Enzymatic upgrading of heteroxylans for added-value chemicals and polymers

Xylan is one of the most abundant, natural polysaccharides, and much recent interest focuses on upgrading heteroxylan to make use of its unique structures and chemistries. Signiﬁcant progress has been made in the discovery and application of novel enzymes for debranching and modifying heteroxylans. Debranching enzymes include acetylxylan esterases, a - L - arabinofuranosidases and a - D glucuronidases that release side groups from the xylan backbone to recover both biochemicals and less substituted xylans for polymer applications in food packaging or drug delivery systems. Besides esterases and hydrolases, many oxidoreductases including carbohydrate oxidases, lytic polysaccharide monooxygenases, laccases and peroxidases have been also applied to alter different types of xylans for improved physical and chemical properties. This review will highlight the recent discovery and application of enzymes for upgrading xylans for use as added-value chemicals and in functional polymers.


Introduction
Current and anticipated environmental regulations increasing demand ecologically friendly alternatives to petroleum-derived products.Both forest and agricultural industries are responding to the demand by adopting cleaner technologies that transform sustainable bioresources to bio-based products with high economic value.Xylans are major biomass components that remain comparatively underused and so represent an important bioresource for sustainable product development.[1].By contrast, heteroxylans contain a b-(1 !4)-linked Xylp backbone that can be substituted at C2 and/or C3 positions with a-L-arabinofuranose (Araf), 4-O-methyl-a-D-glucuronic acid (MeGlcpA), a-D-glucuronic acid (GlcpA), and other neutral sugar units such as a/b-D-xylose, and a-D/L-galactose [2 ].Other functional groups including acetyl groups and ferulic acid can further substitute heteroxylans [3].Heteroxylans are the predominant form of hemicellulose in terrestrial plants; they generally account for 10-35% of the total dry weight in hardwoods, up to 10% of the total dry weight in softwoods, and up to 30% of cereal dry mass [2 ,4-6,7 ].
Xylan-rich fractions generated by agricultural industries are often used in low-value animal feed, whereas those from forestry industries are often un-used or recovered for energy.Alternatively, established pathways have been deployed to transform xylans to xylitol [8]; enzymatic conversion of xylans to fermentable sugars has also been extensively studied [9 ].Unfortunately, co-fermentation of xylose and other C5 and C6 sugars requires intensive engineering of fermenting microorganisms [10].The discovery of enzymes that modify xylan structures opens new possibilities to upgrade xylans for use in value-added products beyond commodity chemicals and fuels.
Several extraction methods have been developed to isolate xylans from agricultural and wood fiber, including alkali extraction, organic solvent extraction, ionic liquid extraction, hot water extraction and steam explosion [7 ].For instance, using mild alkali and low temperature conditions, over 80% of polymeric arabinoxylan could be extracted from barley husks [26].In addition to co-product formation from the extracted xylan, preextraction of xylan before pulping can benefit subsequent pulping processes [27].Organic solvent extraction also recovers xylans with high uronic acids [28].Whereas hot water extraction and steam explosion provide options for aqueous based extraction [7 ], these approaches typically produce short xylan forms [29].Accordingly, the choice of appropriate isolation method will depend on the intended end use of the isolated xylans as well as end uses of the cellulose and lignin fractions.

Heteroxylans also bring multiple opportunities
Current practices aim to recover value from xylans through their pre-extraction from forest and agricultural residues (Figure 2).The corresponding xylose can then be chemically converted to furfural and xylitol.Furfural has a wide range of applications [7 ] and it is obtained solely from lignocellulosic biomass, particularly from xylose via chemical dehydration, as currently there is no commercial synthetic route for furfural production.Xylitol, obtained by catalytic hydrogenation of xylose, is mainly used in food industries as an alternative sweetener and preservative [33].Xylans possess low oxygen permeability, aroma permeability and high light transmittance, making them suitable for packaging applications, particularly edible inner packaging for low-moisture foods or inner layers of a multilayer film protected from moisture by a hydrophobic outer layer [34].Therefore, polymeric xylans have been used to prepare films and hydrogels for food packaging and drug delivery [7 ,35,36].For instance, bagasse xylans with high molecular weight, as well as a low substitution and lignin content, were used to form films with high tensile strength and high modulus of elasticity [36].However, as xylans are hydrophilic, they are good barriers towards oils and fats, but not water.
Enzymatic upgrading of heteroxylans Vuong and Master 53 Major biochemicals and biopolymers from heteroxylans.Several chemical reactions (red texts) used in xylan refineries can be replaced with enzymatic alternatives (green texts).For instance, three groups of enzymes including glycoside hydrolases, carbohydrate esterases and lytic polysaccharide monooxygenases (LPMOs [30 ]) are used to break covalent bonds of heteroxylans instead of chemical hydrolysis, to release two groups of co-products: polymers and platform chemicals.As an alternative to chemo-catalytic oxidation, the conversion of methyl glucuronic acid, a major side group of glucuronoxylan, to methyl glucaric acid is performed by oxidoreductases [31,32].Examples of platform chemicals derived from the dehydration of xylose include furfuryl alcohol (FA), tetrahydrofurfuryl alcohol (THFA) and tetrahydrofuran (TFH).

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Current Opinion in Biotechnology 2022, 73:51-60 Carboxymethylation of alkali-extracted xylan has been used to modify the oxygen and water permeability as well as mechanical properties of xylan films [37,38].
Other strategies for chemical derivatization of polysaccharides that could be applied to xylans include oxidation, esterification and etherification [39].Xylan-based hydrogels for oral drug delivery and controlled release are of interest due to the fact that some xylans are resistant to digestion in the human stomach and are broken down by enzymes that are only present in the human colon [40]; moreover, some xylan types also display in vivo prebiotic effects [41].For example, covalent binding of alkaline-extracted corn cob xylan with 5-fluorouracil reportedly improved 5-fluorouracil delivery to human colorectal cancer cell lines, compared to the free form of the drug [42].However, hydrogels produced by chemical crosslinking methods might be incompatible for direct use in pharmaceutical applications due to traces of residual chemicals [43].Non-toxic and biocompatible methods for production of xylan hydrogels and films are needed to widen their applications, particularly in food and pharmaceuticals.

Enzymatic upgrading xylans
Debranching xylans without degrading the main xylan chain is an attractive approach to alter physico-chemical properties of xylan-based films and hydrogels (

Enzymatic technology for xylan-based polymers
By enzymatically controlling the Araf:Xylp ratio of arabinoxylan, films with tailor-made properties can be created.For instance, removal of Araf by m2,3 a-L-arabinofuranosidase that acts on (1 !2)-linked and (1 !3)-linked Araf units on monosubstituted Xylp residues generated xylan films with increased degree of crystallinity and decreased oxygen permeability [21].The moderately unsubstituted film with an Araf:Xylp ratio of 0.37 exhibited stress/strain behavior similar to synthetic semicrystalline polymers [21].In addition to controlling mechanical properties, a-L-arabinofuranosidases alone or with other enzymes have been used to alter the bioactivity of arabinoxylans [49,50].The natural ability of xylans to adsorb onto cellulose surfaces makes them suitable additives for producing biocomposite materials; for example, the addition of softwood and hardwood xylans to cellulose hydrogels increased the elongation at break under tension of corresponding composites [58].Also, in situ selective hydrolysis of xylans by GH54 a-L-arabinofuranosidase, GH115 a-D-glucuronidase and their mixture increased the adsorption of treated arabinoglucuronoxylans onto cotton lint [57].Enzymatic debranching of wheat arabinoxylan with GH62 and GH43 a-L-arabinofuranosidases also benefited subsequent grafting with glycidyl methacrylate [55].Additional recent examples of enzymes for xylan-based polymer engineering are provided in Table 1.

Making use of released side groups from enzymatic xylan debranching
The potential to create co-products from xylan side groups that are released during enzymatic upgrading of xylans would increase the economic and environmental benefit of valorizing underused xylan sources.The coproducts from xylan-side groups should ideally leverage the particular chemistry of the side groups and target markets that remain challenging to meet through fermentation of major sugars (e.g.xylose and glucose).One of the main substituents of both glucuronoxylan and arabinoglucuronoxylan is MeGlcpA (and to a lesser extent, GlcpA).MeGlcpA could be removed from xylans by GH115 a-glucuronidases [45,46,59] and then be enzymatically converted to the corresponding glucaric acid using a gluco-oligosaccharide oxidase (EC 1.1.3.-) from Auxiliary Activities (AA) family AA7 [31] or using uronate dehydrogenases as in case of GlcpA [32].This dicarboxylic acid is a component of detergents and a key intermediate for the production of biodegradable polymers [60].
As heteroxylans display different side groups depending on botanical source and extraction process, enzymatic combinations are likely needed for their selective isolation or modification.For instance, two a-L-arabinofuranosidases from GH51 and GH62 families enhanced MeGlcpA release from arabinoglucuronoxylan by a GH115 a-D-glucuronidase by up to 50% [46].Moreover, the recent structural and functional characterizations of glucuronoyl esterases are generating new tools for xylan recovery and use [61][62][63][64], including enhanced enzymatic recovery of MeGlcpA from glucuronoxylans [65].Likewise, the usage of an unclassified carbohydrate esterase that cleaves not only singly acetylated Xylp and doubly 2,3-O-acetyl-Xylp, but also internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues, boosted the enzymatic recovery of MeGlcpA from hot water-extracted glucuronoxylan by up to nine times [13 ].
The combinational usage of these enzymes allows the effective release of two co-products: a sought after platform chemical (i.e.(Me)GlcpA) and a less substituted xylan.

Enzymatic oxidation of xylan backbone/side groups
Besides the application of GHs and CEs to selectively remove xylan side groups, the chemical functionality of xylans can be controlled using oxidoreductases, particularly those from AA families (www.cazy.org)(Figure 3).Laccase (family AA1, EC 1.10.3.2) and peroxidase (family AA2, EC 1.11.1.-)are already used in the preparation of hydrogels through oxidative cross linking of feruloylated arabinoxylans and glucuronoarabinoxylans, forming dimers and even trimers of ferulic acid [23 ,24 ,51,52].Because of the natural source of arabinoxylans from cereals, cross-linking feruloylated arabinoxylans could act as texturizing and stabilizing agents in food systems [6,54].The macroporous structure of gels from native feruloylated arabinoxylan makes them an interesting feedstock for the preparation of hydrophilic matrixes for the controlled release of macromolecules and cells.Accordingly, laccase was used to create Current Opinion in Biotechnology Enzymatic oxidation of xylans for biopolymers and biochemicals.A simplified model of highly substituted corn fiber glucuronoarabinoxylan [2 ] is given here as an example.(a) Feruloylated arabinoxylans are cross-linked to form hydrogels for drug delivery [35], or grafted with different functional groups using laccases or peroxidases [52].(b) Galactopyranosyl residues are oxidized by galactose oxidases from AA family 5 [71], creating aldehyde positions for further derivatization.(c) Released MeGlcpA is oxidized by a gluco-oligosaccharide oxidase [31] to produce a co-product methyl glucaric acid while reserving the polymeric structure of xylan.(d) A lytic polysaccharide monooxygenase introduces carboxyl groups on xylan that is bound to cellulose [30 ], whereas carboxyl groups could also be introduced to the reducing end of xylan chains by a gluco-oligosaccharide oxidase from AA family 7 [67] (e), creating new functional sites for subsequent enzymatic or chemical modification.
Current Opinion in Biotechnology 2022, 73:51-60 www.sciencedirect.comcovalent microspheres via enzymatic cross-linking of ferulic acid esterified to arabinoxylan chains that were loaded with insulin leading to a delivery system with significant hypoglycemic effects and improved insulin bioavailability [35].
Other AA family members are able to introduce new functions to different types of xylans.Lytic polysaccharide monooxygenases (LPMOs, AA14, EC 1.14.99.-) that act on xylan bound to cellulose have been reported [30 ].Such C1-oxidizing LPMOs could be used to create negatively charged cellulosic fiber or else introduce new chemical functionalities to cellulose fiber that can serve as reactive handles for further modification [66].Similarly, an AA7 gluco-oligosaccharide oxidase that oxidizes soluble xylans at the reducing end [67] permitted the addition of clickable chemical groups to the end of xylan fragments [68].Some heteroxylans are also decorated with galactopyranosyl residues [2 ] and so are suitable for oxidation by AA5 galactose oxidases (EC 1.1.3.9) that introduce an aldehyde functionality at the C6 position of terminal D-galactose in polysaccharides [69,70].These enzymes have been used to oxidize various types of galactose-containing polysaccharides [69,71], allowing to produce functional cellulosic fiber surfaces [72] for paper and textile applications.

Conclusion and outlook
Maximizing use of all components of forestry and agricultural feedstocks while reducing waste has heightened interest in upgrading xylans, which are often underused fractions of current biorefinery processes.Chemical approaches to convert xylans into biochemicals and functional polymers are available [73]; however, to expand the applications of xylan-based polymers, particularly in food packaging, food coating, drug delivery and other pharmaceutical applications, enzymatic approaches are preferable.Recent discoveries of new glycoside hydrolase, carbohydrate esterase and oxidoreductase enzymes [13 ,30 ,46] provide exact instruments to tailor the properties of xylan-based films and hydrogels.
Beyond those enzyme activities described in this review, novel xylan-active enzymes are needed.For example, enzymes that introduce carbonyls at C2 and/ or C3 positions of xylose backbone units would permit the formation of intra-chain and inter-chain hemiacetals, stabilizing hydrogels.Notably, several AA3 pyranose dehydrogenases are already able to oxidize linear and substituted xylo-oligosaccharides at C1, C2 and C3 positions [74,75].Engineering such enzymes or the discovery of previously unknown proteins that oxidize polymeric xylan would offer new tools to diversify bio-based materials from polymeric xylans.
The success of enzyme technologies for the modification of xylans will depend on parallel improvements to process technologies for xylan extraction.For example, to preserve functional phenolic acids of arabinoxylan, a subcritical water extraction has been optimized [76].Incorporating this extraction method with enzymatic pretreatment transformed wheat bran into several co-products, including feruloylated arabinoxylan [77], which was used to generate bioactive barrier films with antioxidant properties [78 ].The integration of xylan extraction with other biorefinery processes could be even more economically viable when potentially degraded xylan fractions are also valorized; for instance, through enzyme treatments that permit the reassembly of xylo-oligosaccharide fragments [68].
Techno-economic assessments for the extraction and enzymatic upgrading of xylans from different feedstocks are critically needed to inform the integration of these key processes for a given biorefinery set up.

Table 1
Selective demonstrations of enzymatic upgrading heteroxylan reported within the past five years Current Opinion in Biotechnology 2022, 73:51-60 www.sciencedirect.comEnzymatic upgrading of heteroxylans Vuong and Master 55