3.3 transglycosylation donor/acceptor selectivity of mutants
Donor/acceptor selectivity for transglycosylation was investigated by incubating wild-type and mutant enzyme with malto-oligosecharides with degree of polymerization ranging from one to five (glucose to maltopentose). In this reaction,malto-oligosecharides served as both a donor and an acceptor molecule for transglycosylation . It was observed from a TLC chromatogram that only malto-oligosecharides with degree of polymerization of three (maltotriose) and higher were transglycosylates by all of the mutant and wild-type enzyme however , HPLC analysis showed that each mutant exhibited a distinct pattern of transglycosylation products when different donor/acceptor were used. From the HPLC data, the proportion of hydrolysis was calculated as the percentage of malto-oligosecharides with degree of polymerization lower than the donor/acceptor , while the proportion of transglycosylation was calculate as the percentage of malto-oligosecharides with degrees of polymerization higher than the donor/acceptor . The T380v mutation did not significantly affect MAG1 activities. As shown in,the levels of hydrolysis and transglycosylation activities of T380V were similar to wild-type. This showed that the Thr380 residue was not involves in capturing or positioning the water molecule for hydrolysis , as mentioned in the previous subsection.