World Conference on biotechnology for the Fats and Oils Industry, Hamburg, Germany, September 27-October 2, 1987. American Oil Chemists′ Society, Champaign, Ill, USA, pp 17–22
Author information
Authors and Affiliations
Additional information
Offprint requests to: J. S. Rhee
Rights and permissions
About this article
Cite this article
Chung, G.H., Lee, Y.P., Yoo, O.J. et al. Overexpression of a thermostable lipase gene from Pseudomonas fluorescens in Escherichia coli . Appl Microbiol Biotechnol 35, 237–241 (1991). https://doi.org/10.1007/BF00184694
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00184694