Expression, purification and characterization of a bifunctional α-L-arabinofuranosidase/β-D-xylosidase from Trichoderma koningii G-39

Chin Feng Wan, Cheng Ta Chen, Lina Huang, Yaw-Kuen Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

A gene of α-L-arabinofuranosidase (Abf) from Trichoderma koningii G-39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cation-exchanged chromatography. The purified enzyme exhibits both α-L-arabinofuranosidase and β-D-xylosidase (Xyl) activities with p-nitrophenyl-α-L- arabionfuranoside (pNPAF) and 2,4-dinitrophenyl-β-D-xylopyanoside (2,4-DNPX) as substrate, respectively. The stability and the catalytic feature of the bifunctional enzyme were characterized. The enzyme was stable for at least 2 h at pH values between 2 and 8.3 at room temperature when assayed for Abf and Xyl activities. Enzyme activity decreased dramatically when the pH exceeded 9.5 or dropped below 1.5. The enzyme lost 35% of Abf activity after incubation at 55 °C for 2 h, but retained 95% of Xyl activity, with 2,4-DNXP as substrate, under the same conditions. Further investigation of the active site topology of both enzymatic functions was performed with the inhibition study of enzyme activities. The results revealed that methyl-α-L- arabinofuranoside inhibition is noncompetitive towards 2,4-DNPX as substrate but competitive towards pNPAF. Based on the thermal stability and the inhibition studies, we suggest that the enzymatic reactions of Abf and Xyl are performed at distinct catalytic sites. The recombinant enzyme possesses both the retaining transarabinofuranosyl and transxylopyranosyl activities, indicating both enzymatic reactions proceed through a two-step, double displacement mechanism.

Original languageEnglish
Pages (from-to)109-116
Number of pages8
JournalJournal of the Chinese Chemical Society
Volume54
Issue number1
DOIs
StatePublished - 1 Jan 2007

Keywords

  • Family 54
  • Inhibition
  • Transglycosidase activity
  • α-L-arabinofuranosidase/β-D-xylopyranosidase

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