Thermostable Pyrococcus woesei beta-D-galactosidase - high level expression, purification and biochemical properties

• Autorzy: Wanarska M. , Kur J. , Pladzyk R. , Turkiewicz M.
• Języki publikacji: EN

Abstrakty

EN

The gene encoding β-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei β-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-β-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei β-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type β-D-galactosidase (51100 U/mg). The activity of P. woesei β-D-galactosidase was enhanced by thiol compounds, Mg2+ ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca2+ ions had no effect.

Słowa kluczowe

EN

expression system; beta-D-galactosidase; Pyrococcus woesei; plasmid construction; gene encoding; purification; Escherichia coli

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2005
• Tom: 52
• Numer: 4
• Opis fizyczny: p.781-787,fig.,ref.

Twórcy

autor

Wanarska M.

• Gdansk University of Technology, Gdansk, Poland

autor

Kur J.

autor

Pladzyk R.

autor

Turkiewicz M.

Bibliografia

• Boon MA, Janssen AEM, van’t Riet K (2000) Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme Microb Technol 26: 271–281.
• 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: 248–254.
• Craven GR, Steers E, Enfinsen CB (1965) Purification, composition and molecular weight of the β-galactosidase of Escherichia coli K12. J Biol Chem 240: 2468–2477.
• Crittenden RG, Playne MJ (1996) Production, properties and applications of food-grade oligosaccharides. Trends Food Sci Technol 7: 353–361.
• Dąbrowski S, Kur J (1999) Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. Protein Expr Purif 16: 96–102.
• Dąbrowski S, Maciuńska J, Synowiecki J (1998) Cloning and nucleotide sequence of the thermostable β-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Mol Biotechnol 10: 217–222.
• Dąbrowski S, Sobiewska G, Maciuńska J, Synowiecki J, Kur J (2000) Cloning, expression and purification of the His6-tagged thermostable β-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Protein Expr Purif 19: 107–112.
• Gekas V, Lopez Leiva MH (1985) Hydrolysis of lactose: a literature review. Process Biochem 20: 2–12.
• Jurado E, Camacho F, Luzón G, Vicaria JM (2002) A new kinetic model proposed for enzymatic hydrolysis of lactose by a β-galactosidase from Kluyveromyces fragilis. Enzyme Microb Technol 31: 300–309.
• Ladero M, Santos A, Garcia-Ochoa F (2000) Kinetic modeling of lactose hydrolysis by an immobilized β-galactosidase from Kluyveromyces fragilis. Enzyme Microb Technol 27: 583–592.
• Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
• Lopez Leiva MH, Guzman M (1995) Formation of oligosaccharides during enzymic hydrolysis of milk whey permeates. Process Biochem 30: 752–762.
• Mahoney RR (1998) Galactosyl-oligosaccharide formation during lactose hydrolysis: a review. Food Chem 63: 147–154.
• Papayannakos N, Markas G, Kekos D (1993) Studies on modeling and simulation of lactose hydrolysis by free and immobilized β-galactosidase from Aspergillus niger. Chem Eng J 52: B1–B12.
• Portaccio M, Stellato S, Rossi S, Bencivenga U, Mohy Eldin MS, Gaeta FS, Mita DG (1998) Galactose competitive inhibition of β-galactosidase (Aspergillus oryzae) immobilized on chitosan and nylon supports. Enzyme Microb Technol 23: 101–106.
• Reuter S, Rusborg Nygaard A, Zimmermann W (1999) β- Galactooligosaccharide synthesis with β-galactosidases from Sulfolobus solfataricus, Aspergillus oryzae, and Escherichia coli. Enzyme Microb Technol 25: 509–516.
• Rosado JL, Allen LH, Solomons NW (1987) Milk consumption, symptom response and lactose digestion in milk intolerance. Am J Clin Nutr 45: 1457–1460.
• Sako T, Matsumoto K, Tanaka R (1999) Recent progress on research and applications of non-digestible galacto-oligosaccharides. Int Dairy J 9: 69–80.
• Santos A, Ladero M, Garcia-Ochoa F (1998) Kinetic modeling of lactose hydrolysis by a β-galactosidase from Kluyveromyces fragilis. Enzyme Microb Technol 22: 558– 567.
• Shukla TP (1975) Beta-galactosidase technology: a solution to the lactose problem. CRC Crit Rev Food Technol 5: 325–356.