Acacia Xylan as a Substitute for Commercially Available Xylan and Its Application in the Production of Xylooligosaccharides.

Over the past two decades, birchwood and beechwood xylans have been used as a popular substrate for the characterization of xylanases. Recently, major companies have discontinued their commercial production. Therefore, there is a need to find an alternative to these substrates. Xylan extraction from Acacia sawdust resulted in 23.5% (w/w) yield. The extracted xylan is composed of xylose and glucuronic acid residues in a molar ratio of 6:1 with a molecular mass of ∼70 kDa. The specific optical rotation analysis of extracted xylan displayed that it is composed of the d-form of xylose and glucuronic acid monomeric sugars. The nuclear magnetic resonance analysis of extracted xylan revealed that the xylan backbone is substituted with 4-O-methyl glucuronic acid at the O2 position. Fourier transform infrared analysis confirmed the absence of lignin contamination in the extracted xylan. Xylanase from Clostridium thermocellum displayed the enzyme activity of 1761 U/mg against extracted xylan, and the corresponding activity against beechwood xylan was 1556 U/mg, which confirmed that the extracted xylan could be used as an alternative substrate for the characterization of xylanases.

Introduction

Lignocellulosic biomass (LCB) is one of the most abundant and economically viable feedstocks having high sugar content that can serve as a sustainable raw material supply for the production of chemicals, fuels, and biopolymers for the fulfillment of the needs of modern industrial societies. LCB is composed of 25–55% cellulose, 25–40% hemicellulose, and 10–35% lignin component.[1] Hemicelluloses are the second most abundant and underutilized biopolymers.[2] Hemicelluloses are chemically heterogeneous in nature, with a low degree of polymerization in the range of 80–200. Unlike cellulose, the hemicellulose is an amorphous polymer, which includes xylan, arabinoxylan, glucuronoxylan, glucomannan, galactomannan, and xyloglucan. The hardwood contains xylans, and softwood contains glucomannans as the predominant hemicellulose.[3] The most predominant polysaccharide present in hemicellulose is xylan, which accounts for 10–35% content of total dry weight in hardwoods and 10–15% in softwoods content of total dry weight.[4] Xylans are heterogeneous and branched in nature, which are structurally composed of β-d-1,4-linked xylopyranosyl residues. Based on the xylan source and extraction process, their chemical composition and structure may vary and found to be substituted at O2 or O3 positions with 4-O-methyl-α-d-glucuronic acid, α-d-glucuronic acid, and some neutral sugar units such as α-l-arabinose, α-d-galactose, and α-d-xylose. Different functional groups such as acetyl groups, phenolic acids, and ferulic and coumaric acids can also be found as substitution.[5,6]

The hardwoods such as beechwood, birchwood, and aspen wood mostly contains 4-O-methyl glucuronoxylan, which is composed of β-1-4-d-xylose residues in the main chain and substituted with 4-O-methyl-α-d-glucuronic acid at the O2 position by an α-1-2 linkage.[7] However, the cereal plants such as wheat, rye, and barley mainly contain arabinoxylan (AX), while sorghum, rice, and maize contains glucurono(arabino)xylan (GAX) as a structural component.[8] The arabinoxylan contains linear chains of β-1-4-d-xylose residues and substituted with l-arabinose at O2, O3, or both positions through α-1-2, α-1-3, and α-1-5 linkages.[8] The GAX is more complex in structure and contains the substitution of glucuronic acid at the O2 position of the xylan backbone and one additional substitution of l-arabinose at the O3 position along with the substitution at the O2 position.[9]

Xylans have been used in various industrial applications such as the food industry, feed industry, pharmaceutical industry, and packaging materials.[10] The degradation of xylans by various xylanolytic enzymes play a crucial role in several bioprocesses such as biofuel production, paper and pulp bleaching, digestion of animal feed, and xylooligosaccharide production. These xylanolytic enzymes, namely, endo-β-xylanase, glucuronoxylan hydrolase, arabinofuranosidase, etc., need to be fully characterized by using different xylan substrates before applying to the bioprocess industries mentioned above. Xylans with different structures and compositions such as beechwood xylan, birchwood xylan, 4-O-methyl glucuronoxylan from beechwood, and arabinoxylans from wheat and rye are the major commercially available substrates for the biochemical characterization of xylanolytic enzymes. These polysaccharides are supplied by several chemical companies such as Sigma chemical company, USA, Megazyme Ltd., Ireland, and Carbosynth, UK. However, the demands of efficient and highly active xylanolytic enzymes are increasing day by day, but in the last few years, the xylans from larchwood, beechwood, and birchwood have become commercially unavailable with major suppliers. The strong interaction between lignin networks of xylan and extensive hydrogen bonding between xylan and cellulose make the process of xylan extraction from beechwood more tedious, giving very low yield, which subsequently enhances the cost of production.[11] Currently, 4-O-methyl glucuronoxylan available in the market is only from beechwood, which is sold by Sigma Chemical Company, USA, Megazyme Ltd., Ireland, and Carbosynth, UK at high prices. Similar to the linear xylan from beechwood or birchwood, the availability of 4-O-methyl glucuronoxylan may also vary over the time owing to the difficulty of its extraction process and its economic viability. Therefore, there is a need to find an alternative to these commercially unavailable substrates with similar chemical composition and structure. Xylans have been extracted and characterized from different sources such as from rye straw,[12] wheat bran,[13] cotton stalk,[14] corn,[15] and sugarcane.[16] Arabinoxylans from rye and wheat are in use as a commercial substrate owing to their simpler structure. However, the xylan from corn contains glucuronic acid and arabinose substitutions, which makes it of a highly complex structure and thus restricts its use as a commercial substrate.

Acacia nilotica, commonly known as babool, is one of the broadly distributed plants in the northern states of India. Babool has been widely used in various applications such as in the healthcare industry, in the timber industry for the manufacturing of furniture, and as packing materials in the transport industry; therefore, it is a multipurpose plant. The chemical composition analysis of babool as earlier reported by Han[17] displayed 41.99% glucose, 15.46% xylose, 1.72% mannose, 1.37% arabinose, and 0.49% galactose. Similarly, Pinto et al.[18] also reported that Acacia contains ∼12% xylose, 0.2% arabinose, 1.0% mannose, 0.6% galactose, and 7% uronic acid. The chemical composition analysis of Acacia mangium wood revealed that it contains 85.99% holocellulose content followed by 36.15% hemicellulose content.[19] Similarly, the composition analysis of Acacia nilotica displayed 43.90% α-cellulose, 17.70% hemicellulose, and 18.36% lignin content.[20] These studies suggested that the chemical composition of Acacia varies from species to species and geographic location.

In the furniture industry during the furniture manufacturing process, a large amount of the Acacia sawdust is produced. Therefore, in the present study, the Acacia sawdust is available as waste, and based on its relative abundance, it was explored for the chemical composition analysis followed by xylan extraction. The extracted xylan was characterized for its chemical composition, structure, and thermal stability. The enzyme activity of xylanases from different families was evaluated against extracted xylan and compared with commercial xylans for confirming its potential as a substitute for commercial xylans. In recent times, people across the globe have suffered from several gut-related diseases, and these can be overcome by using food supplements.[10] The xylooligosacharides possess a great potential as a food supplement due to their utilization as prebiotics and antioxidative agents. Owing to utilization of XOS as a food supplement, their global market is expected to reach USD 120 million by 2022.[10] Therefore, the extracted xylan was used for the production of enzyme-mediated xylooligosaccharides.

Results and Discussion

Extraction, Composition, and Molecular Mass Analysis of Extracted Xylan

The estimated total moisture content of Acacia sawdust was found to be 5.4 ± 0.2%. The Acacia sawdust contains 77.3 ± 1.2% holocellulose, 29.57 ± 0.25% hemicellulose, and 25.13 ± 0.42% total lignin content. The delignification process gave 43.5 ± 0.83 g of delignified Acacia sawdust. The xylan extraction process yielded 5.4 g of xylan from 20 g of delignified Acacia sawdust, resulting in 23.5% (w/w) recovery yield (based on initial extractive-free sawdust) after lyophilization (Figure A). The extraction process of xylan from Acacia sawdust gave very high yield compared to those reported earlier. The hot-water-mediated xylan extraction from delignified beechwood resulted in a xylan yield of 13.4% (w/w) of initial extractive-free wood.[11] Alkaline peroxide-assisted arabinoxylan extraction from rye straw resulted in the final yield of 21.8% (w/w).[12] A total of 14% (w/w) glucuronoxylan (total wood weight) was extracted from Acacia mangium wood.[18] The DMSO-assisted xylan extraction from bleached corn stover gave 8.7% yield.[21] The alkali extraction of xylan gave the yield of 12.3% (w/w) from bambara and 13.6% (w/w) from cowpea biomass.[22] The composition analysis by HPLC analysis of TFA-hydrolyzed extracted xylan displayed the presence of glucuronic acid and xylose with a retention time of 12.70 and 14.95 min, respectively (Figure B). The retention time of glucuronic acid and xylose sugar matches well with the retention time of glucuronic acid (Figure B) and xylose (Figure B) standards. The presence of xylose and glucuronic acid content was determined using the standard sugar concentration and found to be 85% (w/w) xylose and 15% (w/w) glucuronic acid units. The presence of xylose units in a huge amount confirmed that its main chain is composed of d-xylose monomers linked by the xylosidic bonds and glucuronic acid as a side chain substitution.

Figure 1

(A) Digital image of extracted xylan from Acacia, (B) HPLC profile of TFA-hydrolyzed Acacia xylan, (C) HP-SEC profile of extracted xylan, (D) Hendricks plot analysis of a dextran standard, and (E) DLS profile of extracted xylan.

The majority of the hardwood xylans are substituted with 4-O-methyl-α-d-glucuronic acid residue (MeGlcA) at the O2 position of the xylan backbone and are termed as 4-O-methyl-α-glucurono-d-xylan.[9] The ratio of Xyl/4-O-MeGlcA in 4-O-methyl-α-glucurono-d-xylan varies from 4:1 to 16:1, depending on the source and extraction conditions with an average ratio of about 10:1.[9] In the present study, the molar ratio of Xyl/4-O-MeGlcA was 6:1, which is similar to the results reported earlier.[23,24] The HP-SEC analysis of extracted xylan displayed a single peak (Figure C) with a molecular mass of ∼70 kDa (Figure D). The molecular mass of extracted xylan from Acacia sawdust is similar to glucuronoxylan isolated from the apical portion of Neolamarckia cadamba, which is 70.8 kDa,[25] from neem sawdust is 65 kDa (unpublished results), from the woody part of eucalyptus is 56.60 kDa,[26] and from Cassia obtusifolia seeds is 49 kDa.[27] Several other studies reported the lower molecular mass of extracted xylan, 20.84 kDa from beechwood[28] and 15 kDa from viscose fiber.[24] The DLS analysis of the extracted xylan confirmed that the isolated polysaccharide is monodispersed in nature with a hydrodynamic diameter of 311 nm (Figure E). The monodispersed state obtained from DLS analysis corresponded with a single peak obtained by mass by HP-SEC analysis and further confirms that the extracted polysaccharide is pure and devoid of any contamination.

Specific Optical Rotation and Elemental Composition Analysis of Extracted Xylan

The estimated specific optical rotations of extracted xylan from Acacia sawdust was −72.65°. The commercial xylan, namely, beechwood xylan and xylan from Carbosynth, displayed the specific optical rotation of −68.40 and −70.36°, respectively. The specific optical rotation of extracted xylan matches well with the commercial xylan and confirms that the extracted xylan is composed of α-linked d-glucuronic acid and β-linked d-xylose monosugar. Similar results for different xylans extracted from wood were reported by Gutmann and Timell.[29] The elemental composition analysis of extracted xylan showed that it contains 36.70% carbon content and 3.92% hydrogen content, while the nitrogen and sulfur content are absent in the extracted xylan, confirming that the extracted xylan is highly pure.

FTIR Analysis of Extracted Xylan

The FTIR spectrum of extracted xylan displayed a distinct hydroxyl (OH) group stretching vibration peak at 3436 cm–1 (Figure A), which is similar to the peak obtained by OH stretching vibration for other extracted xylans.[21,30] The small peak at 2929 cm–1 in extracted xylan revealed symmetric CH stretching, which corroborates earlier reports.[31] The peak at 1643 cm–1 indicated water absorption and matched well with earlier reports.[16,24] The symmetric and asymmetric stretching mode of the carboxyl group at 1415 and 1572 cm–1 revealed the presence of the glucuronic acid component and corroborated previous reports.[32−34] The β-glycosidic linkage absorption peak is at 897 cm–1 and considered as the anomeric region.[24,35] The FTIR spectrum region between 1200 and 1000 cm–1 was dominated by the ring vibration overlapped by the C–O–C glycosidic vibrations and the stretching vibration of the OH side group. The peak at 1049 cm–1 represented the β-1,4 backbone of extracted xylan as reported earlier.[35]

Figure 2

(A) Fourier transform infrared (FTIR) spectroscopic analysis of extracted xylan, (B) 1H NMR (600 MHz) analysis , (C) 13C NMR (600 MHz) analysis, and (D) 2D HSQC NMR analysis of extracted xylan.

NMR Analysis of Extracted Xylan

The structure of extracted xylan from Acacia sawdust was determined by recording the 1H (Figure B), 13C (Figure C) and two-dimensional (2D) total correlation spectroscopy (TOCSY)-heteronuclear single quantum coherence (HSQC) (Figure D) NMR spectra at 600 MHz (Table ).

Table 1

NMR Analysis of Xylan Extracted from Acacia Sawdust

	chemical shift (ppm)
linkages	1	2	3	4	5eq	5ax	–OCH3
→4)-β-d-Xylp-(1→
1H	4.33	3.15	3.42	3.64	3.95	3.22
13C	101.67	72.99	72.18	76.23	62.67	62.67
→4)-β-d-Xylp-2-O-GlcpA-(1→
1H	4.47	3.41	3.61	3.69	3.98	3.29
13C	101.23	76.05	71.86	76.66	62.8	62.8
4-O-Me-α-d-GlcpA-(→
1H	5.13	3.43	3.64	3.34	4.34/4.37		3.33
13C	97.44	71.86	72.99	71.16	73.15		59.77

The characteristic signals of the chemical shift were assigned by combining the data obtained from 1H, 13C, and 2D TOCSY-HSQC NMR spectra analysis. 1H NMR spectrum analysis of extracted xylan confirmed the presence of an α-anomeric proton at 5.14 ppm for 4-O-methyl glucuronic acid (G) and β-anomeric protons at 4.33 for xylose (X) and 4.53 for 4-O-methyl glucuronic acid-linked xylose residue (XG). The chemical shifts for X, XG, and G obtained from 1H NMR analysis are consistent with previous studies.[16,27,36] The integration analysis of the 1H NMR spectrum revealed that the ratio of xylose/4-O-methyl glucuronic acid is 6:1. The corresponding anomeric carbon atoms identified from TOCSY-HSQC analysis were found at δ 101.58, 101.31, and 97.44 for X, XG, and G, respectively. The chemical shifts at 101.58 and 4.33 identified from the TOCSY-HSQC spectrum revealed the β(1→4) linkage of the non-substituted d-xylopyranosyl (X) units. The chemical shifts at δ 3.95 (H5eq), 3.64 (H4), 3.42 (H3), 3.22 (H5ax), and 3.15 ppm (H2) correspond with the cross chemical shifts of non-substituted xylose carbon atoms obtained from TOCSY-HSQC at 62.67 (C5eq), 76.21 (C4), 72.18 (C3), 62.67 (C5ax), and 72.99 (C2), respectively. The results obtained from 1H and 13C NMR analysis are in close agreement with earlier reports[16,27,36] and further confirm the β(1→4) linkage of residue X. The anomeric carbon chemical and proton shifts of residue 4-O-methyl glucuronic acid linked xylose (XG) were at δ 101.23 and 4.47, respectively, and also revealed the β-linkage between X and XG (Figure B). The chemical shifts of XG carbon atoms at δ 76.05, 71.86, 76.66, and 62.8 corresponded to C2, C3, C4, and C5 of glucuronic acid-substituted xylose (→4)-β-D-Xylp-2-O-GlcpA-(1→), respectively. The downfield intensity signal at δ 76.05 for the C2 position of residue XG as compared with the signal of the C2 position of X residue indicated the substitution of the XG residue. The corresponding proton NMR peaks of XG residues are at 3.41 (H2), 3.61 (H3), 3.69 (H4), 3.98 (H5eq), and 3.29 (H5ax) assigned to the β-d-xylopyranosyl units substituted with 4-O-methyl glucuronic acid at the O2 position, and these chemical shift signals are well consistent with the earlier reports.[27,37] Similarly, G residue showed the chemical shifts for anomeric carbon and protons at δ 97.44 (C1) and 5.14 (H1), respectively, indicating the α-linkage of glucuronic acid with the xylan main chain. The chemical shifts of other carbon at δ 71.86, 72.99, 71.15, 73.15, and 176.76 corresponded to C2, C3, C4, C5, and C6 of α-GlcpA-(1→, respectively. The chemical shift at δ 59.77, corresponding to the methoxy group (−OCH3), confirmed the methylation of glucuronopyranosyl residues at the O4 position. The chemical shift peaks of G matched well with the earlier report of NMR analysis of xylan from palm of Phoenix dactyliferaL.[38] The cross chemical shift peaks of protons present in glucuronopyranosyl residues were identified and designated at 3.43 (H2), 3.64 (H3), 3.34 (H4), 4.32/4.34 (H5), and 3.33(−OCH3). The NMR results of extracted xylan from Acacia sawdust corroborated the previous reports of xylan extraction from sugarcane bagasse,[16]Cassia obtusifolia seeds,[27] and pericarp seeds of Opuntia ficus-indica prickly pear fruits.[36]

FE-SEM and TG Analysis of Extracted Xylan

The surface and morphological properties of extracted xylan powder from Acacia sawdust by FE-SEM analysis revealed that the surface of isolated polysaccharides is rough and highly porous in nature. The roughness and highly porous nature of the surface may be due to the stacking or arrangement of irregular granules in a layer form (Figure ). In a previous study, the xylan extracted from corncob,[15] pineapple peel waste,[31] and from rice bran and finger millet[39] displayed a similar rough and irregular surface morphology, further confirming the universal characteristic of xylan polysaccharides.

Figure 3

Field emission scanning electron micrograph (FE-SEM) analysis of extracted xylan.

The thermal stability and thermal decomposition properties of extracted xylan from Acacia sawdust were studied by differential thermogravimetric analysis (DTG) and thermogravimetric analysis (TGA). The TGA of extracted xylan from Acacia displayed the weight loss resulted from the formation of ash content by pyrolysis with an increase in temperature (Figure ). The decomposition of the extracted xylan occurred in three stages at different temperatures.

Figure 4

Thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTG) displaying thermal degradation temperature (Td) of 266 °C.

The first stage of decomposition displayed the initial 8% weight loss between the range of 65 and 160 °C, and it corresponds to the loss of moisture content or water content present in the extracted xylan (Figure B), and the obtained results are similar to earlier reports.[40,41] The second stages of sudden weight loss started at 170 °C, and 40% weight loss was observed at 266 °C by fragmentation of the extracted xylan into smaller molecules like CH4, CO, CO2, CH3COOH, and HCOOH and further decomposition as suggested by Bian et al.,[16] Zhao et al.,[25] and Deumaga et al.[41] The third stage of decomposition revealed that the extracted xylan converted into ash by the pyrolysis process and retained 35% of initial weight. DTGmax analysis displayed the maximum thermal degradation (Td) point at a particular temperature, which can be used for analyzing the stability of the sample. The DTG curve of extracted xylan from Acacia displayed a single sharp peak at 266 °C, which confirms that the sample is highly pure and has less substitution in the main chain, and the peak corresponded to the DTGmax or Td.

Evaluation of Extracted Xylan as a Commercial Substrate by Estimating the Activity of Different endo-β-Xylanases

Commercial applicability of extracted xylan from Acacia sawdust as a replacement of beechwood xylan and birchwood xylan was determined by estimating the activity of different endo-β-xylanases. All three enzymes, namely, PsGH10A, CtXyn11A, and CtXynGH30, displayed activity and are reported in Table . The specific activities of PsGH10A, CtXyn11A, and CtXynGH30 on extracted xylan were approximately 12% higher than the beechwood xylan. These activity results confirmed that the extracted xylan could be used as a substrate for the screening and biochemical characterization of xylanases from different glycoside hydrolase families.

Table 2

Enzyme Activity Analysis of Different Xylanases on Extracted Xylan and Commercial Xylans

	enzyme activity (U/mg)
enzyme	beechwood xylan	birchwood xylan	xylan from Acacia
PsGH10A	56.5 ± 1.8	35.2 ± 1.4	61.9 ± 2.1
CtXyn11A	1556.6 ± 31.5	1335.3 ± 25.9	1761.4 ± 36.7
CtXynGH30	32.1 ± 0.9	29.5 ± 1.1	37.6 ± 1.8

Xylanase-Mediated Production and Identification of Xylooligosaccharides

The hydrolytic activity of CtXyn11A against the extracted xylan from Acacia sawdust was found to be higher than the other enzymes and commercial substrates. Therefore, the extracted xylan can be used as a substitute for the commercially available substrate, such as birchwood xylan and beechwood xylan. CtXyn11A was further used for the production of xylooligosaccharides. The total yield of xylooligosaccharides obtained from the hydrolysis of 10 mL of 1% (w/v) extracted xylan was found to be 46 ± 1.8 mg determined by phenol sulfuric acid analysis. The CtXyn11A-mediated hydrolyzed product was analyzed by thin-layer chromatography (TLC) analysis. The TLC analysis displayed that the CtXyn11A forms the xylooligosaccharides with various degrees of polymerization, ranging from DP2 to DP7 from extracted xylan hydrolysis (Figure A).

Figure 5

Analysis of CtXyn11A-hydrolyzed products of extracted xylan from Acacia sawdust by (A) thin-layer chromatography and (B) mass spectrometry.

The hydrolysis of 4-O-methyl glucuronoxylan by CtXynGH30 and PsGH10A resulted in the release of similar types of xylooligosaccharides including both linear and glucuronoxylooligosaccharides.[42,43] The TLC analysis of hydrolyzed products further revealed that it contains higher xylooligosaccharides, which were not well resolved. Therefore, the mixture of XOS was further analyzed by mass spectrometry to obtain precise information about the degree of polymerization and the presence of any substitution. The electrospray ionization-mass spectrometry analysis of CtXyn11A-hydrolyzed products containing the XOS mixture revealed that it contains a series of linear xylooligosaccharides ranging from DP2 to DP8 and higher species of 4-O-methyl glucuronic acid-substituted xylooligosaccharides. The mixture included xylobiose (m/z, 305.01), xylotriose (m/z, 437.0), xylotetraose (m/z, 569.0), xylopentaose (m/z, 701.0), xylohexaose (m/z, 833.0), xyloheptaose (m/z, 965.0), and xylooctaose (m/z, 1097.0). The XOS mixture also contained 4-O-methylglucuronoxylotriose (m/z, 627.0), 4-O-methylglucuronoxylotetraose (m/z, 759.0), and 4-O-methylglucuronoxylopentaose (m/z, 891.0) (Figure B). The molecular mass of neutral and acidic xylooligosaccharides matches well with the earlier studies.[44,45] The matrix-assisted laser desorption/ionization time-of-flight analysis of xylooligosaccharides produced by mild acid-mediated hydrolysis of extracted glucuronoxylan from olive pulp and olive seed hulls displayed the production of series of neutral and acidic xyloligosaccharides.[44] Similarly, in another study, the acetylated glucuronoxylan was hydrolyzed by GH10-xylanase and acetyl xylan esterase enzymes to determine the mode of action. The resulting hydrolyzed product mixture contained a variety of xylooligosaccharides, which include linear xylooligosaccharides, methylated glucronoxylooligosaccharides, and acetylated oligosaccharides.[45] The enzyme-assisted production of xylooligosaccharides and their various applications such as food supplements,[46] antioxidative agents,[47] and in healthcare as antiproliferative agents[48] has increased in recent times. Therefore, the extracted xylan, apart from its role as a substitute for commercial substrates, can also be used for the enzyme-assisted production of xylooligosaccharides for the abovementioned applications.

Conclusions

This study demonstrated the alkali-assisted extraction of xylan from waste biomass, i.e., Acacia sawdust, and its utilization as an alternative to the commercial substrate. Carbohydrate composition and NMR-based structural analysis displayed that the extracted xylan is composed of d-xylose and substituted with 4-O-methyl-d-glucuronoxylan. The endo-β-xylanases displayed a higher activity on extracted xylan than the commercial substrate, beechwood xylan. The CtXyn11A-mediated hydrolysis of the extracted xylan produces ∼47 mg of xylooligosaccharides, ranging from DP2 to DP8. These results demonstrate that the extracted xylan can be used as an alternative to the commercial substrate for xylanase characterization and for xylooligosaccharide production.

Materials and Methods

Materials

Acacia sawdust was procured from the wood cutting workshop of the local market. Glacial acetic acid, ethanol, hydrochloric acid, trifluoroacetic acid, and D2O were procured from Merck India Pvt. Ltd. The chemicals sodium hydroxide, sodium chlorite, and boric acid and dialysis bags were purchased from Himedia Pvt. Ltd., India. Beechwood xylan, birchwood xylan, xylose, and glucuronic acid were procured from Sigma Chemical Company, USA.

Extraction of Xylan from Acacia Sawdust

Acacia sawdust (1 g) was kept in a hot-air oven at 105 °C for 24 h, and thereafter, the residual sawdust was weighed to determine the moisture content. The holocellulose, hemicellulose, and lignin content present in acacia sawdust was estimated by following the standard methods described earlier by Browning and TAPPI.[49,50] The xylan extraction from Acacia sawdust was performed by following the protocol described earlier with slight modification.[51] The moisture content of Acacia sawdust was removed by keeping it at 80 °C for 24 h. Then, the lignin content was removed by incubating 50 g of dried Acacia sawdust with 500 mL of solution containing 60 g of sodium chlorite solution and 2.5 mL of glacial acetic acid at 60 °C in the water bath for 3 h. The delignified sawdust was filtered using a muslin cloth and washed with deionized water and dried in a hot-air oven at 80 °C for 24 h. After that, the xylan was extracted by soaking 20 g of delignified Acacia sawdust in 200 mL of an alkaline solution of 1% (w/v) boric acid containing 10% (w/v) sodium hydroxide filled in a 1 L conical flask, and the flask was kept in stirring conditions at 2000 rpm and 60 °C for 3 h. The residual biomass was removed by filtering the suspension through the muslin cloth. The xylan from the filtrate was completely precipitated by the addition of 3 volumes of 600 mL of ice-cold ethanol (95% v/v) and 10% (v/v) acetic acid and incubated overnight at 4 °C. The precipitant was further washed 2 times with ethanol to remove the reducing sugar and impurities, and then, the xylan was recovered by centrifugation at 10000g for 10 min. Other reagents and the salt traces from the precipitated xylan were removed by dialyzing against 5 L of Milli-Q water by changing the water three times. The dialyzed xylan was lyophilized for further analysis and characterization.

Carbohydrate Composition Analysis of Extracted Xylan from Acacia Sawdust

The monosugars present in the extracted xylan from Acacia sawdust were determined by trifluoroacetic acid (TFA)-mediated hydrolysis in a boiling water bath for 3 h, as reported earlier.[52] The residual TFA was evaporated from the hydrolyzed xylan sample by keeping the sample in a hot-air oven at 80 °C for 16 h. The dried hydrolyzed sample was dissolved in 500 μL of degassed Milli-Q water and then filtered through the PVDF membrane (0.22 μm pore size) using a syringe filter for the removal of any unhydrolyzed xylan part of the sample. The filtrate was subjected to HPLC analysis using a high-performance liquid chromatography (HPLC) instrument (UFLC, Prominence, Shimadzu, Japan) equipped with an RI detector for the determination of monosaccharide composition. The hydrolyzed sample (10 μL) was loaded on a Phenomenex Rezex ROA (H+) (specification, 300 mm × 7.8 mm) connected with a guard column (specification 50 mm × 7.8 mm), and the sample was eluted using 0.005 N H2SO4 as the mobile phase at a flow rate of 0.5 mL/min. The concentration of the monosaccharide sugars, i.e., xylose and glucuronic acid, was estimated using their commercial standards.

Average Molecular Mass Analysis of Extracted Xylan

The high-performance size exclusion chromatography (HP-SEC) analysis of extracted xylan from Acacia sawdust was performed for the determination of average molecular mass. Extracted xylan (10 μL, 1 mg/mL) and dextran standard molecular weight markers, i.e., 10, 20, 40, 70, 100, 200, 270, and 500 kDa, at a concentration of 1 mg/mL were loaded on a Phenomenex Polysep-GFC-P6000 column coupled with a guard column (Phenomenex Polysep-GFC-P) connected to an HPLC system equipped with an RI detector. The loaded samples were eluted at a flow rate of 0.5 mL/min using a 100 mM NaNO3 solution as a mobile phase. The molecular mass of extracted xylan was calculated by a Hendricks plot using dextran standards.

Particle Size Determination of Extracted Xylan

The hydrodynamic radius or particle size distribution of extracted xylan from Acacia sawdust was estimated by using a dynamic light scattering (DLS) instrument (Litesizer 500, Anton Paar, GmbH, Austria). The 0.1% w/v extracted xylan sample was dissolved in degassed Milli-Q water by heating at 70 °C and then filtered using a 0.45 μm PVDF membrane on a syringe filter. The temperature of the DLS system was maintained at 25 °C by a Peltier temperature controller. A total of 30 numbers of scans were recorded, and the average was taken by using Kalliope software for the particle size analysis.

Specific Optical Rotation and Elemental Composition Analysis of Extracted Xylan

The 0.5% (w/v) extracted xylan from Acacia sawdust and commercial xylans, namely, beechwood xylan and xylan from Carbosynth, were dissolved separately in 2 mL of 50 mM NaOH solution by heating at 60 °C. The residual undissolved polysaccharide was removed by centrifuging the sample at 13000g for 15 min, and thereafter, the clear sample was transferred into a polariscope tube. The specific optical rotation of the extracted xylan and commercially available xylans was recorded by a digital polarimeter (MCP150, Anton Paar, GmbH, Austria) at 25 °C maintained by the Peltier-based temperature controller. The elemental composition analysis of extracted xylan from Acacia sawdust was performed to estimate the purity by determining the carbon, hydrogen, nitrogen, and sulfur (CHNS) content by a CHNS analyzer (EuroEA3000 elemental analyzer, EuroVector, Italy).

Fourier Transform Infrared (FTIR) Spectroscopic Analysis of Extracted Xylan

The functional groups present in extracted xylan of Acacia sawdust were determined by FTIR spectroscopy. Approximately 1 mg of extracted xylan and 100 mg of KBr in a ratio of 1:100 were mixed using the mortar and pestle and converted into a fine powder, and then, the thin pellet of the xylan–KBr mixture was prepared using a 15 ton hydraulic press. The FTIR spectra for extracted xylan was recorded using the FTIR spectrophotometer (Perkin Elmer, Spectrum Two, USA) in transmission mode from 4000–400 cm–1 with a spectral resolution of 4 cm–1.

NMR Analysis of Extracted Xylan from Acacia Sawdust

Extracted xylan (15 mg) from Acacia sawdust was dissolved in 600 μL of D2O (99.96%) (Merck, Germany). 1H, 13C, and 2D TOCSY-HSQC high-resolution NMR spectra were recorded at 25 °C using a 600 MHz nuclear magnetic resonance (NMR) spectrometer (Bruker, ASCEND 600, Karlsruhe, Germany) fitted with a 5 mm probe. 1H, 13C, and 2D TOCSY-HSQC spectra were processed and analyzed by using TopSpin v4.07 NMR software (Bruker, Karlsruhe, Germany).

FE-SEM Analysis of Extracted Xylan

Surface properties of extracted xylan from Acacia sawdust were analyzed using a field emission scanning electron microscope (FE-SEM, Carl Zeiss, Model Gemini 300, Germany). Approximately 200 μg of extracted xylan powder from Acacia sawdust was placed and uniformly distributed on carbon tape adhered to the stub surface, and then, gold coating was performed. The gold-coated sample was placed in a vacuum chamber to remove the moisture content before surface imaging by FE-SEM.

Thermogravimetric (TG) Analysis

The extracted xylan was subjected to TGA to evaluate the thermal degradation properties (thermogravimetric and differential thermal properties) by measuring the change in enthalpy and weight loss using a thermal analyzer (Netzsch, model STA449F3A00). Approximately 6 mg of extracted xylan was filled in an alumina crucible, and the sample was heated in the range of 25–500 °C with a heating rate of 10 °C min–1, and the apparatus was continuously flushed with argon at a flow rate of 60 mL/min at the atmospheric pressure.

Evaluation of Extracted Xylan as a Commercial Substrate by Estimating the Activity of Different endo-β-Xylanases

The potential of extracted xylan as a substitute for commercially available xylans was evaluated by estimating the enzyme activity of endo-β-1,4-xylanases with different substrate specificities of different families, namely, family 10, 11, and 30 glycoside hydrolases. endo-β-Xylanases from Pseudopedobacter saltans (PsGH10A)[43] and from Clostridium thermocellum (CtXyn11A and CtXynGH30)[42,53,54] were expressed and purified by following earlier reported protocols. The hydrolytic activities of PsGH10A, CtXyn11A, and CtXynGH30 enzymes were determined using their optimum conditions. The specific activities of PsGH10A, CtXyn11A, and CtXynGH30 enzymes were determined by estimating the reducing sugar released by following the method of Nelson[55] and Somogyi.[56]

endo-β-1,4-Xylanase from Clostridium thermocellum (CtXyn11A) was expressed and purified by following an earlier reported protocol.[53] Extracted xylan (1% w/v) from Acacia sawdust was dissolved in 10 mL of 50 mM sodium phosphate buffer, pH 7.5, and mixed with 5 μg of CtXyn11A. Then, the reaction mixture mentioned above was incubated at 65 °C for 12 h. Thereafter, 3 volumes of ice-cold 95% (v/v) ethanol were added to terminate the reaction, and the residual unhydrolyzed extracted xylan was separated by centrifugation at 10000g for 5 min. The ethanol content was evaporated by keeping the ethanol–xylooligosaccharide mixture at 80 °C for 16 h. The dried product was dissolved in 200 μL of deionized water, and the xylooligosaccharide yield (total carbohydrate content) was determined using the phenol-sulfuric acid method.[57] The qualitative estimation and identification of xylooligosaccharides were performed by running 0.5 μL of the concentrated sample on the TLC plate using chloroform/acetic acid/water in a ratio of 6:7:1 as a mobile phase. The TLC was developed by staining with a visualizing solution containing 0.5% w/v α-naphthol dissolved in a solution of methanol and sulfuric acid at a ratio of 95:5. A xylooligosaccharide standard with the degree of polymerization (DP) of 2–5 (1 mg/mL) was used as the standard for identifying the hydrolyzed products. The presence of xylooligosaccharide species in the XOS mixture was identified by performing the mass spectrometry (ESI-MS) analysis as reported earlier.[43]