A novel Aspergillus oryzae diglycosidase that hydrolyzes 6-O-α-L-rhamnosyl-β-D-glucoside from flavonoids

A novel Aspergillus oryzae diglycosidase that hydrolyzes 6-O-α-L-rhamnosyl-β-D-glucoside from... α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) hydrolyzes the glycosidic linkage between the disaccharide 6-O-α-L-rhamnosyl-β-D-glucoside (rutinose) and the aglycone. We identified a hypothetical protein (annotated as AO090012000917) encoded in the Aspergillus oryzae genome that exhibits sequence similarity with Aspergillus niger rutinosidase. The recombinant enzyme was expressed in Pichia pastoris GS115 and purified as a glyco-protein with apparent molecular mass of 65–75 kDa by SDS-PAGE. After N-deglycosylation, we observed a 42- and 40-kDa band, representing proteins before and after N-terminal signal peptide processing, respectively. Optimal enzymatic activity was observed at pH 4.0 and temperature of 45 °C. This enzyme is also significantly thermo-stable, with 90% activity retained after 1 h at 45 °C and 70% activity retained after 4 h, even at 50 °C. Biochemical characterization revealed that the enzyme has higher substrate specificity for 3-O-linked flavonoid β-rutinosides like rutin and kaempferol-3-O-rutinoside, than for 7-O-linked flavonoid β-rutinoside like hesperidin. However, no activity was found with naringin, diosmin, monoglycosylated chromogenic substrates, and polymeric laminarin substrate. Kinetic analyses showed that K m value toward rutin was higher than those toward hesperidin and kaempferol-3-O-rutinoside. Meanwhile, k cat value toward hesperidin was lower than those toward kaempferol-3-O-rutinoside and rutin. Overall, the catalytic efficiency (k cat/K m ) was highest for kaempferol-3-O-rutinoside. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Microbiology and Biotechnology Springer Journals

A novel Aspergillus oryzae diglycosidase that hydrolyzes 6-O-α-L-rhamnosyl-β-D-glucoside from flavonoids

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Publisher
Springer Berlin Heidelberg
Copyright
Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
ISSN
0175-7598
eISSN
1432-0614
D.O.I.
10.1007/s00253-018-8840-9
Publisher site
See Article on Publisher Site

Abstract

α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) hydrolyzes the glycosidic linkage between the disaccharide 6-O-α-L-rhamnosyl-β-D-glucoside (rutinose) and the aglycone. We identified a hypothetical protein (annotated as AO090012000917) encoded in the Aspergillus oryzae genome that exhibits sequence similarity with Aspergillus niger rutinosidase. The recombinant enzyme was expressed in Pichia pastoris GS115 and purified as a glyco-protein with apparent molecular mass of 65–75 kDa by SDS-PAGE. After N-deglycosylation, we observed a 42- and 40-kDa band, representing proteins before and after N-terminal signal peptide processing, respectively. Optimal enzymatic activity was observed at pH 4.0 and temperature of 45 °C. This enzyme is also significantly thermo-stable, with 90% activity retained after 1 h at 45 °C and 70% activity retained after 4 h, even at 50 °C. Biochemical characterization revealed that the enzyme has higher substrate specificity for 3-O-linked flavonoid β-rutinosides like rutin and kaempferol-3-O-rutinoside, than for 7-O-linked flavonoid β-rutinoside like hesperidin. However, no activity was found with naringin, diosmin, monoglycosylated chromogenic substrates, and polymeric laminarin substrate. Kinetic analyses showed that K m value toward rutin was higher than those toward hesperidin and kaempferol-3-O-rutinoside. Meanwhile, k cat value toward hesperidin was lower than those toward kaempferol-3-O-rutinoside and rutin. Overall, the catalytic efficiency (k cat/K m ) was highest for kaempferol-3-O-rutinoside.

Journal

Applied Microbiology and BiotechnologySpringer Journals

Published: Feb 23, 2018

References

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