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Biochemical characterization of Aspergillus oryzae recombinant α-l-rhamnosidase expressed in Pichia pastoris
- Ishikawa, Mai, Shiono, Yoshihito, Koseki, Takuya
- Journal of bioscience and bioengineering 2017 v.124 no.6 pp. 630-634
- Aspergillus oryzae, Pichia pastoris, alpha-L-rhamnosidase, catalytic activity, enzyme activity, genes, hesperidin, kinetics, molecular weight, naringin, pH, polyacrylamide gel electrophoresis, rutin, temperature
- An α-l-rhamnosidase-encoding gene from Aspergillus oryzae, which belongs to the glycoside hydrolase family 78, was cloned and expressed in Pichia pastoris. SDS-PAGE of the purified recombinant α-l-rhamnosidase protein revealed smeared bands with apparent molecular mass of 90–130 kDa. After N-deglycosylation, the recombinant enzyme showed a molecular mass of 70 kDa. The enzyme exhibited optimal activity at a pH of 5.0 and a temperature of 70 °C. Specific activity of the enzyme was higher toward hesperidin than toward naringin, which consist of α-1,6 and α-1,2 linkages, respectively. The activity was also higher toward hesperidin than toward rutin, which consist of 7-O- and 3-O-glycosyl linkages of flavonoids, respectively. Kinetic analysis of the enzyme showed that the Michaelis constant (Km) was lowest toward rutin, moderate toward naringin, and higher toward p-nitrophenyl-α-l-rhamnopyranoside and hesperidin. Its high catalytic efficiency (kcat/Km) toward rutin was results of its low Km value while its high catalytic efficiency toward hesperidin was results of a considerably high kcat value.