A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity

Constantinos Katsimpouras,Nikolaos S Thomaidis,Grigorios Dedes,Evangelos Topakas

doi:10.1186/s13068-019-1455-2

Abstract

BackgroundThe main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity.ResultsThe expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-d-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well.ConclusionsHereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

Highlights

The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases
Xylanases belonging to GH10 and GH11 families have been widely investigated in literature with GH10 xylanases exhibiting less substrate specificity than GH11 xylanases
We reported the cloning of the T. thermophila ex30a gene and its heterologous expression using the P. pastoris expression system

Summary

Introduction

The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity. The enzymes that are responsible for the degradation of xylan main chain are endoxylanases (E.C. 3.2.1.8) and they have been grouped in glycoside hydrolases families 5, 8, 10, 11, 16, 30, 43, 51, 98, and 141 of the CAZy database (http://www.cazy.org; Lombard et al [3]) based on amino acid similarities and structural characteristics. The former enzymes are able to catalyze the hydrolysis of a wide spectrum of xylans, while the latter are known to preferentially cleave unsubstituted regions of arabinoxylan [4]

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