Recombinant α-NAcetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens.
A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426T (α-AlNaGal) in Escherichia coli cells. The yield of α-Al- NaGal attains 10 mg/ml with activity of 49.7 ± 1.3 U at 16°C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify α-AlNaGal were applied. α-AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50°C with a temperature range optimum activity of 20-37°C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that α-AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. α-AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of α-AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Vladivostoka Ave., 159, 690022, Vladivostok, Russia ; Far Eastern Federal University, Sukhanova Str., 8, 690950, Vladivostok, Russia.
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Balabanova
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Vladivostoka Ave., 159, 690022, Vladivostok, Russia.
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Golotin
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Vladivostoka Ave., 159, 690022, Vladivostok, Russia.
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Bakunina
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Vladivostoka Ave., 159, 690022, Vladivostok, Russia.
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Slepchenko
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Vladivostoka Ave., 159, 690022, Vladivostok, Russia.
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Isakov
Far Eastern Federal University, Sukhanova Str., 8, 690950, Vladivostok, Russia.
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Podvolotskaya
Far Eastern Federal University, Sukhanova Str., 8, 690950, Vladivostok, Russia.
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Rasskazov
1H NMR spectroscopy; Arenibacter latericius; conversion of A erythrocytes; glycoside hydrolase GH109