Recombinant α-NAcetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens

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Abstract


A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426 T (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.


L. A. Balabanova

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences; Far Eastern Federal University

Author for correspondence.
Email: balaban@piboc.dvo.ru

Russian Federation

V. А. Golotin

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences

Email: balaban@piboc.dvo.ru

Russian Federation

I. Yu. Bakunina

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences

Email: balaban@piboc.dvo.ru

Russian Federation

L. V. Slepchenko

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences

Email: balaban@piboc.dvo.ru

Russian Federation

V. V Isakov

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences

Email: balaban@piboc.dvo.ru

Russian Federation

A. B. Podvolotskaya

Far Eastern Federal University

Email: balaban@piboc.dvo.ru

Russian Federation

V. А. Rasskazov

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences

Email: balaban@piboc.dvo.ru

Russian Federation

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Copyright (c) 2015 Balabanova L.A., Golotin V.А., Bakunina I.Y., Slepchenko L.V., Isakov V.V., Podvolotskaya A.B., Rasskazov V.А.

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