Abstrakt

INTRODUCTION. β-Galactosidase [EC 3.2.1.23] is an enzyme that catalyzes the hydrolysis of O-glycosidic linkages in galactosides. It is commercially used in dairy industry for the production of milk with reduced lactose content. Potentially, the best method for lactose removal under cooling conditions should be carried out with a cold-adapted enzyme. AIM. The aim of this study was to determine the taxonomic affiliation of the isolate labeled as Spitzbergen VII-4, and obtain the sequence of the gene encoding cold-active β-galactosidase and the construction of expression system, biosynthesis and biochemical characterisation of the enzyme. MATERIALS AND METHODS. Research methods included the use of the PCR technique, cloning, and expression of the gene. The protein was purified to homogeneity by using ion exchange chromatography and gel filtration. Then an analysis of the enzyme was done. RESULTS. The isolate Spitzbergen VII-4 was classified as Arthrobacter sp. based on 16S rRNA gene sequence. Sequence of the gene encoding β-galactosidase was obtained by construction of genomic DNA library, and its analysis showed that it consists of 2064 bp, and encodes an enzyme belonging to the glycoside hydrolase family 42. The biosynthesis of protein was carried out in Escherichia coli. After purification the enzyme with specific activity of 9.2 U/mg was obtained. The enzyme is a trimer with molecular mass 227 kDa. Optimal conditions for enzyme activity are 32°C and pH 7.5. Cations Mg2+ are activators of β-galactosidase whereas Ca2+, Co2+, Mn2+ and Ni2+ are inhibitors of the enzyme. Moreover, the thiol group-containing compounds such as dithiotreitol, tris(2-carboxyethyl)phosphine, reduced glutathione and cysteine decreased the activity of the glycoside hydrolase. The enzyme exhibits hydrolytic activity with β-D-galactosides, β-D-glucuronide, and β-D-fucoside. CONCLUSIONS. Biochemical properties of β-galactosidase Arthrobacter sp. VII-4 show that enzyme can be potentially used for lactose removal from dairy products, however, comprehensive research must be done.

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