Characterization of α-amylase from mandarin orange honey
Takeshi Nagai, Reiji Inoue, Nobutaka Suzuki, Yasuhiro Tanoue and Norihisa Kai
α-Amylase was purified from mandarin orange honey by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies, and characterized. The molecular weight of the purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, suggesting that the purified enzyme was a monomer. The purified enzyme exhibited optimum activity at pH 5.0 and at 50°C. Inactivation of the enzyme occurred when the pH was lower than 3.0 or higher than 9.0. The enzyme was activated by Co2+, Mn2+, but inhibited by Hg2+, Mg2+, and Fe3+. By TLC analysis, this enzyme was of the α-type that split the interior α-1,4-glycosidic bonds in a random manner. The relative rate of hydrolysis of the polymeric substrate decreased with decreasing percentage of α-1,4-linkages and with increasing percentage of α-1,6-linkages in substrate similar to the results from commercially available honey.