PRODUCTION OF RECOMBINANT LACTASE FROM Lactobacillus helveticus VA FOR APPLICATION IN ADVANCED MILK PROCESSING TECHNOLOGIES
Main Article Content
Authors
Y.A. Shamsiyeva
“National Center for Biotechnology” Ltd., 13/5 Korgalzhyn road, Astana, 010000, Kazakhstan
A.T. Kostanova
“National Center for Biotechnology” Ltd., 13/5 Korgalzhyn road, Astana, 010000, Kazakhstan
S.Seifullin Kazakh Agrotechnical Research University, 62 Zhenis av. Astana, 010011, Kazakhstan
A.E. Tursunbekova
“National Center for Biotechnology” Ltd., 13/5 Korgalzhyn road, Astana, 010000, Kazakhstan
S.Seifullin Kazakh Agrotechnical Research University, 62 Zhenis av. Astana, 010011, Kazakhstan
B.B. Khassenov
“National Center for Biotechnology” Ltd., 13/5 Korgalzhyn road, Astana, 010000, Kazakhstan
“GenLab” Ltd, 19/1 M. Gabdullin str., Astana, 010000, Kazakhstan
Zhiger Akishev
“National Center for Biotechnology” Ltd., 13/5 Korgalzhyn road, Astana, 010000, Kazakhstan
“GenLab” Ltd, 19/1 M. Gabdullin str., Astana, 010000, Kazakhstan
Abstract
β-Galactosidases (lactases, EC 3.2.1.23) are widely applied in the production of lactose-free dairy products and whey processing. The aim of the study is to obtain recombinant β-galactosidase and to determine its biochemical and kinetic parameters.
In the present work, eight strains were isolated from goat, sheep, mare, and camel milk, as well as cheeses and fermented dairy products from the Akmola and Almaty regions: Lactobacillus plantarum NAR, Lactobacillus plantarum VGM1, Lactobacillus paracasei VK2, Lactococcus lactis ZCM, Lactococcus lactis IS2, Lactobacillus curvatus ICH3, Lactobacillus rhamnosus ICH5, and Lactobacillus helveticus VA.
Screening for lactase activity revealed that the cheese-isolated strain Lactobacillus helveticus VA exhibited the highest activity compared to the other strains, reaching 212.63 U/mg. The β-galactosidase gene from Lactobacillus helveticus was cloned into the pET-28c(+) vector, and the recombinant lactase rLacLM was produced via plasmid expression in the Escherichia coli BL21(DE3) strain. Using immobilized metal affinity chromatography (IMAC), rLacLM was purified from the induced recombinant culture. The recombinant β-galactosidase exhibited maximum activity at pH 6.5–7.0 and a temperature of 50°C. The specific activity of the enzyme was 76.1 U/mg, while the Km, max, kcat, and kcat/Km values were 21.76 mM, 1015 µM·min⁻¹, 141.48 s⁻¹, and 6.5 × 10³ M⁻¹·s⁻¹, respectively. Thin-layer chromatography confirmed efficient lactose hydrolysis yielding glucose and galactose.
These results indicate that the recombinant β-galactosidase from Lactobacillus helveticus VA possesses biochemical and catalytic characteristics compatible with milk processing conditions, rendering it a promising enzyme for the production of lactose-free dairy products and the further development of domestic enzyme preparations.
Keywords
milk, lactose, β-galactosidase, hydrolysis
Article Details
References
Zhou Y., Liu Y., Gao F., Xia Z., Zhang Z., Addai F. P., Zhu Y., Chen J., Lin F., Chen D. β-Galactosidase: Insights into source variability, genetic engineering, immobilization and diverse applications in food, industry and medicine// International Journal of Dairy Technology.- 2024. -Vol.77.-No 3.-P. 651-679. http://Crossref
Ruiz-Ramírez S., Jiménez-Flores R. Invited review: Properties of β-galactosidases derived from Lactobacillaceae species and their capacity for galacto-oligosaccharide production // Journal of Dairy Science. -2023. – Vol.106. -No.12. -P. 8193-8206. Crossref
Misselwitz B., Butter M., Verbeke K., Fox M. R. Update on lactose malabsorption and intolerance: pathogenesis, diagnosis and clinical management// Gut. -2019. – Vol.68. – No.11. – P.2080-2091. Crossref
Prazeres A. R., Carvalho F., Rivas J. Cheese whey management: a review // J Environ Manage. - 2012. – Vol. 110. P. 48-68. Crossref
Bourdichon F., Casaregola S., Farrokh C., Frisvad J. C., Gerds M. L., Hammes W. P., Harnett J., Huys G., Laulund S., Ouwehand A., Powell I. B., Prajapati J. B., Seto Y., Ter Schure E., Van Boven A., Vankerckhoven V., Zgoda A., Tuijtelaars S., Hansen E. B. Food fermentations: microorganisms with technological beneficial use // Int J Food Microbiol. - 2012. – Vol.154. -No.3. – P. 87-97. Crossref
Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding // Anal Biochem. - 1976. – Vol. 72. P.248-54. Crossref
Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors// Proc Natl Acad Sci USA. - 1977. – Vol.74. – 12. - P. 5463-7. Crossref
Kittibunchakul S., Pham M. L., Tran A. M., Nguyen T. H. β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry // Int J Mol Sci. - 2019. -Vol. 20. – No.4. - 947 Crossref
Juers D. H., Matthews B. W., Huber R. E. LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance // Protein Sci, - 2012. – Vol.21. – No.12. – P. 1792-807. Crossref
Foroutan A., Guo A. C., Vazquez-Fresno R., Lipfert M., Zhang L., Zheng J., Badran H., Budinski Z., Mandal R., Ametaj B. N., Wishart D. S. Chemical Composition of Commercial Cow's Milk // J Agric Food Chem. - 2019. – Vol.67. – No.17. – P. 4897-4914. Crossref
Nguyen T. T., Nguyen H. A., Arreola S. L., Mlynek G., Djinović-Carugo K., Mathiesen G., Nguyen T. H., Haltrich D. Homodimeric β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization // J Agric Food Chem. - 2012. – Vol.60. – No.7. – P. 1713-21. Crossref
Ruiz-Ramírez S., Jiménez-Flores R. Isolation and characterization of a β-galactosidase from Lactobacillus helveticus for industrial processing //JDS Communications. - 2025. – Vol.6. – No.1. -P.19-23. Crossref