• Journal of Microbiology and Biotechnology
• /
• v.10 no.2
• /
• pp.244-250
• /
• 2000

An ${\alpha}-glucosidase$ gene (aglA) from thermophilic Bacillus sp. DG0303 was cloned, sequenced, and expressed in Escherichia coli. The aglA was localized to the 2.1-kb PvuI-XmnI region within the 5.9-kb DNA insert of the gybrid plasmid pAG1. The gene consisted of an open reading frame of 1,686 bp with an unusual GTG initiation codon and TGA termination codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 562 amino acid residues with a M, of 66,551 dalton. A comparative amino acid sequence analysis revealed that DG0303 ${\alpha}-glucosidase$ is related to bacillary oligo-1, 6-glucosidases. The Bacillus sp. DG0303 ${\alpha}-glucosidase$ showed a high sequence identity (36-59%) to the B. flavocaldarius, B. cereus, and B. thermoglucosidasius oligo-1, 6-glucosidases. The number of prolines in theses four ${\alpha}-glucosidases. was observed to increase with increasing thermostability of these enzymes. The cloned ${\alpha}-glucosidase was purified from E. coli $DH5{\alpha}$ bearing pAG1 and characterized. The recombinant enzyme was identical with the native enzyme in its optimum pH and in its molecular mass, estimated by sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The temperature optimum of the cloned ${\alpha}-glucosidase$ was lower than that of the native enzyme.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.3
• /
• pp.270-276
• /
• 1998

Extracellular ${\alpha}$-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable ${\alpha}$-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at $60^{\circ}C$. It had a half-life of 35 min at $60^{\circ}C$. The enzyme was stable at the pH range of 4.5~7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable ${\alpha}$-glucosidase hydrolyzed the ${\alpha}$-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The $K_m$ (mM) for p-nitrophenyl-${\alpha}$-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the $V_{max}$(${\mu}mol{\cdot}min^-1$$mg^-1$) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.

• Korean Journal of Microbiology
• /
• v.43 no.1
• /
• pp.40-46
• /
• 2007

For restoration of disturbed ecosystem in Lake Paro, artificial floating island (AFI) was installed. Even though the lake water was oligo-mesotrophic, the macrophytes, such as Iris ensata, Iris pseudoacorus, Phragmites communis were growing well in the rubberized coconut fiber media. For elucidating this process, total bacterial numbers, active bacterial numbers and exoenzymatic activities of ${\beta}-glucosidase$ and phosphatase of pore water of media and lake water were analyzed. The average of total bacterial numbers, active bacterial numbers and exoenzymatic activities of ${\beta}-glucosidase$ and phosphatase were $28.6{\times}10^{6}\;cells/ml,\;22.7{\times}10^{6}\;cells/ml,\;452.9nM/L/hr,\;and\;16381.9nM/L/hr$ which were 10, 15, 22 and 38 times higher than those of lake water, respectively. Moreover, the total phosphorus and total nitrogen concentration of media showed high values of 0.82 mg/L and 7.0 mg/L, respectively, while those of lake water 0.07 mg/L and 2.3 mg/L. This results suggest that the bacteria was playing an important role for restoration of disturbed ecosystem with newly created microbial ecosystem in media of artificial floating island.