Characterization of a novel GH10 xylanase with a carbohydrate binding module from Aspergillus sulphureus and its synergistic hydrolysis activity with cellulase
Introduction
Presently, trillions of tons of agro-residues, forestry residues, woody materials, and grasses are globally produced annually [[1], [2], [3]]. Corn cob, rapeseed meal, and corn stover biomasses are all agricultural milling by-products comprised of 50–80% C5 and C6 sugar units [4]. Combustion of these biomasses would produce a large quantity of greenhouse gases like methane and CO2, which poses a threat to the environment [3]. Rational development and utilization of biomass can minimize environmental pollution and produce valuable products. Corncob, rapeseed meal, and corn stover biomasses are abundant renewable energy sources. They are composed of lignin (5%–15%), cellulose (37%–50%) and hemicellulose (25%–50%), in which hemicellulose is rich in xylan [5]. Xylan is the major component of hemicellulose and is a cross linker between different cell-wall components that inhibit biomass utilization. Therefore, chemical, or enzymatic methods used to decompose hemicellulose into its component xylan are an effective way to prepare useful polysaccharides [6,7].
Xylan's main chain is made up of β-1,4-glycosidic linkages of D-xylopyranoside with arabinosyl, acetyl, and glucuronosyl groups attached to side chains. So, Endo-β-1,4-xylanase (EC 3.2.1.8) is a key enzyme in the catalysis of β-1,4-glycosidic bonds of hemicellulose to produce a mixture of xylo-oligosaccharides (XOSs) with different degrees of polymerization (DP) [8]. Endo-β-1,4-xylanase mainly belongs to glycoside hydrolase (GH) families 10 and 11 based on amino acid composition, protein structure, and reaction mechanisms [9]. GH10 family xylanases have a higher molecular weight, a lower isoelectric point (pI) than GH11 family xylanases, and a typical structure of (α/β)8 barrel folded [10].
Xylanases are used widely in the food, animal feed, paper and pulping, and biofuel industries. In the food industry, xylanases are used in bread making and brewing beer. Xylanases enhance quality of bread and improve fermentation efficiency of brewing beer [11,12]. In the paper and pulping industry, xylanases strengthen the bleaching effect and reduce environmental pollution [13]. In the animal feed industry, xylanases improve digestibility of feed for livestock such as pigs [14]. Xylanase catalytic product, XOS, is an effective prebiotic that may improve the growth performance and health of pigs and poultry [15].
For XOS production, researchers reported that GH10 xylanases have better hydrolytic capacity and thermostability than GH11 xylanases [16,17]; for example, a thermostable GH10 family xylanase was stable at 60 °C and 70 °C [18] and capable of releasing xylose and xylobiose from agricultural waste biomass. Xylanase can serve as an accessory enzyme that improves the efficiency of hydrolysis lignocellulosic biomass by cellulase and increased the production of XOSs [19]. Thus, the synergistic effect of cellulase and xylanase in industry has attracted extensive attention.
Some xylanases comprise a glycoside hydrolase catalytic domain (CD) and a carbohydrate-binding module (CBM) [20,21]. To date, CBMs existing in carbohydrate-active enzymes are grouped into 87 families. Generally, CBM is connected with CD by a linker, which is a serine-rich or threonine-rich peptide [22]. The CBM contained within a xylanase is important for catalysis of substrate such as crystalline cellulose, non-crystalline cellulose, xylan, and mannan because the CBM plays a role in substrate recognition. A review concluded that CBM with affinity to xylan was essential for the degradation of lignocellulosic biomass [23].
In our previous work, a complete genome sequence of Aspergillus sulphureus JCM01963 was analyzed and a novel GH10 family endo-β-1,4-xylanase gene xyn10A was predicted. The xylanase full-length and truncated CBM AS-xyn10A were heterologously expressed in Komagataella phaffii. This study aims to investigate the effects of CBM truncation on the biochemical properties of the enzyme and the effects of CBM truncation AS-xyn10A hydrolyzed biomasses alone and together with cellulase hydrolyzed biomasses. In this study, we hypothesized that AS-xyn10A exhibited synergistic effects with commercial cellulase in the hydrolysis of biomasses from pretreated corncob, corn stover, and rapeseed meal and that CBM-truncation could lead to the reduction of the synergistic effect.
Section snippets
Strains, plasmids, and reagents
Komagataella phaffii X-33 (Invitrogen, USA) and pPICZαA plasmid were stored in our laboratory. Beechwood xylan (V900513) and Avicel (9004-34-6) were purchased from Sigma-Aldrich (USA), corncob xylan (9014-63-5) from Maclin (China), and Zeocin from Invitrogen (USA). Restriction nuclease, T4 DNA ligase and protein molecular weight standard were purchased from TaKaRa (Japan). Plasmid DNA extraction kit and gel extraction kit were obtained from Omega Corp. (USA). Birchwood xylan and XOSs (xylose
Sequence analysis of A. sulphureus JCM01963 AS-xyn10A
Full-length sequence of xyn10A containing 4 introns is composed of 1427 nucleotides (nts) (Fig. 1s). The predicted mRNA encodes for a protein of 404 amino acids (AA) with a theoretical molecular weight of 43.64 kDa. The AA sequence of AS-xyn10A showed relatively high similarity (88%) with a xylanase from A. steynii IBT 23096 (GenBank accession: XP_024705365). Phylogenetic tree of AS-xyn10A with some xylanases opened in GenBank was shown in Fig. 2s. The 22 AA in the N-terminal of the protein was
Conclusion
In this study, the β-1,4-xylanase gene (xyn10A) and its carbohydrate binding module (CBM)-Truncated variant (xyn10A-dC) were cloned from Aspergillus sulphureus JCM01963 and successfully expressed in P. pastoris. The results indicated that CBM did not affect the optimal conditions of AS-xyn10A, but had an effect on enzymatic properties. Metal ions and EDTA slightly affected the activity of AS-xyn10A. AS-xyn10A could efficiently hydrolyze xylan from alkali-pretreated corncob, corn stover, and
CRediT authorship contribution statement
Yajing Liu: Data curation, Formal analysis, Investigation, Writing-original draft, Visualization. Jian Wang: Formal analysis, Validation. Chengling Bao: Investigation, Visualization. Bing Dong: writing guidance. Yunhe Cao: Methodology, Formal analysis, Funding acquisition, Data curation, Writing- review & editing.
Declaration of competing interest
The authors have no conflicts of interest to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 31572437 and No. 31760673).
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