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.

Optimization of glycosidases production by Pseudoalteromonas issachenkonii KMM 3549(T).

AIMS: The present work aimed to design an optimized medium to yield a higher production of glycosides by Pseudoalteromonas issachenkonii KMM 3549(T). METHODS AND RESULTS: Higher levels of fucoidan hydrolase, alginase, laminaranase and b-N-acetylglucosaminidase production were obtained with peptone concentrations ranging from 2.5 g l(-1) to 10 g l(-1), while the presence of both yeast extract and glucose did not affect enzyme production. The activity of fucoidan hydrolase and laminaranase increased up to 4.83 microM h(-1) mg(-1) and 19.23 microM h(-1) mg(-1) protein, respectively, in growth media containing xylose (1.0 g l(-1)), laminarin (0.5 g l(-1)) or alginate (0.5 g l(-1)), and production of b-N-acetylglucosaminidase substantially increased in the presence of fucoidan (0.5 g l(-1)) or galactose (1 g l(-1)). All polysaccharides tested in concentrations of 0.5 g l(-1) fucoidan and 0.2 g l(-1) fucose induced production of alginase (up to 5.06 microM h(-1) mg-1 protein). CONCLUSIONS: The production of glycosidases is not only stimulated by the presence of algal polysaccharides, but may also be stimulated by monosaccharides (e.g. xylose). SIGNIFICANCE AND IMPACT OF THE STUDY: The production of glycosidases by Pseudoalteromonas issachenkonii KMM 3549(T) was significantly improved by using a simple nutrient medium containing peptone (2.5 g l(-1)) and xylose (5.0 g l(-1)) in 100% natural seawater.

Alpha-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM 701.

An alpha-galactosidase that inactivates the group specificity of B erythrocytes (group III) of human blood and does not affect A erythrocytes (group II) was isolated from the marine bacterium Pseudoalteromonas sp. KMM 701. The enzyme preparation did not contain lectin, hemolytic, sialidase, endoglycanase, or glycosidase activities. The enzyme is stable at 20 degreesC for 24 h, has pH optimum for catalysis within the range of 6.7-7.7, and is stable to high concentrations of NaCl. It is 4-fold more efficient than the alpha-galactosidase from green coffee beans. At pH 7.0 the Km for p-nitrophenyl-alpha-D-galactopyranoside is 0.29 mM. The molecular weight of the enzyme determined by gel-filtration is 195 +/- 5 kD. The alpha-galactosidase is denatured by urea and guanidine hydrochloride. Its activity does not depend on the presence of metal ions. It contains a sulfhydryl group essential for its catalytic activity.