Characteristic of a GH10 Xylanase From The Anaerobic Rumen Fungus Anaeromyces Robustus and Application in Bread Making

The rumen of ruminants contains a variety of fungi capable of producing xylanases to break down plant cell walls. In this study, a new GH10 xylanase gene ArXyn10c20 from anaerobic rumen microorganism Anaeromyces robustus was successfully expressed in Pichia Pastoris GS115, with a protein molecular weight of approximately 42 kDa and showed the similarity by 64.08% with the β-Xylanase form Neocallimastix Californiae. The optimal pH and temperature for ArXyn10c20 was 5.5 at 40℃. ArXyn10c20 was stable in the pH range 5.0–9.0 for 1h which the residual enzyme activity was all above 75%. The activity of recombinant xylanase was significantly enhanced by 1 mM Cu 2+ . The products of ArXyn10c20 hydrolysis of beechwood xylan were xylobiose, xylotriose and xylotetraose by TLC analysis. In food applications, ArXyn10c20 can significantly improve the quality of dough and bread. With the addition of 7.5 mg ArXyn10c20, the hardness, gumminess and chewiness of the bread decreased by 42.24%, 45.33% and 55.36% respectively and the reducing sugar increased by 18.67%. The new discovered xylanase ArXyn10c20 has great potential in food industry.


Introduction
Anaerobic fungi found in the rumen of ruminants are an excellent source of xylanase. Ruminal fungi were first discovered in 1975 and are the only known group of anaerobic fungi. To date, more than 20 species have been successfully isolated, including six genera: Piromyces, Anaeromyces, Neocallimastix, Cyllamyces, Caecomyes, and Orpinomyces. The first genome sequencing of anaerobic rumen fungi was completed in 2013 [1], with Anaeromyces robustus being classified in 2016 [2].
Ruminal anaerobic fungi are highly adapted to fibre degradation, which is important for the degradation of difficult to process biological resources currently encountered in industry [3,4]. Among rumen microorganisms, fungi take a small percentage, but play an important role in the degradation of xylan in plant cell walls by ruminants [5].
Xylan is an important component of plant hemicellulose, which accounts for one third of the total plant carbohydrates and is the second most abundant in nature after cellulose [6]. Xylan is found in the cell walls of terrestrial plants and is present in almost all parts of the plant. The amount of xylan in different plants varies, generally more in hardwoods than in softwoods, accounting for 15%-30% of the dry weight in hardwoods and 7%-10% in softwoods. In some annual plants, such as wheat, sugarcane and cotton, the xylan content is very high, generally up to 30% or more [7].
Xylanase is a general term for enzymes that degrade xylan. β-xylanase, β-1,4endo-xylanase, and α-L-arabinosidase all belong to xylanase. Among all xylanases, β-1,4-endo-xylanase (EC 3.2.1.8) plays the main degradation function. In the hydrolysis process, xylanase mainly breaks the β-1,4-glycosidic bond of xylan to hydrolyze it into xylose and other oligosaccharides [8]. Oligosaccharides are finally degraded into xylose by the action of β-xylosidase, and other enzymes in xylanase such as acetylxylan esterase and other side chain hydrolases usually have synergistic effects on the main chain hydrolases. Through the cooperation of multiple enzymes finally hydrolyze xylan into xylose [9].
Based on a comparison of structures at the catalytic domain level, almost all xylanases to date have been grouped into the glycoside hydrolase family (GH) 5, 8, 10, 11, 43, 62 [10]. Most of xylanases were classified into GH10 and GH11. Xylanase can be produced by plants, animals and microorganisms [11], but the nature xylanase produced differs depending on the type of producer. Bacterial-derived xylanases have an advantage over fungal-derived xylanases in terms of heat resistance [12]. Among all xylanase producing microorganisms, rumen fungi are a very special group and currently, less than 10% of rumen fungi can be isolated by pure culture techniques [13]. Therefore, there is an increasing use of macro-genomic techniques to research rumen fungi [14].
Xylan is widely used in the food, medical and chemical industries. Xylan can be added to food as a dietary additive or used as a raw material in the production of oligosaccharides or xylitol [15]. Its high water-holding capacity allows it to be used in biomedical applications such as drug delivery [16]. In addition, xylan is used as a renewable and biodegradable plant resource in chemical applications such as the production of 5-hydroxymethylfurfural (HMF), furfural, ethanol or other value-added chemicals [17].

Strains and reagents
Pichia pastoris GS115 and Escherichia coli DH5α was preserved in our laboratory. G418

Transformation and expression of ArXyn10c20 in P. pastoris GS115
The pPIC9K-ArXyn10c20 was linearized by SalI and transformed into P. pastoris

Purification and deglycosylation of ArXyn10c20
The culture was centrifuged at 5000×g, 4℃ for 10 min to collect the supernatant and purified by Ni-NTA resin. The protein was eluted with elution buffer (0.25 M NaCl, and 20 mM Tris-HCl buffer, 0.2 M imidazole, pH 6.5). Endo H was used to remove Nlinked glycan of purified ArXyn10c20. SDS-PAGE was performed to detect the degree of ArXyn10c20 glycosylation.

Enzyme activity assay
The enzyme activity of xylanase was determined by the DNS method [18]. The reaction solution contained 90 μL 1% (W/V) beechwood xylan and 30 μL diluted enzyme and incubated at pH 5.5 and 40℃ for 30 minutes. After 180 μL of DNS was added to the reaction, the mixture was boiled for 5 min, 1.2 mL deionized water was added to set the volume to 1.5 mL. The released reducing sugar was quantified by spectrometric method at 540 nm. All determinations of ArXyn10c20 activity were performed in triplicate. The xylanase required to degrade beech xylan to produce 1 μmol xylose per minute at pH 5.5 and 40℃ was defined as one unit of enzyme activity.

Effects of pH and temperature on ArXyn10c20 activity and stability
The optimal pH of purified ArXyn10 was determined at 40℃ in 50 mM different citric acid/sodium citrate buffers at the pH range of 3.0-6.5 and in 50 mM different Tris-HCl buffers at the pH range of 7.0-9.0. The pH stability was determined after treatment for 5-60 minutes at pH range 3.0 to 9.0 and dilution with 0.1 M citric acid/sodium citrate buffer at pH 5.5. Residual activity was determined by activity assay and calculated relative activity using the untreated sample as control (100%).
The optimum temperature of ArXyn10c20 was determined at pH 5.5 in the temperature range 30℃-80℃. The thermostability of ArXyn10c20 was determined by treatment at 50℃, 60℃ and 70℃ for 5-60 minutes, respectively. The residual activity was determined by activity assay and the relative activity was calculated as above.

Effect of metal ions and chemicals on the ArXyn10c20 activity
To determine the effect of metal ions and chemicals on ArXyn10c20 activity, final concentrations of 1 mM and 5 mM of Na + , K + , Ca 2+ , Cu 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Mg 2+ , Co 2+ , SDS, and EDTA were added to the reaction solution, respectively. Reaction solutions without metal ions and chemicals were used as control. The residual activity was determined by activity assay and the relative activity was calculated as above.

Substrate specificity and kinetic of ArXyn10c20
The kinetic parameters of ArXyn10c20 were determined using different concentrations of beechwood xylan (1 mg/mL, 2 mg/mL, 2.5 mg/mL, 3.3 mg/mL, 5 mg/mL, 10 mg/mL) in the reaction system. the Km, Vmax and kcat values of ArXyn10c20 were calculated from Lineweaver-Burk plots, y=Vmax* To test the substrate specificity of ArXyn10c20, beechwood xylan, sodium carboxymethylcellulose (CMC-Na), microcrystalline cellulose, and filter paper (2×1 cm) were used as substrates in the standard activity assay to determine enzyme activity and the relative activity of the other substrates was calculated using the enzyme activity of beechwood as substrate as 100%.

Thin-layer chromatography (TLC) assay
The hydrolysis products were analysed by incubating purified ArXyn10c20 at pH and xylopentaose (X5) was used as standard.

Application of ArXyn10c20 in bread making
The recipe for bread was as follows: 100 g high-gluten wheat flour, 60 mL water, 10 g sugar, 5 g butter, 0.3 g yeast (Instant dry yeast, Saccharomyces cerevisiae, Angel Yeast Co., Ltd), 1 g salt. The bread making process was to knead the dough for 15 min and to rest at 40℃, 80% humidity for 2 h, take out and divide the dough into 50 g for each one, then rest for 1 h, and bake for 30 min(Upper tube at 160℃, lower tube at 180℃).
0 mg (control), 2.5 mg, 5 mg and 7.5 mg of purified ArXyn10c20 was added into dough to make bread, respectively, and specific volume, texture, and reducing sugar content were determined. The specific volume was tested by the rapeseed substitution method [21]: Put the bread into a beaker, fill it with rapeseed, shake it thoroughly to expel the air and record the volume V1, take the bread out and record the volume V2 , the volume of the bread V= V1-V2. The mass of the bread is weighed on the electronic balance as m. Specific volume v=V/m. The texture was measured by texture analyzer (TMS-PRO) and the bread was sliced to a thickness of 10 mm. The reducing sugar content was determined using the supernatant of the dough by DNS method as above.
The supernatant was obtained by dissolving 10 g of dough in 100 ml of water, mixing thoroughly and centrifuging.

Effect of ArXyn10c20 on microstructure of dough
The dough was pressed onto a slide and observed and photographed on an optical microscope (apparatus model Olympus BX63, Yangtze University, Jingzhou, China) using a 100× oil microscope.
The dough was freeze-dried and exposed to gold sputtering. The samples were then observed and photographed with a scanning electron micrograph (apparatus model TM4000, Shanghai Academy of Agricultural Sciences, Shanghai, China) at an accelerating voltage of 5 kV.

Sequence and structure analysis
This is the first time that ArXyn10c20 has been isolated from an anaerobic rumen fungus and heterologously expressed in P. pastoris GS115. Through sequence alignment, the β-xylanase from Neocallimastix californiae was the most similar to Arxyn10c20 with 64.08% identity (GenBank: ORY30865.1), indicating that ArXyn10c20 produced by Anaeromyces robustus is a new GH10 xylanase. A phylogenetic tree was constructed using xylanases with 35%-64.08% identity to ArXyn10c20 (Fig. 1.). It showed in the tree that ArXyn10 evolved as an independent branch, and it was most close to the branch including the xylanases from N. californiae.
It was the first discovered xylanase identified from A. robustus, which enriched the source of xylanases and may had important meanings for the study of xylanases from anaerobic origins.
The multiple sequence comparisons of several GH10 xylanases produced by rumen microorganisms revealed that their catalytic sites are highly consistent and their relative positions in the GH10 domain was conservative (Fig. 3.). In the model of ArXyn10c20, the catalytic sites located at the inner edge of the depression of the bowlshaped structure formed by the (α/β)8 TIM-barrel structure. E144 and E260 were speculated as catalytic sites in ArXyn10c20, were the key amino acids for xylanase activity in this study.

Expression and purification of ArXyn10c20
Analysis of the protein sequence showed that 18 amino acids at the N-terminus of the protein were signal peptides, amino acid (AA) 16-332 was GH10 domain. E144 and E260 were catalytical active sites of ArXyn10c20. ArXyn10c20 possessed five possible N-glycosylation sites, N36, N267, N304, N337 and N346, and two possible O-glycosylation sites, T233 and T365.
ArXyn10c20 encoding gene was obtained and cloned into pPIC9K, resulting the recombinant plasmid pPIC9K-ArXyn10c20. ArXyn10c20 comprised 1113 bp with 370 amino acids. After expression in P. pastoris GS115 and purification, the protein sample was analyzed by SDS-PAGE. A single band of ArXyn10c20 was observed near 42 kDa in SDS-PAGE, which was approximately the calculated theoretical size of ArXyn10c20.
By determination of the SDS-PAGE and enzyme activity assay, the recombinant protein of ArXyn10c20 was successfully expressed, purified and identified. After digestion by Endo H, the molecular weight of ArXyn10c20 did not change significantly, indicating that ArXyn10c20 was not glycosylated (Fig. 4.).

Characterization of the purified ArXyn10c20
As shown in Fig. 5., The optimal pH of the purified ArXyn10c20 was 5.5, and ArXyn10c20 maintained more than 90% of the enzyme activity between pH 5.5-6.0, showing it was an acid xylanase. ArXyn10c20 had a wide range of pH stability. After holding at pH 5.0-9.0 for 1h respectively, the residual enzyme activity was all above 75%.
The optimum temperature of ArXyn10c20 was 40℃, and could maintain high enzyme activity at 35-40℃, indicating that ArXyn10c20 is a medium temperature enzyme. In the case of high temperature treatment, after holding at 50℃ for 10 minutes, 21.99% enzyme activity remained. After holding at 60℃ for 5 min, the enzyme activity was only 10.08% remained. When treated at 70°C, less then 5% enzyme activity remained, indicating that the protein is not resistant to high temperatures.
The optimum temperature of ArXyn10c20 is consistent with the fermentation temperature of yeast and is ideal for applications in food, such as bread making. For other applications, ArXyn10c20 needs to be modified as it has good pH stability but poor thermal stability.
Compared to other xylanases, ArXyn10c20 lacks the CBM domain. Importantly, the presence of some CBM domains enhances the thermal stability of the protein.
After deleting the C-terminal CBM, the mutant obtained greater substrate affinity and catalytic efficiency, and the thermal stability was also improved [23]. To improve the enzymatic activity and stability of ArXyn10c20, the addition of a thermally stable CBM domain could be considered. Setting the enzyme activity for beechwood xylan as 100%, the enzyme activities of filter paper was 4.38%, and it showed no activity on soluble starch、CMC-Na and microcrystalline cellulose, exhibited a high degree of substrate specificity. Although GH10 family xylanases are able to hydrolyse a wider range of substrates than GH11 family xylanases [24], they do not have cellulase activity, whereas GH11 family xylanases have cellulase activity.
The kinetic parameters of ArXyn10c20 were obtained by Lineweaver-Burk plot.
Vmax and Km were 250.00 U/mg and 6.975 mg/mL, Kcat was 175.40 s -1 , and Kcat/Km was 25.15 s -1 ·(mg/mL) -1 . As shown in Table 2, the Km, Kcat and Kcat/Km of ArXyn10c20 are higher than those of other GH10 family xylanases, showing excellent catalytic activity and efficiency.
Based on food applications, oligosaccharides with a polymerisation degree of 2-4 were preferred [29], making ArXyn10c20 suitable for use in food applications. In addition, xylooligosaccharides (X2-X7) are known to improve the intestinal environment [30], inhibit pathogenic bacteria, prevent constipation, protect liver function, lower serum cholesterol and blood pressure, and effectively promote the activity of Bifidobacteria [31]. Xylooligosaccharides are becoming increasingly important in areas such as human health, anti-cancer and clean energy conversion [32].

Application of ArXyn10c20 in bread making
The specific volume of the bread was 2.2 without the addition of ArXyn10c20, while the specific volume of the bread was 2.63, 2.59 and 2.80 with the addition of 2.5 mg, 5.0 mg and 7.5 mg ArXyn10c20 respectively. It is showed that ArXyn10c20 could improve the dough quality and increase the volume of the bread. By measuring the texture of the bread, the hardness, gumminess and chewiness of the bread decreased with the increase of ArXyn10c20 addition (Table 3), and the taste of the bread was improved. After the addition of 2.5mg ArXyn10c20, the hardness, gumminess and chewiness of the bread decreased by 25.86%, 32.00% and 13.10% respectively, while the addition of 7.5 mg decreased by 42.24%, 45.33% and 55.36% respectively, indicating that ArXyn10c20 can significantly improve the quality of dough and bread.
Comparing the effects of different xylanases on the chewiness and hardness of bread, ArXyn10c20 significantly reduced the chewiness and hardness of bread at a low addition level ( Table 4).
As shown in Fig. 7, with the addition of 2.5 mg ArXyn10c20, gluten network formation was minimal and not significantly different from the control. Gluten network formation started at 5.0 mg of ArXyn10c20 addition. With the addition of 7.5 mg ArXyn10c20, gluten network was formed on the whole surface of the dough, and water-unextractable arabinoxylan (WUAX) was hydrolysed to small molecules like waterextractable arabinoxylan (WEAX) and xylooligosaccharides.
Bread quality is mainly determined by the dough's ability to produce and hold air. significantly influences dough characteristics and quality [40]. WUAX accounts for about 90% of AX [41]. WUAX is able to adsorb large amounts of water and can directly interfere with gluten formation [42], being broken down increases the free water in the dough and makes it softer. WEAX and xylooligosaccharides produced by the breakdown of WUEX can bind to the proteins in the dough and form gluten [43].
As more gluten network is formed, the dough's ability to hold air increases and therefore the quality of the bread is better [44]. The gluten network gives the dough its unique viscoelastic properties, which in turn determines the quality of the final product such as baked bread and steamed bread [45]. The bubble walls can expand like a small balloon without tearing until the bread is deformed. When carbon dioxide exerts more pressure than the fermenting dough can withstand, the gluten structure weakens, releasing gas and deflating the over-fermented dough, hence the characteristics of a high quality dough that produces a large volume [46], with uniformly fine pores and fluffy.
In addition, the content of reducing sugars in the bread with 2.5 mg, 5.0 mg and 7.5 mg of ArXyn10c20 increased by 6.95%, 16.91% and 18.67% respectively compared to the control. The increase in reducing sugars shows the production of oligosaccharides.
These oligosaccharides can be used by yeast to improve the air production capacity of the dough. On the other hand, these oligosaccharides not fully used by yeast can enter the human intestine to be used by Bifidobacterium. As an important probiotic in the human body, Bifidobacterium has a variety of functions for human health, such as biological barrier, anti-tumour, immune enhancement and improvement of gastrointestinal function [47], making bread healthier for the human body.

Conclusion
In this paper, it is the first time that ArXyn10c20 has been isolated from an anaerobic rumen fungus and successfully expressed in P. pastoris GS115. Analysis of SDS-PAGE showed the monomeric form of the recombinant protein in a size consistent with predicted molecular weight of 42 kDa. The xylanase ArXyn10c20 was characterized by determination of optimal temperature and pH, thermal and pH stability, substrate specificity and kinetic constants were also determined. In practical application, the optimum temperature of ArXyn10c20 is consistent with the fermentation temperature of yeast, making ArXyn10c20 suitable for use in food applications. As the amount of ArXyn10c20 added increases, the reducing sugar content within the dough increases and TLC analysis shows that the hydrolysis products of ArXyn10c20 are mainly X2, X3 and X4, and xylooligosaccharides are now becoming a common health supplement and becoming increasingly popular. The addition of ArXyn10c20 at 7.5 mg reduced the hardness and chewiness of the bread by 42.24% and 55.39% respectively.
The addition of ArXyn10c20 makes the bread fluffier and softer, with an improved texture.            (e,j)dough with 7.5 mg ArXyn10c20 after fermentation .    Phylogenetic tree of ArXyn10c20 based on protein sequence homology.