BIOCHEMICAL PROPERTIES OF RECOMBINANT Β-GALACTOSIDASE FROM STREPTOCOCCUS THERMOPHILUS
Main Article Content
Authors
K.K. Baltin
National Center for Biotechnology,13/5, Korgalzhyn hwy, Astana, Kazakhstan
Zh.D. Akishev
National Center for Biotechnology,13/5, Korgalzhyn hwy, Astana, Kazakhstan
S.K. Abeldenov
National Center for Biotechnology,13/5, Korgalzhyn hwy, Astana, Kazakhstan
D.V. Silayev
National Center for Biotechnology,13/5, Korgalzhyn hwy, Astana, Kazakhstan
B.B. Khassenov
National Center for Biotechnology,13/5, Korgalzhyn hwy, Astana, Kazakhstan
Abstract
Recombinant β-galactosidase from Streptococcus thermophilus was successfully expressed in Escherichia coli, purified, and biochemically characterized. The gene encoding β-galactosidase was amplified from the genomic DNA of St. thermophilus and cloned into the expression vector pET-28c (+). Using the recombinant vector, a BL21 (DE3)/pLacZST strain-producer was obtained with overexpression of the gene. Optimal culture parameters for producing recombinant β-galactosidase were determined. The recombinant β-galactosidase had an activity of 19 units/mg. Biochemical characterization of recombinant β-galactosidase showed that the enzyme had maximum activity at pH 9.0 and temperature of 60°C. Analysis of the kinetics of lactose hydrolysis gave a Michaelis constant Kmof 10.12 ± 2.5 mM and a limiting value of the initial rate of the enzymatic reaction Vmaxof 0.47 ± 0.027 mM/min. The β-galactosidase has been used in experiments that simulate commercial production conditions, to producea glucose-galactose syrup.
Keywords
β-galactosidase, Streptococcus thermophilus, genomic DNA, lactose
Article Details
References
Arribas J.C., Herrero A.G., Martin-Lomas M., Canada F.J., He S., Withers S.G. Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase.Eur J Biochem., 2000,vol. 267, no. 24, pp. 6996-7005.
Braunstein A. Recommendations of the International Biochemical Union on the nomenclature and classification of enzymes, as well as on the units of enzymes and symbols of the kinetics of enzymatic reactions. Nomenclature of enzymes., 1979, pp. 320.
Panesar P.S., Kumari S., Panesar R. Potential Applications of Immobilized beta-Galactosidase in Food Processing Industries.Enzyme Res., 2010, vol. 2010, pp. 473-137.
Husain Q. Beta galactosidases and their potential applications: a review.Crit Rev Biotechnol., 2010,vol. 30, no. 1, pp. 41-62.
Heyman M.B. Lactose intolerance in infants, children, and adolescents.Pediatrics., 2006,vol. 118, no. 3, pp. 1279-1286.
V. Gekas M.L.-L. Hydrolysis of lactose: a literature review.Process Biochemistry, 1985,vol. 20, pp. 2-12.
Pisani F.M., Rella R., Raia C.A., Rozzo C., et al. Thermostable beta-galactosidase from the archaebacteriumSulfolobussolfataricus. Purification and properties.Eur J Biochem., 1990,vol. 187, no. 2, pp. 321-328.
Ohtsu N., Motoshima H., Goto K., Tsukasaki F., Matsuzawa H. Thermostable beta-galactosidase from an extreme thermophile, Thermus sp. A4: enzyme purification and characterization, and gene cloning and sequencing.BiosciBiotechnolBiochem., 1998,vol. 62, no. 8, pp. 1539-1545.
Hidaka M., Fushinobu S., Ohtsu N., et al. Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermusthermophilus A4 and the structure of its complex with galactose.J Mol Biol., 2002,vol. 322, no. 1, pp. 79-91.
Wong-Madden S.T., Landry D. Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas.Glycobiology, 1995, vol. 5, no. 1, pp. 19-28.
Nakagawa T., Fujimoto Y., Ikehata R., Miyaji T., Tomizuka N. Purification and molecular characterization of cold-active beta-galactosidase from Arthrobacterpsychrolactophilus strain F2.ApplMicrobiolBiotechnol., 2006,vol. 72, no. 4, pp. 720-725.
Fernandes S., Geueke B., Delgado O., Coleman J., Hatti-Kaul R. Beta-galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis.ApplMicrobiolBiotechnol., 2002,vol. 58, no. 3, pp. 313-321.
Song C., Chi Z., Li J., Wang X. beta-Galactosidase production by the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica and lactose hydrolysis.Bioprocess Biosyst Eng., 2010,vol. 33, no. 9, pp. 1025-1031.
Hirata H., Negoro S., Okada H. High Production of Thermostable beta-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis.Appl Environ Microbiol., 1985,vol. 49, no. 6, pp. 1547-1549.
Sriphannam W., Lumyong S., Niumsap P., et al. A selected probiotic strain of Lactobacillus fermentum CM33 isolated from breast-fed infants as a potential source of beta-galactosidase for prebiotic oligosaccharide synthesis.J Microbiol., 2012,vol. 50, no. 1, pp. 119-126.
Liu G.X., Kong J., Lu W.W., Kong W.T., et al. beta-Galactosidase with transgalactosylation activity from Lactobacillus fermentum K4.J Dairy Sci., 2011,vol. 94, no. 12, pp. 5811-5820.
Benavente R., Pessela B.C., Curiel J.A., et al. Improving Properties of a Novel beta-Galactosidase from Lactobacillus plantarum by Covalent Immobilization.Molecules., 2015,vol. 20, no. 5, pp. 7874-7889.
Acebron I., Curiel J.A., de Las Rivas B., Munoz R., Mancheno J.M. Cloning, production, purification and preliminary crystallographic analysis of a glycosidase from the food lactic acid bacterium Lactobacillus plantarum CECT 748(T).Protein Expr Purif., 2009,vol. 68,no., pp. 177-182.
Schroeder C.J., Robert C., Lenzen G., McKay L.L., Mercenier A. Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences.J Gen Microbiol., 1991,vol. 137, no. 2, pp. 369-380.
Vincent V., Aghajari N., Pollet N., et al. The acid tolerant and cold-active beta-galactosidase from Lactococcuslactis strain is an attractive biocatalyst for lactose hydrolysis. Antonie Van Leeuwenhoek. Journal of Microbiology, 2013,vol. 103, no. 4, pp. 701-712.
Magalhes F., Aguiar T.Q., Oliveira C., Domingues L. High-level expression of Aspergillus niger b-galactosidase in Ashbyagossypii.BiotechnolProg., 2014,vol. 30, no. 2, pp. 261-268.
Oliveira C., Teixeira J.A., Lima N., Da Silva N.A., Domingues L. Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus nigerbeta-galactosidase production.J BiosciBioeng., 2007,vol. 103, no. 4, pp. 318-324.
Hu X., Robin S., O'Connell S., Walsh G., Wall J.G. Engineering of a fungal beta-galactosidase to remove product inhibition by galactose.ApplMicrobiolBiotechnol., 2010,vol. 87, no. 5, pp. 1773-1782.
Molecular cloning. A laboratory manual / T. Maniatis E., J. Sambrook.New York,Cold Spring Harbor Laboratory, 1982, 545 p.
Greenberg N.A., R. M. R. Production and Characterization of β-Galactosidase from Streptococcus thermophiles.Food Science, 1982,vol. 47, no. 6, pp. 1824-1835.