Balabanova L A, Golotin V A, Bakunina I Y, Slepchenko L V, Isakov V V, Podvolotskaya A B, Rasskazov V A
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.
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Vladivostoka Ave., 159, 690022, Vladivostok, Russia.
Acta Naturae. 2015 Jan-Mar;7(1):117-20.
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.
构建了一种基于pET-40b的质粒，用于在大肠杆菌细胞中合成海洋细菌后滨岸沙雷氏菌KMM 426T的重组α-N-乙酰半乳糖胺酶（α-AlNaGal）。在16°C、诱导剂浓度2 mM、培养12 h的条件下，α-AlNaGal的产量达到10 mg/ml，活性为49.7±1.3 U。应用了阴离子交换、金属亲和及凝胶过滤色谱等技术纯化α-AlNaGal。α-AlNaGal是一种分子量为164 kDa的同型二聚体。该酶在高达50°C时稳定，最适活性温度范围为20-37°C。此外，其活性与孵育介质中金属离子的存在无关。1H NMR光谱显示，α-AlNaGal催化O-糖苷键的水解，同时保留异头立体化学，其作用机制与第109家族的其他糖苷水解酶相同。α-AlNaGal在pH 7.3时可降低A红细胞的血清学活性。α-AlNaGal的这一特性可能用于将A和AB红细胞酶促转化为O型红细胞。
