Comparative analysis of enzymatic hydrolysis of Miscanthus xylan using Aspergillus niger, Hypocrea orientalis, and Trichoderma reesei xylan-degrading enzymes

Abstract

Xylan-degrading enzymes from Aspergillus niger and Hypocrea orientalis were characterized by enzyme activity assays and protein profiling with SDS-PAGE and LC-MS/MS. The hydrolysis of Miscanthus xylan by xylan-degrading enzymes from A. niger, H. orientalis, and Trichoderma reesei were comparatively studied by HPLC analysis. It was found that the glycoside hydrolase families 10 xylanase was the main xylanase secreted by H. orientalis and A. niger when using corn cob and wheat bran as inducers. Compared to the enzymes from T. reesei,the enzymes from A. niger showed better efficiency in the hydrolysis of Miscanthus xylan into monosaccharides. Nevertheless, the enzymes from H. orientalis were more preferable for the hydrolysis of Miscanthus xylan into xylo-oligosaccharides (XOS), especially xylobiose and xylotriose. Miscanthus xylan degradation was significantly influenced by the activities of β-xylosidase and α-L-arabinofuranosidase. Xylan-degrading enzymes with high ratios of β-xylosidase and α-L-arabinofuranosidase are necessary for the efficient conversion of Miscanthus xylan into monosaccharides. However, xylan-degrading enzymes with low β-xylosidase activity and high α-L-arabinofuranosidase activity were required for producing XOS.

Full Article

Comparative Analysis of Enzymatic Hydrolysis of Miscanthus Xylan using Aspergillus niger, Hypocrea orientalis, and Trichoderma reesei Xylan-degrading Enzymes

Hailong Li, Jian Liu, Jinlian Wu, Yong Xue, Lihui Gan, and Minnan Long*

Xylan-degrading enzymes from Aspergillus niger and Hypocrea orientalis were characterized by enzyme activity assays and protein profiling with SDS-PAGE and LC-MS/MS. The hydrolysis of Miscanthus xylan by xylan-degrading enzymes from A. niger, H. orientalis, and Trichoderma reeseiwere comparatively studied by HPLC analysis. It was found that the glycoside hydrolase families 10 xylanase was the main xylanase secreted by H. orientalis and A. niger when using corn cob and wheat bran as inducers. Compared to the enzymes from T. reesei, the enzymes from A. niger showed better efficiency in the hydrolysis of Miscanthus xylan into monosaccharides. Nevertheless, the enzymes from H. orientalis were more preferable for the hydrolysis of Miscanthus xylan into xylo-oligosaccharides (XOS), especially xylobiose and xylotriose. Miscanthus xylan degradation was significantly influenced by the activities of β-xylosidase and α-L-arabinofuranosidase. Xylan-degrading enzymes with high ratios of β-xylosidase and α-L-arabinofuranosidase are necessary for the efficient conversion of Miscanthus xylan into monosaccharides. However, xylan-degrading enzymes with low β-xylosidase activity and high α-L-arabinofuranosidase activity were required for producing XOS.

Keywords: Aspergillus niger; Hypocrea orientalis; Trichoderma reesei; Xylan-degrading enzymes; Hydrolysis; Xylo-oligosaccharides

Contact information: College of Energy, Xiamen University, Xiamen 361005, P. R. China;

* Corresponding author: longmn@xmu.edu.cn

INTRODUCTION

The efficient enzymatic conversion of cellulose and hemicellulose polymers into monosaccharides and xylo-oligosaccharides (XOS) is a topic of current interest. Hemicelluloses play an important role in biomass enzymatic digestion (Xu et al. 2012; Zhang et al. 2012; Li et al. 2013a). Xylan, the major hemicellulosic polysaccharide present in plant cell walls, has a backbone of β-1,4-linked xylose residues and side chains of different substituents (Vázquez et al. 2002). Enzymatic hydrolysis of xylan involves the synergistic action of several main chain- and side group-cleaving enzymes, including endo-β-1,4-xylanases (EC 3.2.1.8), β-xylosidases (EC 3.2.1.37), α-arabinofuranosidases (EC 3.2.1.55), α-glucuronidases (EC 3.2.1.139), acetyl xylan esterases (EC 3.1.1.72), and feruloyl esterases (EC 3.1.1.73) (de Vries and Visser 2001). Endo-β-1,4-xylanases as the main xylan-degrading enzymes randomly cleave the internal β-1,4-glycosyl bonds in the xylan main chain producing xylooligomers (Zhang et al. 2011), and β-xylosidases convert xylooligomers to xylose monomers (Knob et al 2009). Species of Trichoderma and Aspergillus are used as producers of enzymes that deconstruct lignocellulosic biomass by various companies (Banerjee et al. 2010). Trichoderma reesei has a highly efficient set of enzymes involved in cellulose degradation (Selig et al. 2008), while Aspergillus nigermostly produces enzymes that degrade hemicelluloses (Stricker et al. 2008).

Xylan-degrading enzymes with negligible amounts of exo-xylanase or β-xylosidase activity are required for the production of high-value added XOS because exo-xylanase and β-xylosidase produce xylose as the main product and inhibit the production of XOS (Vázquez et al. 2002). However, high-activity xylanase and β-xylosidase should be supplemented in saccharification processes for total hydrolysis of lignocellulosic biomass into monosaccharides because xylan clearly inhibited the hydrolysis of cellulose by cellulase and xylooligomers are stronger inhibitors of cellulase activity than are glucose and cellobiose (Selig et al. 2008; Qing et al. 2010; Zhang et al. 2012). Currently, most biotechnological processes are based on the use of crude enzymes. Strains with specificity toward xylan-degrading enzyme activity are of great application potential for the bioconversion of lignocellulosic biomass into monosaccharides or high-value added XOS.

Miscanthus with high biomass yield and low nitrogen and water requirement is considered as one of leading feedstocks for biofuel and value-added chemicals production (Xu et al. 2012; Li et al. 2013a). The aim of this work was to investigate the application potential of xylan-degrading enzymes from A. niger BE-2 and Hypocrea orientalis EU7-22 for the bioconversion of Miscanthus into monosaccharides and XOS. In this paper, xylan-degrading enzymes obtained from A. niger BE-2 and H. orientalis EU7-22 using corn cob and wheat bran as substrates were characterized by enzyme activity assays and SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the hydrolysis of Miscanthus xylan by xylan-degrading enzymes from A. niger BE-2, H. orientalis EU7-22, and T. reesei were comparatively studied by HPLC analysis.

EXPERIMENTAL

Materials

Xylohexaose (X₆), xylopentaose (X₅), xylotetraose (X₄), xylotriose (X₃), and xylobiose (X₂) were obtained from Megazyme (Bray, Ireland). Glucose (Glu), xylose (Xyl), arabinose (Ara), and HPLC-grade acetonitrile were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-phenyl-3-methyl-5-pyrazolone (PMP) was obtained from Acros Organics (Geel, Belgium). Beechwood xylan, p-nitrophenyl-β-D-xylopyranoside (pNPX), and p-nitrophenyl-α-L-arabinofuranoside (pNPA) were purchased from Sigma-Aldrich (USA).

Commercial xylan-degrading enzyme preparation (xylanase activity, 696,345 IU/g; β-xylosidase activity, 469 IU/g; α-arabinofuranosidase activity, 281 IU/g) produced by T. reesei was purchased from Zaozhuang Jienuo Enzyme Co., Ltd (Shandong, China). Miscanthus xylan substrate (79.28% xylose, 13.08% arabinose, and 7.64% glucose) was isolated from Miscanthus floridulus as described previously (Li et al. 2013b).

Microorganism Xylan-degrading Enzyme Production

The xylan-degrading enzyme-producing strains A. niger BE-2 (GenBank accession No. JQ867187) and H. orientalis EU7-22 (GenBank accession No. KC751873) were preserved in the laboratory. Enzyme production was carried out in 250-mL flasks with 50 mL of medium. Each flask included 5 g/L tryptone, 0.4 g/L CaCl₂, 0.4 g/L FeSO₄, 0.8 g/L MgSO₄, 2.5 g/L KH₂PO₄, 1.5% (w/v) corncob, and 1.5% (w/v) wheat bran. The pre-cultured H. orientalis EU7-22 and A. niger BE-2 mycelium (10%, v/v) were inoculated into each flask and then incubated at 37 ^oC in a rotary shaker (180 rpm) for 72 h. The enzymes were obtained by centrifugation at 5000 g for 10 min to remove the mycelium and solid medium. Then, the enzymes were aliquoted into tubes and stored at -80 ^oC.

Enzyme Assays

Xylanase activity was assayed using 1% beechwood xylan as the substrate in sodium citrate buffer (50 mM, pH 5.0) at 50 ^oC for 10 min (Bailey et al. 1992). The amount of released sugar was determined by the dinitrosalicylic acid method described by Miller et al. (1960). One unit of xylanase activity was defined as the amount of enzyme that liberated 1 μmol of reducing saccharides per min from the substrate. β-xylosidase and α-arabinofuranosidase enzyme activities were measured using a final concentration of 5 mM pNPX and 2.5 mM pNPA as the substrates in citrate buffer (50 mM, pH 5), respectively. After incubation at 50 ^oC for 30 min, the reaction was terminated by addition of 1 mL of 0.5 M Na₂CO₃, and the absorbance was measured at 405 nm. One unit of β-xylosidase or α-arabinofuranosidase enzyme activity was defined as the amount of enzyme that liberated 1 μmol of p-nitrophenyl per min from the synthetic substrate. Thermostability assays were determined by measuring residual xylanase and β-xylosidase activities after pre-incubation of enzymes at 50 ^oC for 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h. The activity of the enzyme without pre-incubation was defined as 100%.

Enzymatic Hydrolysis

Miscanthus xylan was hydrolyzed by crude enzymes from H. orientalis EU7-22, A. niger BE-2, and T. reesei as described previously (Li et al. 2013b). Miscanthus xylan (2 g) was suspended in 100 mL of citrate buffer (50 mM, pH 5.0) containing 0.02% azide, and enzymes were added at 100 to 200 IU/g (xylanase activity) dry matter. Hydrolysis was performed in shake-flasks at 50 ^oC for 0.5 h, 1 h, 2 h, 3 h, and 6 h. Hydrolysis samples were boiled for 10 min and then centrifuged at 8000 g for 5 min, filtered, and stored at 4 ^oC for further analysis.

HPLC Analysis

Monosaccharides and oligo-saccharides were analyzed by HPLC using a PMP pre-column derivatization method described previously (Li et al. 2013b). The analysis of PMP derivatives of saccharides was carried out on an Agilent 1200 HPLC system (U.S.) equipped with a diode array detector. The analytical column used was a CAPCELL PAK C₁₈ MG column (3.0 mm i.d. × 250 mm, 5μm, Shiseido, Japan). Elution was carried out at a flow rate of 0.5 mL/min at 30 ^oC, with a sodium phosphate buffer (40 mM, pH 8.0)/-acetonitrile (79:21, v/v). The wavelength for UV detection was 245 nm.

SDS-PAGE and LC-MS/MS Analysis

SDS-PAGE on 15% polyacrylamide was performed using the method of Laemmli (1970). Protein bands were visualized by Coomassie Brilliant Blue R-250 staining. The most abundant protein bands from H. orientalis EU7-22 and A. niger BE-2 were excised from Coomassie-stained gels and subjected to in-gel trypsin digestion. Tryptic peptides were extracted from gels using 0.15% formic acid/67% acetonitrile. Dried peptides were dissolved in 0.1% formic acid/2% acetonitrile and separated on a fused silica capillary emitter (inner diameter, 75 µm; length, 15 cm; New Objective, Woburn, MA) packed in-house with 5 µm of C18 resin and analyzed on an AB SCIEX TripleTOF 5600 system. The resulting peak lists were used to query the Swiss-Prot database using the Mascot program. Only significant hits as defined by mascot probability analysis were considered.

RESULTS AND DISCUSSION

Analysis of Xylan-degrading Enzymes from A. niger BE-2 and H. orientalis EU7-22

The xylan-degrading enzymes obtained from A. niger BE-2 and H. orientalis EU7-22 using corncob and wheat bran as substrates were evaluated. The enzyme activity of xylanase, β-xylosidase, and α-L-arabinofuranosidase from A. niger BE-2 and H. orientalis EU7-22 after 72 h of cultivation are shown in Table 1.

Table 1. Enzyme Activity of Xylan-degrading Enzymes from A. niger BE-2 and H. orientalis EU7-22

All evaluations of samples were carried out in triplicate. The uncertainties represent the standard errors of three experiments. The differences in xylan-degrading enzymes between A. niger and H. orientaliswere analyzed by the percentage of the increased or decreased level at pair: subtraction of two samples divided by means of two values at pair.

H. orientalis EU7-22 expressed higher xylanase activity, but much lower β-xylosidase and α-L-arabinofuranosidase activities, than the A. niger BE-2 strain. The xylanases from H. orientalis EU7-22 and A. niger BE-2 exhibited better thermo-tolerance than the xylanases from T. reesei at 50 ^oC and 60 ^oC (Fig. 1a, c). β-xylosidase plays a key role in the complete hydrolysis of XOS into xylose (Knob et al 2009). As shown in Fig. 1b and 1d, the β-xylosidase from A. niger BE-2 retained more than 90% enzyme activity after 24 h of incubation at 50 ^oC and 60 ^oC, which is more thermo-tolerance than β-xylosidase from either H. orientalis EU7-22 or T. reesei. XOS are stronger inhibitors of cellulase activity (Qing et al 2010). These results show that the thermo-tolerant β-xylosidase from A. niger BE-2 has promise for use in lignocellulosic biomass hydrolysis processes.

Enzymatic Hydrolysis of Miscanthus Xylan

The time course of enzymatic hydrolysis of Miscanthus xylan by different xylan-degrading enzymes at 50 ^oC was studied. When equal amounts of xylanase activity units were used, the enzymes from A. niger BE-2 degraded crude xylan into primarily xylose, arabinose, xylobiose, xylotriose, and xylohexaose, with trace amounts of xylotetraose and glucose, within 0.5 h (Fig. 2a). The enzymes from H. orientalis EU7-22 and T. reesei produced primarily xylobiose, xylotriose, and xylotetraose, with small amounts of xylohexaose, xylopentaose, xylose, arabinose, and glucose, within 0.5 h (Fig. 2b, 2c).

Fig. 1. The thermostability of xylan-degrading enzymes of H. orientalis EU7-22 and A. niger BE-2 : (a) thermostability of xylanase activity at 50 ^oC, (b) thermostability of β-xylosidase activity 50 ^oC, (c) thermostability of xylanase activity at 60 ^oC, (d) thermostability of β-xylosidase at 60 ^oC;

All tests were done in triplicate. The error bars represent the standard errors of three experiments.

Fig. 2. Analysis of enzymatic hydrolysates of Miscanthus xylan by HPLC (100 IU/g, 0.5 h).

(a) A. niger BE-2, (b) H. orientalis EU7-22, (c) T. reesei. Xylohexaose (X₆), xylopentaose (X₅), xylotetraose (X₄), xylotriose (X₃), xylobiose (X₂), glucose (Glu), xylose (Xyl), and arabinose (Ara).

The presence of arabinose as a side group on xylan limits the access of xylanase to the main chain of xylan, and the cleavage of arabinose from xylan provides more binding sites for xylanase (Sakamoto et al. 2011). Most arabinose from Miscanthus xylan was released within 0.5 h by the enzymes from A. niger BE-2 (Fig. 3a, b).

However, the yield of arabinose kept increasing in hydrolysates of H. orientalis EU7-22 (Fig. 3c, 3d) and T. reesei (Fig. 3e, 3f). This shows that the enzymes from A. niger BE-2 exhibit high catalytic activity for arabinosyl side-chains of Miscanthus xylan. Despite A. niger BE-2 having the highest amount of β-xylosidase, the yields of xylose and XOS produced by the enzymes from A. niger BE-2 were both higher than the enzymes from H. orientalis EU7-22 and T. reesei at 0.5 h (Table S1). This indicated that the cleavage of arabinose side chains at the initial hydrolysis phase enhanced the conversion of Miscanthus xylan into XOS. When the hydrolysis time was extended, the enzymes fromA. niger BE-2 with a lower ratio of xylanase to β-xylosidase (EX/BX) efficiently degraded xylan into xylose (Fig. 3a, b). The enzymes from H. orientalis EU7-22 and T. reesei with higher EX/BX ratios also degraded xylan into XOS (Fig. 3c, d, e, f). The yields of xylose produced by the enzymes (200 IU/g) from H. orientalis EU7-22 and T. reesei (Fig. 3d, f) were much lower than that produced by the enzymes (100 IU/g) from A. niger BE-2 (Fig. 3a). These results revealed that Miscanthus xylan degradation was significantly influenced by the activity of β-xylosidase.

Fig. 3. Time course of enzymatic hydrolysis of xylan by xylan-degrading enzymes from A. niger BE-2 (a 100 IU/g and b 200 IU/g), H. orientalis EU7-22 (c 100 IU/g and d 200 IU/g), and T. reesei (e 100 IU/g and f 200 IU/g) when different amounts of xylanase activity units were added.

The saccharide yield was quantitated by the peak response (mAU).

SDS-PAGE and LC-MS/MS Analysis of Xylan-degrading Enzymes

The yield of xylotetraose decreased and the yield of xylohexaose increased when the enzymes from H. orientalis EU7-22 were used, while the yield of xylotetraose increased and little xylohexaose was found when the enzymes from T. reesei were used (Fig. 3c, d, e, f). In addition, compared to the enzymes from T. reesei, more xylobiose and xylotriose were produced by the enzymes from H. orientalis EU7-22, even though a smaller dosage of enzymes was used (Fig. 3c, f). The results indicated that the action patterns of xylan-degrading enzymes from H. orientalis EU7-22 and T. reeseion Miscanthus xylan are different. The two major inducible endo-1,4-beta-xylanases expressed by T. reesei are xyn1 and xyn2, accounting for more than 90% of the xylan-degrading activity of this fungus (Torronen et al. 1992; Torronen et al. 1994). H. orientalis is in the genus of Trichoderma, which shows high homology with T. reesei. The deduced amino acid sequences of xyn1 (GenBank accession No. AFD50198.1) and xyn2 (GenBank accession No. AFD50199.1) from H. orientalis showed 95% and 96% homology to that of T. reesei xyn2 (GenBank accession No. P36217.1) and xyn1 (GenBank accession No. P36218.1), respectively.

Based on the amino acid sequences in the catalytic domain, most fungal xylanases are clustered into glycoside hydrolase families 10 and 11 (GH10 and GH11). Members of GH10 have a molecular weight greater than 30 kDa and are highly specific for small xylo-oligosaccharides, while members of GH11 show a low molecular weight and high activity on long chains of xylo-oligosaccharides (Biely et al. 1997; Pollet et al. 2010). Both xyn1 (19 kDa) and xyn2 (21 kDa) from T. reesei belong to GH11. Xyn3 (32 kDa) in T. reesei, which belongs to GH10, was found expressed in a mutant strain (T. reeseiPC-3-7), but silenced in the most studied strain of T .reesei, QM9414 (Xu et al. 2000). SDS-PAGE analysis showed that a major band from 19 to 21 kDa was found in commercial xylanase preparation, but a major band from 32 to 33 kDa was found in H. orientalis EU7-22 and A. niger BE-2 (Fig. 4). The results revealed that GH11 xylanase may be the dominant protein in the commercial xylan-degrading enzyme preparation; nevertheless, GH10 xylanase may be the major xylanase expressed in H. orientalis EU7-22, which resulted in different hydrolyzates from T. reesei.

Table 2. Mascot Protein Identification of Major Proteins Produced by A. niger BE-2, H. orientalisEU7-22, and T. reesei

The most abundant protein bands from A. niger BE-2. H. orientalis EU7-22, and T. reesei were excised from Coomassie-stained gels and analyzed by LC-MS/MS. As shown in Table 2, the mass spectrum identified the protein band A as GH10 endo-1,4-beta-xylanase C from A. niger, the protein band B as GH10 endo-1,4-beta-xylanase 3 from T. reesei, and the protein band C as GH11 endo-1,4-beta-xylanase from T. reesei. These results showed that corn cob and wheat bran are fine substrates for H. orientalis EU7-22 and A. niger BE-2 to produce GH10 endo-1,4-beta-xylanase. SDS-PAGE and LC-MS/MS analysis confirmed the presumption that a GH10 xylanase is expressed by H. orientalis EU7-22. Compared to GH11 xylanases, GH10 xylanases with broader substrate specificities show better synergistic effects to boost the hydrolytic potential of cellulases with pretreated lignocellulosic substrates (Zhang et al. 2011; Hu et al. 2013).

CONCLUSIONS

1. Miscanthus xylan degradation was significantly influenced by the activity of β-xylosidase and α-L-arabinofuranosidase. Xylan-degrading enzymes with a high ratio of β-xylosidase and α-L-arabinofuranosidase were necessary for the efficient conversion of Miscanthus xylan into monosaccharides. However, xylan-degrading enzymes with low β-xylosidase activity and high α-L-arabinofuranosidase activity were required for producing XOS from Miscanthus.
2. SDS-PAGE coupled with LC-MS/MS is a powerful tool for the identification of the most abundant protein from crude enzymes. The GH10 xylanase was the primary xylanase expressed by H. orientalis EU7-22 and A. niger BE-2 when corn cob and wheat bran were used as substrates.
3. Compared to commercial xylan-degrading enzymes produced by T. reesei, the enzymes from A. niger BE-2 showed higher catalytic efficiency for complete hydrolysis of xylan into monosaccharides, while the enzymes from H. orientalis EU7-22 exhibited better potential for the production of XOS, especially xylobiose and xylotriose.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (Grant No. 31170067, No. 21303142), the National Basic Research Program of China (973 Program), (Grant No: 2010CB732201) and the research fund of Fujian Provincial Natural Science Foundation (Grant No: 2012J05029).

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Article submitted: November 9, 2013; Peer review completed: December 22, 2013; Revised version received and accepted: February 15, 2014; Published: March 3, 2014.

APPENDIX: SUPPLEMENTARY INFORMATION

Table S1. Yield of Xylose and XOS Produced by Enzymatic Hydrolysis of Miscanthus xylan (100 IU/g, 0.5 h)

The saccharide yield was quantitated by the peak response (mAU). The yields of XOS were the sum of the peak response of X₂~X₆.