A novel thermophilic xylanase from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing and other industries

Highlights

• First report of a thermophilic xylanase of family 10 in Achaetomium.

• Good activity and stability in broad pH and temperature ranges.

• Resistant to all tested chemicals and potential in detergent industry.

• Simple xylan products and no cellulose activity and useful in biorefinery.

• Efficient in mash filtration and valuable in brewing industry.

Abstract

Thermophilic xylanases are of great interest for their wide industrial application prospects. Here we identified a thermophilic xylanase (XynC01) of glycoside hydrolase (GH) family 10 in a thermophilic fungal strain Achaetomium sp. Xz-8. The deduced amino acids of XynC01 showed the highest identity of ≤52% to experimentally verified xylanases. XynC01 was functionally expressed in Pichia pastoris, showed optimal activity at pH 5.5 and 75 °C with stability over a broad pH range (pH 4.0–10.0) and at temperatures of 55 °C and below. XynC01 had the highest catalytic efficiency (k_cat/K_m, 3710 mL/s/mg) ever reported for all GH 10 xylanases, and was resistant to all tested metal ions and chemical reagents. Its hydrolysis products of various xylans were simple, mainly consisting of xylobiose and xylose. Under simulated mashing conditions, XynC01 alone had a comparable effect on filtration improvement with Ultraflo from Novozymes (20.24% vs. 20.71%), and showed better performance when combined with a commercial β-glucanase (38.50%). Combining all excellent properties described above, XynC01 may find diverse applications in industrial fields, especially in the brewing industry.

Introduction

Cellulose, hemicellulose and lignin are the major components of plant cell walls. Xylan, a xylose polymer linked by β-1,4-glycosidic bonds, is the predominant component of hemicellulose and accounts for approximately 33% of all renewable organic carbon on earth [1], [2]. Its complete degradation is a key to efficient biomass utilization. Several enzymes are involved in xylan degradation; of them, endo-1,4-β-xylanase (EC 3.2.1.8) plays the major role in cleaving the xylan backbone into short xylooligosaccharides of variable lengths [3]. Based on their primary structures of the catalytic domains, xylanases have been classified into two main families of glycosyl hydrolase (GH), i.e. GH 10 and GH 11 [3]. Some enzymes with xylanase activity are also found in GH 5, 7, 8, 16, 26, 43, 52 and 62 (http://www.cazy.org/fam/acc_GH.html) [4]. Compared to the xylanases of other families, those from GH 10 typically have a highly conserved 8-fold α/β-barrel structure with relatively small variations in loops and helices surrounding the inner β-strands, have a high molecular weight of ≥30 kDa, a low pI value, and a lower specificity or larger versatility, and hydrolyze heteroxylans into simple products [5], [6].

More and more researches are being conducted to explore novel xylanases with specific potentials for application in the brewing, animal feed, bread-making, paper and pulp, waste treatment and bioethanol industries [7]. A majority of microorganisms, including bacteria, yeasts and fungi, produce many different kinds of xylanases; however, most xylanases fail to function under specific conditions, especially the high temperature [8]. Because elevated temperatures are usually applied to industrial processes owing to the advantages of high reaction rate, increased substrate solubility and decreased viscosity [9], thermostable and thermotolerant xylanases from thermophilic fungi are more favorable than mesophilic enzymes for industrial applications [10]. Considering the lower substrate specificity of GH 10 xylanases in extensive degradation of xylan, those having activities at high temperatures are desired in the brewing, detergent and biofuel industries [11].

Species of fungal genera that are known to produce xylanases include Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium, Chaetomium, Trichoderma, etc. And commercial xylanases are mainly produced by Aspergillus and Trichoderma [8]. The genus of Achaetomium, belonging to the family of Chaetomiaceae, was first described by Rai et al. with three species, Achaetomium globosum (type species), Achaetomium strumarium and Achaetomium luteum [12]. Until now, there are only 25 strains of Achaetomium described (http://www.indexfungorum.org/Names/Names.asp), most of which are from soil. Among them, only one thermophilic species, Achaetomium thermophilum, was isolated from leaf litter [13]. Over the past half-century, little research has been conducted on the metabolites or enzymes of Achaetomium. In a previous study, we isolated a thermophilic fungal strain, Achaetomium sp. Xz-8, from a desert sand sample and observed substantial xylanase activity in it [14]. Two family 11 xylanases were then identified, which had excellent properties, such as high catalytic efficiency and application potentials in the brewing industry. Therefore strain Xz-8 may represent a good thermophilic xylanase producer. The aim of the present study was to obtain an excellent xylanase of family 10 from it. This enzyme should have superior properties like high temperature activity, broad pH and temperature adaptability and stability and great application potentials in many industrial fields.