Paecilomyces variotii xylanase production, purification and characterization with antioxidant xylo-oligosaccharides production

Paecilomyces variotii xylanase was, produced in stirred tank bioreactor with yield of 760 U/mL and purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.29-fold purification with 34.47% activity recovery. The enzyme purity was analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirming its monomeric nature as single band at 32 KDa. Zymography showed xylan hydrolysis activity at the same band. The purified enzyme had optimum activity at 60 °C and pH 5.0. The pH stability range was 5–9 and the temperature stability was up 70 °C. Fe2+and Fe3+ exhibited inhibition of xylanase enzyme while Cu2+, Ca2+, Mg2+ and Mn2+ stimulated its activity. Mercaptoethanol stimulated its activity; however, Na2-EDTA and SDS inhibited its activity. The purified xylanase could hydrolyze beechwood xylan but not carboxymethyl cellulose (CMC), avicel or soluble starch. Paecilomyces variotii xylanase Km and Vmax for beechwood were determined to be 3.33 mg/mL and 5555 U/mg, respectively. The produced xylanase enzyme applied on beech xylan resulted in different types of XOS. The antioxidant activity of xylo-oligosaccharides increased from 15.22 to 70.57% when the extract concentration was increased from 0.1 to 1.5 mg/mL. The enzyme characteristics and kinetic parameters indicated its high efficiency in the hydrolysis of xylan and its potential effectiveness in lignocellulosic hydrolysis and other industrial application. It also suggests the potential of xylanase enzyme for production of XOS from biomass which are useful in food and pharmaceutical industries.


SDS-PAGE and zymogram analysis.
After ultra-filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) zymography was applied 20 . SDS-PAGE was carried out using 10% polyacrylamide in the gel, β-mercaptoethanolwas not added to the samples 21 . On completion of electrophoresis, the gel was cut in two parts. One part was used for Coomassie brilliant blue staining and the other was used for zymography.
Zymography was done according to Kumar et al. 22 . The gel was soaked inside 50 mM sodium phosphate buffer (pH 7.0) containing 25% isopropanol 25% for 30 min at 4 °C. Then, the gel was removed and placed in the same buffer containing 1.0% beechwood xylan substrate at 37 °C for 30 min. 0.1% Congo red and 1 M NaCl were used for staining and de-staining of the gel, respectively. Decolorized bands of the red background indicated xylanase activity.
Measurement of xylanase and protein activity. Xylanase activity was assayed using xylan from beech wood (Sigma-Aldrich, Egypt) as substrate. 0.95 mL of 1% (xylan) in 0.05 M citrate buffer, (pH 5) was incubated with 0.05 mL of diluted crude enzyme extract at 50 °C for 15 min. Then the reaction was stopped using 0.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent. The contents are boiled on water bath for 5 min. The absorbance was measured at 575 nm after cooling. The absorbance was compared to that of a substrate control1 23 . One international unit (IU) of xylanase activity is defined as the amount of enzyme catalyzing the release of 1 μmol/min of reducing sugar equivalent to xylose under the specified assay condition 24 . Using a protein assay kit (BioRad Laboratories, USA) with bovine serum albumin as the standard, the total protein concentration was evaluated 25  Analysis of xylo-oligosaccharides produced by xylanase enzyme hydrolysis of xylan. Fifty µL of xylanase was incubated with 1% w/v beechwood xylan in 0.05 M citrate buffer (pH 5) at 55 °C. Aliquots were withdrawn at intervals and boiled for 5 min. Control without enzyme was performed in parallel. Samples were analyzed by HPLC (HPLC, Shimadzu Class-VPV 5.03 (Kyoto, Japan) on an HPX-87H column (300 mm × 7.8 mm). The eluent was HPLC grade DI-water with a flow rate of 0.5 mL/min at 70 °C. Sugars were detected by a refractive index detector (Shodex, RI-101). Xylo-oligosaccharides (xylobiose, X2; xylotriose, X3; xylotetraose, X4; xylopentaose, X5; xylohexaose, X6) and xylose were used as standards for the analysis of the reaction products 27 .
Antioxidant activity of xylo-oligosaccharides. Using the scavenging effect of radicals on DPPH, antioxidant activity can be monitored. 1 mL of the beechwood hydrolysis products (xylo-oligosaccharides) was mixed with 1 mL of 0.04 mg/mL DPPH solution. The reaction was monitored after 15 min. Absorbance at 517 nm was used to calculate radical scavenging activity (% of inhibition) with the formula. Inhibition (%) = 1 -Ab sample − Ab blank /Ab control − Ab blank × 100.
where Ab sample was the absorbance of the reaction in presence of sample (sample + DPPH solution), Ab blank was the absorbance of the blank for each sample dilution (sample + DPPH solvent) and Ab control was the absorbance of control reaction (sample solvent + DPPH solution). Then, this value obtained for every concentration was plotted to obtain IC50 values in each time point 28 . Statistical analysis. All data were subjected to analysis of variance (ANOVA). Three samples of each item were analyzed and the main values as well as the SD were given. Significance of the variable mean differences was determined using Duncan's multiple range tests (p ≤ 0.05). All analyses were carried out using SPSS 16 software.
Ethical statements. The manuscript does not contain experiments using human study.

Results and discussion
Production and purification of xylanase enzyme. Table 1 shows that the activity of xylanase enzyme produced in stirred tank bioreactor was 760 U/mL. The enzyme was purified by precipitation using 70% saturated ammonium sulfate solution. The precipitated enzyme was concentrated by ultra-filtration (Amicon Ultra-3 kDa, Millipore). The purification protocol led to a 3.29-fold increase in purity with 34.47% xylanase yield (recovery). The specific enzyme activity of the purified enzyme fraction was 3968 U/mg protein. It is one of the highest specific activities ever reported for xylanase 29-32 . Characterization of xylanase. Determination of the molecular weight. Purified xylanase migrated as a single band on SDS-PAGE suggesting that the purified xylanase was a monomer consisting of a single polypep- www.nature.com/scientificreports/ tide chain. The molecular weight of the xylanase was 32 kDa. The activity of purified xylanase was confirmed through zymography, which showed decolorization of the red background at 32 kDa, which confirmed the enzyme activity at that band ( Fig. 1). According to (Wang et al.,) 33 , who reported that low molecular weight xylanase is at the range of 21-34 kDa, this xylanase is considered to low molecular weight for xylanases. The low molecular weight of our purified xylanase was identical to the enzyme reported from an Aspergillus mutant strain 34 , Bacillus subtilis 35 , and Trichoderma inhamatum 9 . A higher molecular weight xylanase was reported for Bacillus pumilus 36 , for Bacillus sp. GRE7 37 and for Paenibacillus campinasensis BL11 38 . For the pulp and paper industry, low molecular weight xylanases are favored as they can penetrate pulp fibers more easily than higher molecular weight 39 . Effect of temperature on xylanase activity and stability. The enzyme activity increased as temperature increased reaching a maximum activity at 60 °C, then activity declined at 70 and 80 °C, reaching 57% of its maximum activity at 80 °C (Fig. 2). The observed optimum temperature lies in the same range (50-60 °C) of most reported xylanases 35 .
Regarding thermal stability, 80% of enzyme activity was retained after incubation for 30 min at 60 °C. Only 68% and 55% of activity was retained after incubation for 30 min at 70 and 80 °C, respectively (Fig. 3). Our strain is relatively stable compared to xylanases from other Aspergillus species. Xylanase purified from A. phoenicis 40 and A. giganteus 41 exhibited half-lifes of only 25 and 13 min at 50 °C, respectively. Thermal stability is correlated to intermolecular bonds such as hydrogen and disulfide bonds and molecular interactions such as electrostatic and hydrophobic interactions. Studying these stabilizing factors is beneficial in re-engineering of mesophilic enzymes to more stable a enzymes 42 . Temperature stability is required in industrial applications, especially in biomass hydrolysis, which is carried out under high temperature 43 . Effect of pH on xylanase activity and stability. The pH range of 5 to 6 was suitable for enzyme activity with optimum at pH 5 ( Fig. 4) nearly similar to A. kawachii (pH 5.5) 44 , A. nidulans (pH 6.0) 45 and A. foetidus (pH 5.3) 3 . This result confirmed the suitability of P. variotii xylanase for use in juice manufacture in which acidic pH is favorable 46 .
Xylanase enzyme retained 100% activity at pH 5-6, while 87%, 75% and 62% of activity was retained when assayed at pH 7, 8 and 9 respectively, after 24 h incubation (Fig. 5). The pH stability of xylanase from Aspergillus were different it ranged from pH (2 to 7) in case of A. ochraceus 47 , (4.5 to 6) in case of A. niveus, pH (6.0 to 8.0) in case of A. fumigatus 48 and pH (7 to 9) in case of A. carneus M34 49 . The pulp bleaching process is usually      ., 2015). These metals may serve as a cofactor in the enzyme-substrate reaction 6,59,60 . Calcium also protects xylanase from proteinase inactivation and thermal unfolding 61 . The metal ions Co 2+ , Zn 2+ , K + , Fe 2+ , Fe 3+ reduced xylanase activity by 8%, 11%, 7%, 37%, and 48%, respectively, at concentrations of 5 mM. The inactivation of xylanase enzyme by the addition of salts of heavy metals such as Fe 2+ and Fe 3+ is well known. Yi et al. 62 reported that Fe 2+ inhibited activity of xylanases of Aspergillus sp. This inhibition may be due to nonspecific salt formation with the enzyme 36,63 . Metal ion interaction with SH or carboxyl groups will alter protein configuration 64 . Table 3 shows the effects of 5 mM solutions of different chemicals on the enzyme activity. β-mercaptoethanol, due to its reducing activity, enhanced enzyme activity by 55%. Moreira et al. 65 also reported enhancement of activity by β-mercaptoethanol for xylanase from A. terrus. This could be attributed to the prevention of oxidation of the thiol group in the enzyme 66,67 . Vieira Cardoso and Ferreira Filho 68 , also related the protection of cysteine residues from oxidation by mercaptoethanol and this    53 . This may be explained by the enhancement of xylanase activity by some metal ions for xylanase activity, as was observed in this study. Since EDTA is a metal chelator, decreased xylanase activity would be expected in the presence of EDTA 66 . SDS reduced activity by 77%. This large decrease in activity by SDS, which is an anionic surfactant, confirms the importance of hydrophobic interactions for stabilization of enzyme structure 69 . Hmida-Sayari et al. 57 also reported that SDS decreased activity of xylanase enzyme purified from A. niger by 80%.

Effect of chemicals on xylanase activity.
Substrate specificity. The greatest activity (5230 U/ml) was observed with beechwood xylan, which contains β-1,4 linkages between D-xylose residues 70 . The residual activity was 7.2% for pectin. This may be due to presence of xylan residues in pectin 71 . The enzyme was completely inactive with starch, avicel and carboxymethyl cellulose sodium salt as substrates ( Table 4). The same results were obtained by Chen et al., 72 who also observed that a xylanase enzyme produced from A. niger had no activity towards starch, Avicel and carboxymethyl cellulose. Bai et al., 73 also reported that xylanase from Bacillus was inactive towards CMC, pectin, and starch.It has been suggested that shape recognition of the polysaccharide chain conformation plays a role in polysaccharidase speci¢city. Xylanase recognize the threefold helical structure of xylan as substrate and didn't recognize the flat ribbon like conformation of cellulose 74 . Determination of kinetic parameters of xylanase. The hydrolytic activity of the purified xylanase was measured using beechwood xylan as a substrate at concentrations of 1, 2, 5, 10 and 20 mg/mL. The xylanase was observed to exhibit Michaelis-Menten Kinetics. The K m and V max values obtained from the Lineweaver-Burk plot for beechwood xylan were 3.33 mg/mL and 5555 U/mg respectively (Fig. 6). Xylanase produced in our study was shown to have low K m 3.33 mg/mL and very high V max 5555 U/mg compared to A. terrus, A. fumigatus, A. kawachii, Penicillium glabrum, Sorangium cellulosum and Saccharopolyspora pathumthaniensis which have K m (2.09, 3.12, 10, 3.1, 26.5 and 3.92) and V max (640,2587,1250,194,7.89 and 256) respectively [29][30][31][32]75 . Table 5 A low K m indicated high affinity to substrate while high V max indicate high enzyme activity. K m and V max values were in the range of reported literature (0.09 to 40.9 mg/L for K m ) and (0.106 to 10,000 U/mg for V max ), respectively.  Fig. 7. The XOS produced with xylan hydrolysis by xylanase secreted by P. variotti mainly were composed of xylobiose (X2), xylotriose (X3), and xylotetrose(X4), together with a small amount of xylopentaose (X5) and xylohexose (X6) and xylose (X1). The accumulation of XOS in the hydrolysate was about 0.8% X1, 14% X2, 27% X3, 23% X4, 18% X5 and 13% X6 after incubation for 0.5 h.The fast accumulation of X3 and X4 during the initial 0.5 h was possibly due to the preferential action of xylanase enzyme on xylan chain termini. The finding was consistent with Lin et al., 76 who stated that higher levels of X3 and X4 were found at the initial stage of hydrolysis reactions. After 3 h, X1 (9%), X2 (45%) and X3 (39%) contents had increased and the content of X4 (5%) decreased. While X5 and X6 disappeared. The disappearance of X5 and X6 was due to rapid hydrolysis of X5 and X6 into smaller oligosaccharides immediately after release. The same findings were obtained by Akpinar et al., 77 who stated that by increasing time of hydrolysis, concentration of XOS with a higher degree of polymerization (DP > 5) decreased. After 6 h, X1 (18%), X2 (56%) had increased, while X3 (30%) and X4 (2%) contents decreased. Since xylose concentration was less than the concentrations of XOS, especially xylotriose and xylobiose, it can be concluding that the purified xylanase was an endo-xylanase that randomly cleaves the internal glycosidic linkages in xylan as a substrate 78 .
In the present study, the XOS obtained were principally composed of xylobiose, xylotriose, and xylotetraose. Therefore, in biotechnological applications, xylanase may have potential applications, because these applications rely on its ability to solubilize hemicellulose rather than complete hydrolysis to xylose.
Antioxidant activity ofXylooligosaccharides. The DPPH radical-scavenging ability of XOS mixtures obtained from enzymatic hydrolysis of xylan using xylanase enzyme after 6 h of enzymatic hydrolysis was shown in Table 6. Xylo-oligosaccharides have a dose dependent antioxidant activity. The antioxidant activity increased with increasing concentration of XOS mixtures. Based on statistical analysis; the maximum scavenging percentage (70.57%) was observed using the concentration of 2 mg/mL (p < 0.05). moreover, the concentration of 0. 1 mg/mL gave the lowest scavenging potency with an average of 15.22% (P < 0.05).The same results were obtained by Bian et al., 79 who stated that increasing XOS concentration, increased antioxidant activity. IC 50 , of XOS mixture obtained after 6 h enzymatic hydrolysis using xylanase enzymes was 0.7154 mg/mL. XOS has a branched structure comprising b-(1,4) bonded 2-7 xylose units and a number of substituents such as acetyl groups, uronic acids and arabinose units 80 . Structure of XOS vary in degree of polymerization (DP), monomeric units, and types of linkages depending on the source of xylan used for XOS production 81 . In addition to xylose residues, xylan is usually found in combination with other side groups such as α-D-glucopyranosyl uronic acid or its 4-O-methyl derivative, acetyl groups, or arabinofuranosyl residues 82 . Rao and Muralikrishna 83 demonstrated that the existence of uronyl or acetyl sugars imparts strong antioxidant activity to polysaccharides. Carboxyl groups have also been reported to enhance the antioxidant function of polysaccharides 84 . The antioxidant mechanism may be also due to the supply of hydrogen by polysaccharides, which combines with radicals and forms a stable radical to terminate the radical chain reaction. The other possibility is that polysaccharides can combine with the radical ions which are necessary for radical chain reaction; then the reaction is terminated 85 . These findings showed that beech xylan XOS has outstanding radical-scavenging activity of DPPH and can be useful against oxidative damage.

Conclusion
Overall, Paecilomyces variotii strain produced low molecular weight thermophilic alkalophilic endoxylanase enzyme. It exhibited high stability at relatively high temperature and alkaline pH. It has a high affinity for xylan, very high V max and one of the highest specific activities ever reported for xylanase. The properties of the purified xylanase make it an ideal candidate in hydrolysis of lignocellulosic materials and other industries. It also suggests the potential of xylanase enzyme for production of XOS with potent antioxidant activities which are useful in food and pharmaceutical industries.