Expression, Purification and Characterization of a Bifunctional α‐L‐Arabinofuranosidase/β‐D‐Xylosidase from Trichoderma Koningii G‐39

Abstract

A gene of a-L-arabinofuranosidase (Abf) from Trichoderma koningii G-39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cationexchanged chromatography. The purified enzyme exhibits both a-L-arabinofuranosidase and b-D-xylosidase (Xyl) activities with p-nitrophenyl-a-L-arabionfuranoside (pNPAF) and 2,4-dinitrophenyl-b-Dxylopyanoside (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-a-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.

On-Site Enzyme Production by Trichoderma asperellum for the Degradation of Duckweed

“…Interestingly, a GH54 α-L-arabinofuranosidase (ID 4138) was up regulated on wheat bran, but down regulated on duckweed, underlining substrate dependent regulation. A GH54 α-arabinofuranosidases belonging Trichoderma koningii is reported to contain β-D-xylosidase activity [51], while the ones from GH62 are more strictly α-arabinofuranosidases [52]. This indicates that we saw a specialized up and down regulation, where α-arabinofuranosidases with activity towards xylan were up regulated on wheat bran, having a higher xylan content than duckweed.…”

A novel fungal metal-dependent α-l-arabinofuranosidase of family 54 glycoside hydrolase shows expanded substrate specificity

“…Such activity may be related to some modification of the active site of these enzymes, and resolution of their structures would provide a better understanding of this enzymatic activity toward different substrates, as only one structure in the family has been resolved to date 24 . At the time of writing the present report, 19 enzymes had been characterized in this family, among which only 6 have been tested on different types of synthetic substrates 4 , 19 , 30 , 31 . In addition, as demonstrated via in silico analyses, there are several unstudied proteins of this family harboring domains related to the hydrolysis of galactose, among other types of sugars.…”

Novel fungal metal-dependent GH54 α-L-arabinofuranosidase: expanded substrate specificity and potential use for plant biomass degradation

“…It is made The copyright holder for this preprint this version posted December 21, 2020. ; https://doi.org/10.1101/2020.12.18.423520 doi: bioRxiv preprint 8 been only two studies addressing this topic 4,19 . At the time of writing the present report , 19 enzymes had been characterized in this family, among which only 6 have been tested on different types of synthetic substrates 4,19,30,31 . In addition, as demonstrated via in silico analyses, there are several unstudied proteins of this family harboring domains related to the hydrolysis of galactose, among other types of sugars.…”