An Aspergillus nidulans GH26 endo-β-mannanase with a novel degradation pattern on highly substituted galactomannans
Introduction
β-Mannan (hereafter mannan) is the second most abundant hemicellulose in nature. Mannans are composed of a linear backbone containing d-mannopyranosyl residues (linear mannans) or d-mannopyranosyl and d-glucopyranosyl residues in an alternating manner (glucomannans) linked together by β-(1 → 4)-linkages. The backbone can be decorated with α-(1 → 6)-linked d-galactopyranosyl residues (galactomannans or galactoglucomannans) and acetyl groups [1], [2], [3]. Large amounts of mannans are found in the secondary plant cell wall of softwood (coniferous trees), where acetylated glucomannans and galactoglucomannans, comprise 25% of the wood dry matter [2], [4]. The significance of softwood galactoglucomannans in biomass processing is a main reason for the interest in mannan degrading enzymes: endo-β(1 → 4)-mannanases (endomannanases, EC 3.2.1.78), β-mannosidases (EC 3.2.1.25) and α-galactosidases (EC 3.2.1.22), and their synergistic action [5], [6]. Pure galactoglucomannan is not widely available, which is why galactomannans are often used to study enzymatic degradation of mannans containing galactopyranosyl substitutions [7]. Guar gum from the seeds of the guar plant (Cyamopsis tetragonolobus) and locust bean gum, from the carob tree (Ceretonia siliqua) are significant sources of galactomannans. Guar gum contains more galactopyranosyl residues (Gal:Man, 1:2) than locust bean gum (Gal:Man, 1:4) [2], [8]. In locust bean gum, the distribution of galactopyranosyl residues is irregular with a high proportion of unsubstituted blocks whereas in guar gum, the galactopyranosyl residues are more ordered and found mainly in pairs and triplets with few non-substituted regions [9].
A variety of bacteria, yeasts and filamentous fungi express mannan degrading enzymes [10]. Endomannanases are classified into three glycosyl hydrolase (GH) families: 5, 26 and 113, based on sequence similarity [11]. Endomannanases from these families belong to clan GH-A, share the (β/α)8-TIM barrel fold, and catalyze the hydrolysis of the O-glycosidic bonds in the mannan backbone with retention of the anomeric configuration [12], [13], [14]. “Endo-type” enzymes often use an open active site cleft in contrast to exo enzymes, e.g. β-mannosidases, using a pocket shaped active site region [15]. Characterized endo-mannanases show higher initial rates on locust bean gum compared to guar gum, and it has been proposed that the lower activity on guar gum is caused by the larger amount of galactopyranosyl residues [7], [16], [17]. Reported fungal endomannanases are predominantly found in GH5. Limited knowledge is available relating the sequence similarity classification (GH5 or GH26) to structural and functional differences among the two types of enzymes. Genome analysis has revealed that some organisms have endomannanases from both GH5 and GH26. The potentially different biological roles (substrate preferences) have been addressed for the bacterium Cellvibrio japonicus [18], [19] and the fungus Podospora anserina [20], [21]. A subdivision of fungal endomannanases from GH5 based on sequence similarity and functional characteristics has been proposed [22]. This phenomenon has been studied in Aspergillus nidulans, having a variety of different GH5 endomannanases (AnMan5A, AnMan5B, and AnMan5C) [17], [23], [24]. AnMan5B has been reported to have a high transglycosylation capacity and significantly lower kcat and kcat/km on locust bean gum compared with AnMan5A and AnMan5C [24]. Some fungal GH5 endo-mannanases are modular, typically having a carbohydrate-binding module from family 1 (CBM1), known to confer cellulose binding and to increase the mannan hydrolysis of complex substrates [25], [26]. Fungal GH26 endomannanases may be fused to a CBM35 [21], a CBM family known to bind β-mannans, uronic acids and α-D-galactopyranosyl residues on carbohydrate polymers [27], [28].
The hypothesis of this study is that fungal endomannanases of GH5 and GH26 have different degradation patterns on galactomannans due to structural differences, which affect their ability to accommodate galactopyranosyl residues in the active site cleft. With only a few fungal GH26 endomannanases studied, this work focused on the functional characterization of a novel GH26 endomannanase from A. nidulans (AnMan26A). For comparison, well characterized fungal GH5 and GH26 endomannanases were analyzed as well, including AnMan5A and AnMan5C. The functional characterization included determination of the initial hydrolysis rate and maximal degree of conversion of locust bean gum and guar gum. The DNA sequencer-Assisted Saccharide analysis in High throughput (DASH) method [29] was used to characterize hydrolysis end product profiles on the two substrates. This method has not previously been reported for the analysis of galactomanno-oligo-saccharides. A dominant degradation product on guar gum was elucidated by obtaining the same compound in the degradation of α-61-galactosyl-mannotriose with a known β-mannosidase. Further knowledge about the accommodation of galactopyranosyl residues in the active site cleft was obtained by degradation of α-64-63-di-galactosyl-manno-pentaose by AnMan26A, and by computational methods.
Section snippets
Materials
Locust bean gum (low viscosity; borohydride reduced), guar gum (high viscosity), mannobiose, mannotriose, mannotetraose, mannopentaose, α-61-galactosyl-mannotriose, and α-64-63-di-galactosyl-mannopentaose were purchased from Megazyme (Ireland). All other chemicals were from Sigma (Germany). Mobility markers, dextran ladder, and the DASHboard software for DASH analyses were kindly donated by Prof. Paul Dupree (University of Cambridge, UK).
Structural comparison, homology modeling, and ligand docking
Homology models of AnMan26A, AnMan5A and AnMan5C were
Results and discussion
The fungal endomannanases tested in this study (Table 1) were recombinantly expressed in the fungal host A. oryzae and purified to electrophoretic purity.
Conclusions
A novel GH26 endomannanase from A. nidulans was successfully cloned, purified, and characterized in parallel with known fungal endomannanases from GH5 and GH26. The analyzed GH5 endomannanases were found to be more thermostable than the GH26 endomannanases and neither presence of CBM1 nor CBM35 influenced the thermal stability of the endomannanases. The DASH method was successfully adapted for characterization of endomannanases and their hydrolysis products on galactomannans. The method can be
Authors contribution
Each author has materially participated in the research and the article preparation.
Pernille von Freiesleben, PvF: Participated in the design of the work. Conducted experiments, analysed the data, and co-wrote the manuscript.
Nikolaj Spodsberg, NS: Cloned and recombinantly expressed the enzymes.
Thomas Holberg Blicher, THB: Helped making the docking analysis of the enzymes.
Henning Jørgensen, HJ, Lars Anderson, LA, Henrik Stålbrand, HS, Anne S. Meyer, AM, Kristian B. R. M. Krogh, KBK: Designed and
Acknowledgements
We would like to thank Prof. Paul Dupree (University of Cambridge, UK) for help with implementation and optimization of the DASH method. This study was partially financed by The Danish Agency for Science, Technology and Innovation. Henrik Stålbrand was partially funded by FORMAS (213-2014-1254) and the Swedish Foundation for Strategic Research (14-0046).
References (45)
- et al.
Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood
Bioresour. Technol.
(2011) - et al.
How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation
Curr. Opin. Plant Biol.
(2008) - et al.
β-Mannanase (Man26A) and α-galactosidase (Aga27A) synergism—a key factor for the hydrolysis of galactomannan substrates
Enzyme Microb. Technol.
(2015) - et al.
A simple non-aqueous method for carboxymethylation of galactomannans
Carbohydr. Polym.
(2005) The fine structures of carob and guar galactomannans
Carbohydr. Res.
(1985)- et al.
Structural and sequence-based classification of glycoside hydrolases
Curr. Opin. Struct. Biol.
(1997) Mechanisms of glycosyl transferases and hydrolases
Carbohydr. Polym.
(2001)- et al.
Structures and mechanisms of glycosyl hydrolases
Structure
(1995) - et al.
Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity
Biochim Biophys Acta
(2011) - et al.
Understanding how the complex molecular architecture of mannan-degrading hydrolases contributes to plant cell wall degradation
J. Biol. Chem.
(2014)
Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis
J. Biol. Chem.
Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae
Fungal Genet. Biol.
A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates
J. Biotechnol.
Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes
Biotechnol. Biofuels
The Cellvibrio japonicus mannanase CjMan26C displays a unique exo-mode of action that is conferred by subtle changes to the distal region of the active site
J. Biol. Chem.
Phylogenetic analysis and substrate specificity of GH2 β-mannosidases from Aspergillus species
FEBS Lett.
A new reaction for colorimetric determination of carbohydrates
Anal Biochem.
Modes of action of β-mannanase enzymes of diverse origin on legume seed galactomannans
Phytochemistry
Hydrolytic properties of a β-mannosidase purified from Aspergillus niger
J. Biotechnol.
Action patterns and substrate-binding requirements of β-d-mannanase with mannosaccharides and mannan-type polysaccharides
Carbohydr. Res.
Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp
J. Biotechnol.
Characterisation of the oligosaccharides produced on hydrolysis of galactomannan with β-d-mannanase
Carbohydr. Res.
Cited by (35)
Identification and biochemical characterization of a novel GH113 β-mannanase from acid mine drainage metagenome
2023, Biochemical Engineering JournalMannanases and other mannan-degrading enzymes
2023, Glycoside Hydrolases: Biochemistry, Biophysics, and Biotechnologyβ-Mannanase BoMan26B from Bacteroides ovatus produces mannan-oligosaccharides with prebiotic potential from galactomannan and softwood β-mannans
2021, LWTCitation Excerpt :The analytical separation of low DP (<10) linear and substituted β-mannan-oligosaccharides is important for identification, quantification and characterization of β-mannan endo-hydrolysis products. This is a difficult task considering the expected structural variations in the oligosaccharides formed during hydrolysis of galactomannan backbone (von Freiesleben et al., 2016). Previously implemented HPAEC-PAD methods were evaluated and an improved method for the analytical separation of MOS/GMOS was developed.