Bacillus Thermoamyloliquefaciens KP1071 alpha-Glucosidase II is a Thermostable Mr 540000 Homohexameric alpha-Glucosidase with both Exo-alpha-1,4-Glucosidase and Oligo-1,6-Glucosidase Activities

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Abstract
α-Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477; growth over 30–66°C) was purified to a homogeneous state. Its Mr was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N-terminal sequence of 20 residues was determined as Ala1-Ile-Gln-Pro-Glu-Gln-Asp-Asp-Lys-Thr-Gln-Glu-Asp-Gly-Tyr-Ile-Asp-Ile-Gly-Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo-α-1,4-glucosidase and the monomeric oligo-1,6-glucosidase from the same microorganism. The α-glucosidase II had no antigenic group shared with the latter two enzymes. Analysis of substrate specificity showed that the α-glucosidase II has dual activity towards oligo-1,6-glucosidases and exo-α-1,4-glucosidases, but its preference is for non-reducing terminal α-1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 °C and pH 7.0. Its half-life at pH 6.8 was 10 min at 81 °C, and 5 h at 55 °C in 6.4 M urea, 26% ethanol or 2.5% SDS. We suggest that the α-glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo-α-1,4-glucosidase and the oligo-1,6-glucosidase present in the same strain.

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