Biochemical characterization of Aspergillus oryzae recombinant α-l-rhamnosidase expressed in Pichia pastoris

• Published in: Journal of bioscience and bioengineering 2017-12, Vol.124 (6), p.630-634
• Main Authors: Ishikawa, Mai, Shiono, Yoshihito, Koseki, Takuya
• Format: Article
• Language: English
• Subjects: Aspergillus oryzae; Aspergillus oryzae - genetics; Biocatalysis; Electrophoresis, Polyacrylamide Gel; Flavanones - metabolism; Flavonoids - metabolism; Glycoside hydrolase family 78; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Glycosylation; Hesperidin - metabolism; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; N-glycosylation; Pichia - genetics; Pichia pastoris; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rhamnose - metabolism; Rutin - metabolism; Substrate Specificity; Temperature; α-l-Rhamnosidase
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2017.07.007

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. •An Aspergillus oryzae α-l-rhamnosidase-encoding gene (AorhaA) was cloned and expressed in Pichia pastoris.•Recombinant AoRhaA was a hyper-N-glycosylated protein of 90–130 kDa on SDS-PAGE.•Specific activity of the enzyme was highest toward hesperidin, moderate toward rutin and p-nitrophenyl-α-l-rhamnopyranoside, and lower toward naringin.