Abstrakt

INTRODUCTION: β-Galactosidase is an enzyme which catalyzes the hydrolysis of O glycosidic bond in β-galactosides. Another activity of β galactosidase is a transglycosylation activity. The main industrial use of this protein is the hydrolysis of lactose in milk in a cooling conditions. Synthesis of galactooligosaccharides, which are mostly used as a prebiotics added to some foods or available as dietary supplements, is only one of many applications of transglycosylation activity of β galactosidases. Glycosidase catalysed transglycosylation is a promising alternative to classical chemical glycosylation methods. AIM. The aim of the work was to obtain the sequence of the gene encoding cold-active β galactosidase Arthrobacter sp. 32 cB, construction of the expression system, biosynthesis and biochemical characterization of the protein. MATERIALS AND METHODS. In the first stage of research methods used included Polymerase Chain Reaction (PCR), cloning of DNA fragments and the gene expression. Second stage of research included purification of the protein with a use of ion-exchange and size-exclusion chromatography and biochemical analysis of the protein obtained. RESULTS. Sequence of a β-galactosidase gene from a psychrotolerant bacteria Arthrobacter sp. 32 cB was obtained and expressed in an Escherichia coli expression system. The specific activity of a purified enzyme was 213 U/mg for ONPG at 300C. The molecular mass of native enzyme estimated by gel filtration was 256.6 kDa. Hence, it is assumed that the Arthrobacter sp. 32 cB β galactosidase is a dimeric protein composed of 109.7 kDa subunits. It belongs to glycoside hydrolase family 2. Moreover, the maximum activity of the enzyme was determined at pH 8.0 and 28°C. CONCLUSIONS. Biochemical tests of β galactosidase Arthrobacter sp. 32 cB testify that enzyme can be used for the production of a low lactose milk. However, the enzyme shows high transglycosylation activity therefore main use of it could be production of galactooligosaccharides and heterooligosaccharides.

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