Transglucosylation and Hydrolysis Activity of Gluconobacter oxydans Dextran Dextrinase with Several Donor and Acceptor Substrates
In this paper the reactivity of Gluconobacter oxydans dextran dextrinase (DDase) towards several glucosyl donor and acceptor molecules was studied. The donor/acceptor assay reflecting most accurately the DDase transglucosylation activity, could then be used as an evaluation tool for the optimization of the DDase production by G. oxydans. Different combinations of glucosyl donors (maltodextrin or maltose) and glucosyl acceptors (glucose, maltose or cellobiose) were incubated with a crude G. oxydans cell extract as a biocatalyst. The synthesis of panose revealed to be the most reliable indicator for DDase transglucosylation activity. Measurements of increasing glucose concentrations or decreasing maltose concentrations were less suitable for the quantification of DDase activity, since maltose seemed to be subjected to some hydrolysis during the enzymatic assays. In order to determine whether the hydrolytic activity, revealed in the donor/acceptor assays, originated from the DDase enzyme itself or from another enzyme present in the DDase preparation, the G. oxydans cell extract was subjected to native PAGE and zymogram analysis. The cell extract contained a number of proteins, the majority being smaller than 140 kDa. One protein band could be detected between the MW markers of catalase and ferritin (with a MW of 232 kDa and 440 kDa resp.), corresponding to DDase, which has a MW of 300 kDa, according to Yamamoto et al 1. The zymogram showed an uncoloured zone with the same Rf value as DDase, leading to the conclusion that DDase itself was most probably the enzyme displaying the hydrolytic activity observed in the donor/acceptor assays.
KeywordsNative Page Acceptor Substrate Zymogram Analysis Increase Glucose Concentration Glucosyl Unit
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