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Vuong, Thu V.; Master, Emma R.Enzymatic upgrading of heteroxylans for added-value chemicals and polymers

Published in:CURRENT OPINION IN BIOTECHNOLOGY

DOI:10.1016/j.copbio.2021.07.001

E-pub ahead of print: 01/02/2022

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Please cite the original version:Vuong, T. V., & Master, E. R. (2022). Enzymatic upgrading of heteroxylans for added-value chemicals andpolymers. CURRENT OPINION IN BIOTECHNOLOGY, 73, 51-60. https://doi.org/10.1016/j.copbio.2021.07.001

Enzymatic upgrading of heteroxylans for added-valuechemicals and polymersThu V Vuong1 and Emma R Master1,2

Available online at www.sciencedirect.com

ScienceDirect

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-Dglucuronidases 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.

Addresses1Department of Chemical Engineering and Applied Chemistry, Univer-

sity of Toronto, Toronto, Canada2Department of Bioproducts and Biosystems, Aalto University, Espoo,

Finland

Corresponding author: Master, Emma R (emma.master@utoronto.ca)

Current Opinion in Biotechnology 2021, 73:51–60

This review comes from a themed issue on Energy biotechnology

Edited by Jonathan Dordick and Jungbae Kim

https://doi.org/10.1016/j.copbio.2021.07.001

0958-1669/ã 2021 The Author(s). Published by Elsevier Ltd. This is an

open access article under the CC BY license (http://creativecommons.

org/licenses/by/4.0/).

IntroductionCurrent 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 biore-

sources to bio-based products with high economic value.

Xylans are major biomass components that remain com-

paratively underused and so represent an important bior-

esource for sustainable product development.

Xylan backbones comprises b-D-xylopyranosyl (Xylp)monomers and can be divided into either homoxylan or

www.sciencedirect.com

heteroxylan. The unsubstituted backbone structures of

homoxylans include b-(1 ! 4)-linked Xylp, b-(1 ! 3)-

linked Xylp, and b-(1 ! 4; 1 ! 3)-linked Xylp monomers

and are found mainly in seaweeds [1]. By contrast, hetero-

xylans contain a b-(1 ! 4)-linked Xylp backbone that can be

substituted at C2 and/or C3 positions with a-L-arabinofur-anose (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 func-

tional 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.

Diverse heteroxylan chemistry createschallengesDepending on the abundance of two major monosaccha-

ride substituents sub s, Araf and (Me)GlcpA, heteroxylans

are grouped into three main types: glucuronoxylans, ara-

binoxylans and glucuronoarabinoxylans/arabinoglucuro-

noxylans (Figure 1). Glucuronoxylans are common in

hardwoods and are substituted with MeGlcpA and acetyl

groups with a reported degree of substitution (DS) of

0.04�0.25 and 0.1�0.7, correspondingly [2��,17–19]. Ara-

binoxylans are found in cereal plants and are substituted

with Araf residues with a reported DS between 0.5–1.0

[20,21]. Araf residues can be substituted with ferulic acid

with a reported DS between 0.003�0.01 [6,22,23�,24�].Glucuronoarabinoxylans are found in grasses and cereals

while arabinoglucuronoxylans are found in softwoods; both

are substituted with Araf residues (a reported DS between

0.09�0.28) and (Me)GlcpA residues (a reported DS

between 0.08�0.18) [20,25]. Agricultural glucuronoarabi-

noxylan can be highly substituted and contain diverse

functional groups [2��].

Current Opinion in Biotechnology 2022, 73:51–60

52 Energy biotechnology

Figure 1

(a) Glucuronoxylan

(b) Arabinoxylan

(c) Arabinoglucuronoxylan/Glucuronoarabinoxylan (agricultural feedstocks)

β 4

α2

β 4 β 4 β 4 β 4 β 4 β 4

3Ac 2Ac 2,3Ac

5Fa

3α

2α

α2

α 3

n

β 4

2α

2α

α2

β 4 β 4 β 4 β 4 β 4 β 4 β 4 β 4

4Me

2,3Ac 3Ac 3Ac2Ac

4Me

4Me

α2

4Me

n

β 4

2α

2α

α2

β 4 β 4 β 4 β 4 β 4 β 4 β 4 β 4

4Me

3Ac 2Ac

4Me

3α

α2

5Fa

α3

β2

α2

α2

α 3

n

OO

O

OO

OO

OO

OOH

OOH O

HOO

OO

HOO

OO

OO

OO

HO

O

OO

HOO

OO

HO

OH

OO

OOH

O

nO

HOOH

OH

O3HC

3HCOO

HOOH

HO O O OH

HOOH

OOH

CH3O

OHO

OHHO

O3HCO

O

OH

OHO

HO

O

OH

O

OHO

OH

OOH

HOOH

HOH2C

O

O

HO

OCH3

O

OH

OH

HO

O

OH

OH

HO

OO

OH

OO

OOH

OO

HOO

OO

O

HOOH

OH

O

HOOH

OO

HOO

OO

HOO

OO

O

On

OH3C

OO

OO

O

CH3O

OH3C

O

OH

OH

O

O

HO

OCH3

CH3O

O

HO

HO

OH

O

HO

HO

OH

O

HO

OH

OH

OO

OH

OO

OO

OO

HOO

OHO O

HOO

OO

OO

OO

HOO

OO

HO

O

OO

HOO

O

H3COO

HOOH

HO O

OHO

OHHO

OH3CO

O OH

HOOH

OOCH3

n

OH3C

O CH3

OH3C

O

HO

OH

OO

HOO

O

n

CH3O

CH3O

OHO

HO

OH3CO

α-glucuronidase (GH115)

Feruloyl esterase (CE1)

α-L-galactosidase(GH95)

m2,3 α-L-arabinofuranosidase (GH43, GH51, GH54, GH62)

m,d α-L-arabinofuranosidase (GH43)

d3 α- L-arabinofuranosidase (GH43, GH51,

GH54)Acetylxylan esterase (CE1, CE3-CE6, CE16, FjoAcXE)

Acetylxylan esterase (CE1, CE5, CE6, CE16, FjoAcXE)

Acetylxylan esterase (CE16, FjoAcXE)

L

Current Opinion in Biotechnology

Representative chemical structures of three major types of heteroxylans and enzymes that act on corresponding side groups.

Each heteroxylan was depicted using the chair conformation (above) and the Symbol Nomenclature for Glycans standard [11��] (below). The target

linkages of debranching enzymes from families of glycoside hydrolases (GH) and carbohydrate esterases (CE) are shown by arrows. m2,3 a-L-

Arabinofuranosidases hydrolyze (1 ! 2)-linked and (1 ! 3)-linked a-L-arabinofuranosyl (Araf) units of monosubstituted xylopyranosyl (Xylp) residues

while d3 a-L-arabinofuranosidases release solely (1 ! 3)-linked Araf units from disubstituted Xylp residues, and m,d a-L-arabinofuranosidases are able

to hydrolyze both mono-substituted and di-substituted Araf units [12]. CEs that remove acetyl groups from 2,3-diacetylated Xylp residues are also able

to remove acetyl groups from 2-monoacetylated and 3-monoacetylated Xylp residues. FjoAcXE is an unclassified CE with activity towards 3-O

acetylated Xylp substituted at C2 by MeGlcpA and preference for doubly substituted Xylp residues (2,3-O-acetyl-Xylp) [13�]; the second unclassified

FjoAcXE is an acetylxylan 3-O-deacetylase [14]. (a) Glucuronoxylan or O-acetyl-(4-O-methylglucurono)-xylan with Xylp backbone residues substituted

with MeGlcpA at the C2 position by an a-1-2 linkage, with acetyl groups at the C2 and/or C3 positions. Xylp decorated with 2-O-MeGlcpA can also be

acetylated at the C3 position [13�,14]; n is generally from 1 to 6 [15]. (b) Arabinoxylan with Xylp substituted with Araf at C2, C3, or both positions

through a-1-2, a-1-3 linkages correspondingly, and also substituted C2 and/or C3 positions by acetyl groups; Araf residues could be substituted with

ferulic acid at C5. (c) Glucuronoarabinoxylan/arabinoglucuronoxylan with Xylp substituted with MeGlcpA or GlcpA at C2, and Araf at C2 and/or C3

positions; Xylp residues can be substituted with oligosaccharides comprised of various functional groups including Araf, Xylp, and galactose [2��]; Araf

residues can be feruloylated. Whereas agricultural glucuronoarabinoxylans are typically acetylated [2��], softwood arabinoglucuronoxylans are usually

not acetylated [16].

Current Opinion in Biotechnology 2022, 73:51–60 www.sciencedirect.com

Enzymatic upgrading of heteroxylans Vuong and Master 53

Figure 2

Heteroxylan

Chemical

Xylose

Xylitol

Sweetener

Furfural

FA, TFH, THFA

Biofuel

Arabinose Uronic acid

Glucaricacid

Acetic acid

Polymer

Bioactive material (Antimicrobial, antioxidant...)

Packaging

BiofilmXylan

compositeHydrogel

Drug delivery

Oxidative cross-linking(Laccase/ Peroxidase) Hydrogenation

Sulfation/ Cationization/

Carboxylmethylation

Fermentation

Hydrolysis (Glycoside hydrolase/

Carbohydrate esterase/ LPMO)

Dehydration Oxidation(Gluco-oligosaccharide oxidase/Uronate dehydrogenase)

Current Opinion in Biotechnology

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).

Several extraction methods have been developed to iso-

late 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, pre-

extraction 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 opportunitiesCurrent practices aim to recover value from xylans

through their pre-extraction from forest and agricultural

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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 perme-

ability, aroma permeability and high light transmittance,

making them suitable for packaging applications, partic-

ularly 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.

Current Opinion in Biotechnology 2022, 73:51–60

54 Energy biotechnology

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 polysac-

charides that could be applied to xylans include oxida-

tion, 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 applica-

tions due to traces of residual chemicals [43]. Non-toxic

and biocompatible methods for production of xylan

hydrogels and films are needed to widen their applica-

tions, particularly in food and pharmaceuticals.

Enzymatic upgrading xylansDebranching xylans without degrading the main xylan

chain is an attractive approach to alter physico-chemical

properties of xylan-based films and hydrogels (Table 1)

and can be catalyzed by two groups of enzymes: glycoside

hydrolase (GH) and carbohydrate esterase (CE) (www.

cazy.org). For example, a-L-arabinofuranosidases (EC

3.2.1.55) that are able to remove Araf from polymeric

xylans are found in GH families 43, 51, 54, and 62,

whereas a-D-glucuronidases (EC 3.2.1.131) with demon-

strated ability to remove MeGlcpA side groups from

polymeric xylan are found in family GH115 [44–46]

(Figure 1). Acetylxylan esterases (EC 3.1.1.72) that act

on polymeric xylan are reported in CE families 1, 3–6,

15 and 16. These CE families also contain glucuronoyl

esterases (CE15, EC 3.1.1.-) [47] and feruloyl esterases

(CE1, EC 3.1.1.73) [48]. Some highly substituted glucur-

onoarabinoxylans such as from corn fiber require other

debranching enzymes, including a-xylosidase (GH31,

EC 3.2.1.177) and a-L-galactosidase (GH95, and GH97,

EC 3.2.1.22) [2��] for complete side group removal.

Enzymatic technology for xylan-based polymers

By enzymatically controlling the Araf:Xylp ratio of arabi-

noxylan, films with tailor-made properties can be created.

For instance, removal of Araf by m2,3 a-L-arabinofura-nosidase 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 exhib-

ited stress/strain behavior similar to synthetic semicrys-

talline polymers [21]. In addition to controlling mechani-

cal properties, a-L-arabinofuranosidases alone or with

Current Opinion in Biotechnology 2022, 73:51–60

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 addi-

tives 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 arabi-

noxylan with GH62 and GH43 a-L-arabinofuranosidasesalso benefited subsequent grafting with glycidyl methac-

rylate [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 co-

products from xylan-side groups should ideally leverage

the particular chemistry of the side groups and target

markets that remain challenging to meet through fermen-

tation of major sugars (e.g. xylose and glucose). One of the

main substituents of both glucuronoxylan and arabino-

glucuronoxylan is MeGlcpA (and to a lesser extent,

GlcpA). MeGlcpA could be removed from xylans by

GH115 a-glucuronidases [45,46,59] and then be enzy-

matically 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 dicarbox-

ylic acid is a component of detergents and a key inter-

mediate 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 isola-

tion or modification. For instance, two a-L-arabinofura-nosidases 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 [61–64], including enhanced enzymatic

recovery of MeGlcpA from glucuronoxylans [65]. Like-

wise, 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-Xylplinkages 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

www.sciencedirect.com

Enzymatic

upgrading

of

heteroxylans

Vuong

and

Master

55

Table 1

Selective demonstrations of enzymatic upgrading heteroxylan reported within the past five years

Feedstock Heteroxylan type Enzyme(s) used Process parameters Product properties Reference

Xylan

concentration

(w/v)

Buffer Temperature Incubation

time

Triticale bran Arabinoxylan a-L-arabinofuranosidase – 100 mM sodium

phosphate buffer

pH 7.5

40�C 24 hour Antioxidant and

hypoglycemic xylans

[49]

Wheat chaff Feruloylated arabinoxylan GH51 a-L-

arabinofuranosidase, CE1

feruloyl esterase (and GH11

xylanase)

2% 10 mM acetate

buffer pH 5.0

50�C 24 hour Low-molecular

weight, bioactive

hydrolysates

[50]

Corn bran Feruloylated

glucuronoarabinoxylan

AA1 laccase 2% Water pH 6.5 25�C 1 hour Hydrogels [24�]

Wheat bran Feruloylated arabinoxylan AA1 laccase 2% 50 mM citrate-

phosphate buffer

pH 5.5

25�C 0.5 hour Hydrogels [51]

Sorghum bran Feruloylated arabinoxylan AA2 peroxidase 2.5% Water 25�C 2 hour Protein cross-linked

hydrogels

[52]

Corn bran Feruloylated arabinoxylan AA1 laccase 6% 100 mM acetate

buffer pH 5.5

Room

temperature

and then 4�C

6 hour Microspheres for

insulin encapsulation

and oral delivery

[35]

Beechwood Glucuronoxylan a-D-glucuronidase 5% 50 mM acetate

buffer pH 5.0

40�C 24 hour Xylan hydrogel

-cellulose

composites

[53]

Wheat aleurone-rich flour Feruloylated arabinoxylan AA2 peroxidase 0.2% Water 20�C 3 hour Gels with enhanced

bile acid-binding

capacity, and dough

improvement

[54]

Wheat Arabinoxylan GH62 m2,3 a-L-

arabinofuranosidase and

GH43 d3 a-L-

arabinofuranosidase

1% 50 mM sodium

phosphate pH 6.5

40�C 24�48

hour

Arabinoxylans for

grafting with glycidyl

methacrylate

[55]

Wheat Arabinoxylan AA1 laccase 2% 12.5 mM sodium

citrate buffer pH

5.0

22�C 24 hour Aerogels [56]

Sugarcane, bagasse,

bamboo, patula pine,

and rose gum

Glucuronoxylan and

arabinoglucuronoxylan

GH115 a-D-glucuronidase

and GH54 a-L-

arabinofuranosidase

1% 50 mM acetate pH

4.8

40�C 16 hour Substituted xylan-

cellulose (cotton lint)

composites

[57]

www.sciencedire

ct.c

om

Curre

nt

Opinion

in

Biotechnology

2022,

73:51–60

56 Energy biotechnology

Figure 3

β 4

2α

β 4β 4

β 4β 4

β 4 β 4 β 4 β 4

4Me

3Ac 2Ac 3α

β 4

α2

5Fa

β 4

α2

5Fa

β 4

α2

5Fa

β 4

α2

5Fa

α3

β2

α3

α2

α 3

Feruloylated arabinoxylan

Cellulose

Other functional

groups

OO

HO

OH

OH

OOH

HO

OH OH

O

Gluco-oligosaccharide oxidase (AA7) Gluco-oligosaccharide oxidase (AA7)

H3COO

HO

OH

HO O

OH

O

HOOH

OOH

OCH3

OH

OH

Laccase (AA1)/ peroxidase (AA2)

β 4

α2

5Fa

β 4

α2

5Fa

feruloylated arabinoxylan

Laccase (AA1)/ peroxidase (AA2)

Galactose oxidase (AA5)

Lytic polysaccharide monoxygenase (AA14)

HO

O

HO

OHO

OH

HO

O

HO

OHO

O

β 4 β 4

L

β 4 β 4 β 4

β 4

β 4 β 4 β 4

COOH

α2

(a)

(c) (d) (e)

(b)

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.

effective release of two co-products: a sought after plat-

form 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, particu-

larly 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

Current Opinion in Biotechnology 2022, 73:51–60

preparation of hydrogels through oxidative cross linking

of feruloylated arabinoxylans and glucuronoarabinoxy-

lans, forming dimers and even trimers of ferulic acid

[23�,24�,51,52]. Because of the natural source of arabi-

noxylans from cereals, cross-linking feruloylated arabi-

noxylans 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 hydro-

philic matrixes for the controlled release of macromole-

cules and cells. Accordingly, laccase was used to create

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Enzymatic upgrading of heteroxylans Vuong and Master 57

covalent 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 polysaccha-

ride 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 nega-

tively charged cellulosic fiber or else introduce new

chemical functionalities to cellulose fiber that can serve

as reactive handles for further modification [66]. Simi-

larly, 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 outlookMaximizing use of all components of forestry and agri-

cultural 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 func-

tional polymers are available [73]; however, to expand the

applications of xylan-based polymers, particularly in food

packaging, food coating, drug delivery and other pharma-

ceutical applications, enzymatic approaches are prefera-

ble. Recent discoveries of new glycoside hydrolase,

carbohydrate esterase and oxidoreductase enzymes

[13�,30��,46] provide exact instruments to tailor the prop-

erties 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 posi-

tions [74,75]. Engineering such enzymes or the discovery

of previously unknown proteins that oxidize polymeric

xylan would offer new tools to diversify bio-based mate-

rials from polymeric xylans.

The success of enzyme technologies for the modification

of xylans will depend on parallel improvements to process

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technologies for xylan extraction. For example, to

preserve functional phenolic acids of arabinoxylan, a

subcritical water extraction has been optimized [76]. Incor-

porating this extraction method with enzymatic pre-

treatment 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 bior-

efinery processes could be even more economically viable

when potentially degraded xylan fractions are also valo-

rized; for instance, through enzyme treatments that permit

the reassembly of xylo-oligosaccharide fragments [68].

Techno-economic assessments for the extraction and enzy-

matic upgrading of xylans from different feedstocks are

critically needed to inform the integration of these key

processes for a given biorefinery set up.

Conflict of interest statementNothing declared.

AcknowledgementsThis work was supported by Genome Canada for the project‘SYNBIOMICS - Functional genomics and techno-economic models foradvanced biopolymer synthesis’ (LSARP, grant number 10405), and theEuropean Research Council (ERC) Consolidator Grant (BHIVE- 648925).

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