Characterization of the catalytic and kinetic properties of a thermostable Thermoplasma acidophilum α-glucosidase and its transglucosylation reaction with arbutin

Abstract

Abstract The gene (Ta0298) encoding a putative α-glucosidase from hyperthermophilic archaeon Thermoplasma acidophilum (AglA) was cloned and expressed in Escherichia coli. Gel filtration chromatography of the purified enzyme indicated that the native form was a pentamer with strong maltose (α-1,4 linkage)-hydrolyzing activity. AglA was optimally active at pH 5–6 and 80 °C and had a half-life of 16.8 h and 1.4 h at 80 °C and 85 °C, respectively. The enzyme also hydrolyzes kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6) to a lesser extent. Analysis of the reaction with maltooligosaccharides and panose as substrates show that AglA specifically liberates glucose from the non-reducing end indicating that it is typical of a glycoside hydrolase family 31 (GH31) α-glucosidase. Kinetic analyses revealed that the hydrolytic activity of AglA was greatly affected by the chain length of the substrate and the regiospecificity of the glucosidic linkages. The enzyme showed highest specificity for maltose and decreasing values of catalytic efficiency (kcat/Km) toward higher maltooligosaccahrides, although these still serve as substrates. The inhibition profile of AglA toward aesculin was revealed to be a mixed type of noncompetitive inhibition with a Ki value of 4.30 μM and K ′ i of 12.5 μM, whereas that toward acarbose showed a competitive inhibition pattern with a Ki of 2.99 μM. Structural analyses of two arbutin transglucosylation products using NMR indicated that the glucose unit of maltose was transferred to the C-3 and C-6 position in the glucose moiety of arbutin.