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CHAPTER 6

MICROBIAL XYLANOLYTIC CARBOHYDRATE

ESTERASES

EVANGELOS TOPAKAS AND PAUL CHRISTAKOPOULOS

∗

Biotechnology Laboratory, Chemical Engineering Department, National Technical University

of Athens, Greece

∗

hristako@chemeng.ntua.gr

1. INTRODUCTION

The plant cell wall represents the most abundant reservoir of organic carbon in

the biosphere with 10

tons synthesized annually. The degradation of this macro-

molecular structure by microbial enzymes is a key biological process that is central

to the carbon cycle, herbivore nutrition and host invasion by phytopathogenic

fungi and bacteria. The plant cell wall also represents an important industrial

substrate and microbial enzymes that attack this composite structure are widely

used in the food, beverage, paper and pulp, and detergent sectors, while the

potential utility of these enzymes in the energy industry (the estimated energy

content of sugars released annually from plant cell wall degradation is equiv-

alent to 640 billion barrels of oil) is significant. Xylan is one of the building

blocks of the plant cell wall and is the major constituent of hemicellulose. After

cellulose it is the most abundant renewable polysaccharide in nature. Xylan,

a heterogeneous polymer and highly variable in its structure, is composed of

D-xylopyranosyl units linked by -1,4-glycosidic bonds (Fig. 1). In hardwoods,

the xylan backbone is decorated with side chains, including acetic acid that ester-

ifies the xylose units at the C-2 or C-3 positions and 4-O-methyl-D-glucuronic

acid linked to the xylose units via -1,2-glycosidic bonds. In non-acetylated

softwood xylans, in addition to uronic acids, there are L-arabinofuranose residues

attached to the main chain by -1,2 and/or -1,3-glycosidic linkages. In cereals and

grasses hydroxycinnamic acids esterify the arabinofuranoses. The most abundant

hydroxycinnamic acid is trans-ferulic acid, E-4-hydroxy-3-methoxycinnamic

acid, which is usually esterified at position C-5 or C-2 to -L-arabinofuranosyl

side chains in arabinoxylans and at position C-4 to -D-xylopyranosyl residues

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 83–97.

84 TOPAKAS AND CHRISTAKOPOULOS

Figure 1. The basic structural components found in xylan and the hemicellulases responsible for their

degradation

in xyloglucans. Chains of arabinoxylans are strengthened by cross-linking ferulic

acid dimers which are ester linked to the arabinose sugars. Thus enzymes such as

-L-arabinofuranosidases (EC 3.2.1.55), -glucuronidases (EC 3.2.1.139), acetyl

xylan esterases (EC 3.1.1.72), and feruloyl esterases (EC 3.1.1.73) that remove side

chain substituents from the xylan backbone are required in addition to endo--1,4-

xylanases (EC 3.2.1.8) and -xylosidases (EC 3.2.1.37) for the complete degradation

of xylan (Fig. 1). This battery of enzymes includes two types of microbial carbohy-

drate esterases (EC 3.1.1.1): acetyl xylan esterases (AcXEs) and feruloyl esterases

(FAEs), less well known than related lipases and other esterases.

2. ACETYL XYLAN ESTERASE

The existence of acetyl xylan esterase (AcXE, EC 3.1.1.72) was first reported

in fungal cultures of Schizophyllum commune (Biely 1985; Biely et al., 1986).

AcXE catalyses the hydrolysis of the acetyl side groups in glucuronoxylan, which

is the main component of hardwood hemicellulose. Glucuronoxylan (O-acetyl-4-

O-methylglucuronoxylan) is composed of -1,4-linked D-xylopyranoside residues.

Approximately every 10th xylose unit carries a 4-O-methylglucuronic acid side

chain attached to the 2-position of xylose, and 7 out of 10 xylose residues contain

an O-acetyl side group at the C-2 or C-3 position or both.

AcXEs fall into seven of the fourteen carbohydrate esterase (CE) families estab-

lished by Coutinho and Henrissat (1999), indicating that these enzymes show consid-

erable sequence divergence. Family 1 includes fungal AcXEs from Aspergillus niger

(de Graaff et al., 1992), Aspergillus oryzae (Koseki et al., 2005a) other Aspergillus

species (Koseki et al., 1997; de Graaff et al., 1992; Chung et al., 2002; Koseki

et al., 2005b), S. commune (Halgašová et al., 1994; Biely et al., 1988), Penicillium

purpurogenum AcXE I (Egaña et al., 1996) and bacterial enzymes such as a Ferulic

Acid Esterase (FAE) from Cellvibrio japonicus which contains a cellulose-binding

MICROBIAL XYLANOLYTIC CARBOHYDRATE ESTERASES 85

domain (CBD) (Ferreira et al., 1993), and the catalytic domains of the bifunc-

tional enzymes of anaerobic bacteria (Fontes et al., 1995). Family 2 includes

AcXEs from the fungus Neocallimastix patriciarum (Dalrymple et al., 1997) and the

anaerobic bacterium Clostridium thermocellum (Hall et al., 1988). Family 3 includes

AcXEs from the fungus N. patriciarum (Dalrymple et al., 1997), and the anaerobic

rumen bacteria C. thermocellum (Hall et al., 1988) and Ruminococcus flavefa-

ciens (Zhang et al., 1994). Family 4 includes AcXEs from Streptomyces lividans

(contains a xylan-binding domain (XBD) (Dupont et al., 1996)), Streptomyces

thermoviolaceus (Tsujibo et al., 1997), bifunctional enzymes (having two catalytic

domains along with both CB and XB domains (Laurie et al., 1997; Millward-

Sadler et al., 1995)) from the aerobic bacteria Cellvibrio japonicus, Cellvibrio

mixtus and Cellulomonas fimi, and an endoxylanase-AcXE protein from Clostridium

cellulovorans (Kosugi et al., 2002). Family 5 includes fungal esterases from

Hypocrea jecorina (formerly known as Trichoderma reesei) (Hakulinen et al., 2000)

and P. purpurogenum (Ghosh et al., 2001). Family 6 includes fungal esterases from

the anaerobe N. patriciarum (Dalrymple et al., 1997). Enzymes in carbohydrate

esterase family 7 are unusual in that they display activity towards both acetylated

xylooligosaccharides and the antibiotic cephalosporin C. Members of this family

include AcXEs from Thermoanaerobacterium sp. (Shao et al., 1995), Thermotoga

maritima (Nelson et al., 1999), Bacillus pumilus, (Krastanova et al., 2005) and

the cephalosporin-C deacetylase from Bacillus subtilis (Vincent et al., 2003). It

has been suggested that the AcXE and cephalosporin C deacetylase (EC 3.1.1.41)

enzymes of the CE-7 family represent a single class of proteins with a multifunc-

tional deacetylase activity against a range of small substrates (Vincent et al., 2003).

Three-dimensional structures are known for AcXEs belonging to families 5 and 7.

This is the case for the family 5 enzymes AXE1 of T. reesei (Hakulinen et al., 2000)

and AXEII of

P. purpurogenum (Ghosh et al., 2001). These two enzymes belong to the

superfamilyof the/ hydrolase fold,the coredomain ofwhich hasan // sandwich

fold and a catalytic triad (Ser-His-Asp). Known 3-D structures of carbohydrate family

7 include B. subtilis cephalosporin-C deacetylase (CAH), (Vincent et al., 2003) and

T. maritima AXE (Page et al., 2003). These enzymes are hexameric / hydrolases

with a narrow entrance tunnel which leads to the centre of the molecule where the six

active-centre catalytic triads point towards the tunnel interior and thus are sequestered

away from the cytoplasmic content. By analogy to self-compartmentalising proteases,

the tunnel entrance may function to hinder access of large substrates such as acetylated

xylan to the poly-specific active centre. This also would explain the observation that

the enzyme is active on a variety of small, acetylated molecules. The activity against

cephalosporin-C suggests a possible pharmaceutical application for family 7 AcXEs

in the production of semi-synthetic antibiotics.

3. FERULIC ACID ESTERASE

Ferulic Acid Esterase (FAE, EC 3.1.1.73) comprises a very diverse set of enzymes,

with few sequence and physical characteristics in common. Many FAEs have

been purified and characterized showing differences in physical properties such

86 TOPAKAS AND CHRISTAKOPOULOS

as molecular weight, isoelectric point and optimal reaction conditions (Table 1).

Multiple alignments of sequences or domains demonstrating FAE activity, as well

as related sequences, have been used to construct a neighbour-joining phylogenetic

tree (Crepin et al., 2004a). The result of this genetic comparison, supported also by

substrate specificity data, allows FAEs to be sub-classified into 4 types: A, B, C and

D. Due to the increasing number of FAEs being isolated, a system of nomenclature

has been proposed using the letters of the producer micro-organism followed by

Fae to designate that it is an enzyme with feruloyl esterase activity and then

a letter to designate the proposed sub-class based on the specificity data of the

enzyme (Crepin et al., 2004a). For example, the type-A FAE produced by Fusarium

oxysporum would be termed FoFaeA. Previously reported FAEs do not follow this

nomenclature. Although FAEs appear to have some common roots according to the

phylogenetic tree constructed by Crepin et al. (2004a), they show greater sequence

homology with a variety of other enzymes such as lipases, AcXEs and xylanases.

It is extremely common for esterases to act on a broad range of substrates.

Esterases acting on plant cell walls catalyse similar chemical reactions but they

exhibit different specificities for the aromatic moiety of hydrocinnamates or the

linkage to the primary sugar in feruloylated oligosaccharides and variation in their

ability to release dehydrodimeric forms of ferulic acid from plant cell wall material.

The catalytic specificity shown by FAEs, as defined by the rate of catalysis divided

by the Michaelis constant (k

cat

) which gives the best indication of ‘preferred’

substrates, is a result of the complexity of the plant cell wall material. Type A FAEs

show preference for the phenolic moiety of the substrate that contains methoxy

substitutions, especially at meta- position(s) as occurs in ferulic and sinapinic acids,

while type B FAEs show complementary activity to type A esterases, showing

preference for substrates containing one or two hydroxyl substitutions as found in

p-coumaric or caffeic acid. In contrast to type B esterases, type A FAEs appear to

prefer hydrophobic substrates with bulky substituents on the benzene ring (Kroon

et al., 1997; Topakas et al., 2005b). The high level of sequence identity of AnFaeA

with the lipases from Thermomyces lanuginosus TLL (30% sequence identity)

and Rhizomucor miehei (37% sequence identity) seems to justify the hydrophobic

substrate preference of the esterase. Furthermore, type A and D FAEs in contrast to

type B and C are also able to release small amounts of dehydrodimeric ferulic acid.

Type C and D FAEs show broad specificity against synthetic hydroxycinnamic

acids (ferulic, p-coumaric, caffeic and sinapinic acid) showing differences only

in their ability to release 5-5’ dehydroferulic acid (Crepin et al., 2004a; Crepin

et al., 2004b).

Sufficient specificity studies have been conducted in order to demonstrate the

ability of FAEs in releasing ferulic acid from model substrates synthesized by

chemoenzymatic synthesis (Biely et al., 2002) or from naturally occurring feruloy-

lated oligosaccharides obtained by controlled enzymatic digestion of plant cell

wall material. It seems that there is a correspondence between the FAE classifi-

cation and the affinity of these enzymes for the position of -L-arabinofuranose

feruloylation. Type A esterases such as AnFaeA (Williamson et al., 1998), TsFaeA

MICROBIAL XYLANOLYTIC CARBOHYDRATE ESTERASES 87

Table 1. Physicochemical properties of purified FAEs known to date

Micro-organism Enzyme FAE

type

(kDa)

opt

(



pI Reference

Aspergillus awamori FE - 112 3.7 McCrae et al., 1994

Aspergillus awamori CE - 75 4.2 McCrae et al., 1994

Aspergillus awamori - 35 5.0 45 3.8 Koseki et al., 1998

Aspergillus awamori AwFAEA A 37 5.0 Koseki et al., 2005c

Aspergillus niger FAE-I B 63* 3.0 Faulds and Williamson,

1993

Aspergillus niger FAE-II A 29 3.6 Faulds and Williamson,

1993

Aspergillus niger FAE-III or

AnFaeA

A 36 5.0 55 3.3 Faulds and Williamson,

1994

Aspergillus niger CinnAE

AnFaeB

B*** 75.8* 6.0 50 4.8 Kroon et al., 1996

Aspergillus niger CE - 120 Barbe and Dubourdieu,

1998

Aspergillus oryzae FAE - 30 4.5–6.0 3.6 Tenkanen et al., 1991

Aspergillus tubingensis FaeA - 36 Vries de et al., 1997

Aureobasidium pullulans B 210 6.7 60 6.5 Rumbold et al., 2003

Cellvibrio japonicus XLYD or

CjXYLD

D 59 6.0 Ferreira et al., 1993

Clostridium stercorarium Cor

33 8.0 65 Donaghy et al., 2000

Clostridium thermocellum XynZ - 45 4–7 50–60 5.8 Blum et al., 2000

Fusarium oxysporum FoFAE-I

or FoFaeB

B 31 7.0 55 >9.5 Topakas et al., 2003a

Fusarium oxysporum FAE-II or

FoFaeA

A 27 7.0 45 9.9 Topakas et al., 2003b

Fusarium proliferatum FAE B 31 6.5–7.5 50 Shin and Chen, 2005

Lactobacillus acidophilus

∗∗

- 36 5.6 37 Wang et al., 2004

(Continued)

88 TOPAKAS AND CHRISTAKOPOULOS

Table 1. (Continued)

Micro-organism Enzyme FAE

type

MW(kDa) pH

opt

(



pI Reference

Neocallimastix MC-2 pCAE - 11* 7.2 4.7 Borneman et al., 1991

Neocallimastix MC-2 FAE-I - 69 Borneman et al., 1992

Neocallimastix MC-2 FAE-II - 24 Borneman et al., 1992

Neurospora crassa Fae-1 B 35 6.0 55 Crepin et al., 2003a

Neurospora crassa NcFaeD-3.544 D 32 Crepin et al., 2004b

Penicillium expansum - 57.5 5.6 37 Donaghy and McKay,

1997

Penicillium funiculosum FAE-B or

PfFaeB

B 53 6.0 Kroon et al., 2000

Penicillium pinophilum p-CAE/

FAE

- 57 6.0 55 4.6 Castanares et al., 1992

Piromyces equi EstA D 55 6.7 50–60 Fillingham et al., 1999

Sporotrichum thermophile StFAE-A

or StFaeB

B 33* 6.0 55–60 3.5 Topakas et al., 2004

Sporotrichum thermophile StFaeC C 23* 6.0 55 < 35 Topakas et al., 2005a

Streptomyces olivochromo-

genes

FAE - 29 5.5 30 7.9 Faulds and Williamson,

1991

Talaromyces stipitatus TsFaeA A 35 5.3 Garcia-Conesa et al.,

2004

Talaromyces stipitatus TsFaeB B 35 3.5 Garcia-Conesa et al.,

2004

Talaromyces stipitatus TsFaeC C 66 6–7 60 4.6 Crepin et al., 2003b

* Dimeric proteins (Molecular weight estimated with SDS-PAGE electrophoresis).

** Typical human intestinal bacterium.

*** Phylogenetic analysis of AnFaeB indicated that this enzyme belongs to the type C sub-class (Crepin et al., 2004a).

MICROBIAL XYLANOLYTIC CARBOHYDRATE ESTERASES 89

(Garcia-Conesa et al., 2004) and FoFaeA (Topakas et al., 2003b) (Table 1) are active

only on substrates containing ferulic acid ester linked to the C-5 and not on substrates

containing ferulic acid ester linked to the C-2 linkages of L-arabinofuranose. In

contrast, type B FAEs such as AnFaeB (Williamson et al., 1998), PfFaeB (Kroon

et al., 2000), FAE from A. pullulans (Rumbold et al., 2003), TsFaeB (Garcia-Conesa

et al., 2004), FoFaeB (Topakas et al., 2003a) and StFaeB (Topakas et al., 2004)

(Table 1) are active on substrates containing ferulic acid ester linked to C-5 or C-2 of

L-arabinofuranose, with different preferences depending on the esterase studied. The

inability of type A FAE to hydrolyse the C-2 linkage between ferulic acid and the

L-arabinofuranose residue could be a new criterion for use in the classification of this

subclass of esterases. Type C and D FAEs such as StFaeC (Topakas et al., 2005a),

TsFaeC (Garcia-Conesa et al., 2004) and CjXYLD (Ferreira et al., 1993) are able to

hydrolyse both linkages. The active sites of FAEs from mesophilic and thermophilic

sources have been probed using methyl esters of phenylalkanoic acids (Kroon

et al., 1997; Topakas et al., 2005b). The thermophilic esterases from S. thermophile

(StFaeB and StFaeC) showed k

cat

values for phenylalkanoic and cinnamoyl methyl

esters lower than those of the mesophilic esterases from F. oxysporum (FoFaeA and

FoFaeB). A similar observation was made comparing the k

cat

values for the methyl

esters of hydroxycinnamic acids for StFaeB and FoFaeB. Lengthening or shortening

the aliphatic side chain of phenylalkanoate substrates while maintaining the same

aromatic substitutions of the substrates completely abolished FAE activity, showing

that the distance between the aromatic group and the ester bond is critical for

enzyme catalysis (Kroon et al., 1997; Topakas et al., 2005b). However, Tarbouriech

et al. (2005) reported that substrates with short aliphatic chains (vanillate and

syringate which contain only one carbon atom in the aliphatic chain) also bind to

the active site of XynY FAE indicating that the length between the phenyl group

and methyl ester in these molecules is not crucial, even if it may contribute to the

correct orientation for catalysis.

Recently, the number of reported FAE activities has increased, especially with

the acquisition of related protein sequences in genomic databases (Table 2). Many

of these enzymes are modular, comprising a catalytic domain covalently fused

to a non-catalytic carbohydrate binding module (Fillingham et al., 1999; Ferreira

et al., 1993; Kroon et al., 2000; Laurie et al., 1997). There have also been reports

of FAEs being present in large multidomain structures such as cellulosomes (Blum

et al., 2000). A chimeric enzyme composed of feruloyl esterase A (FAEA) from

A. niger and a dockerin from C. thermocellum was produced in A. niger (Levasseur

et al., 2004). This is the first reported example of a functional fungal enzyme joined

to a bacterial dockerin.

Unlike AcXEs which are distributed across seven different families (CE families

1 to 7), the majority of the FAEs such as the type B esterases of N. crassa

(Crepin et al., 2003a) and P. funiculosum (Kroon et al., 2000) shown in Table 1

are classified in family 1 (Coutinho and Henrissat, 1999). The crystal structures of

FAE, AnFaeA/FAE-III from A. niger (Hermoso et al., 2004; McAuley et al., 2004;

Faulds et al., 2005) and FAE domains, XynY (Prates et al., 2001; Tarbouriech

90 TOPAKAS AND CHRISTAKOPOULOS

Table 2. FAE genes and their accession numbers known to date

Micro-organism Gene Data bank Reference

Vries de et al., 1997

Aspergillus niger faeA Y09330 Juge et al., 2001*

Record et al., 2003

Levasseur et al., 2004**

Aspergillus awamori AwfaeA AB032760 Koseki et al., 2005c

Aspergillus niger faeB AJ309807 Vries de et al., 2002

Aspergillus tubingensis faeA Y09331 Vries de et al., 1997

Butyrivibrio fibrisolvents cinI orcinA U44893 Dalrymple et al., 1996

Butyrivibrio fibrisolvents cinII orcinB U64802 Dalrymple and Swadling, 1997

Cellvibrio japonicus xynD X58956 Ferreira et al., 1993

Clostridium thermocellum XynY X83269 Blum et al., 2000

Clostridium thermocellum XynZ M22624 Blum et al., 2000

Neurospora crassa Fae-1 AJ293029 Crepin et al., 2003a*

Neurospora crassa faeD-3.544 - Crepin et al., 2004b

Penicillium funiculosum faeB AJ291496 Kroon et al., 2000

Piromyces equi estA AF164516 Fillingham et al., 1999

Talaromyces stipitatus faeC AJ505939 Crepin et al., 2003b*

* Heterologous expression of the FAE in the methylotrophic yeast Pichia pastoris.

** Chimeric protein associating FAEA from A. niger and a dockerin domain from C. thermocellum.

et al., 2005), and XynZ (Schubot et al., 2001) of the cellulosomal enzymes included

in the cellulosome complex from C. thermocellum, have been determined. These

FAEs have a common / hydrolase fold and a catalytic triad (Ser-His-Asp)

also present in lipases. For example, the structure of AnFaeA displays an /

hydrolase fold very similar to that of the fungal lipases from T. lanuginosus

(Lawson et al., 1994) and R. miehei (Derewenda et al., 1992) but lacks lipase

activity (Aliwan et al., 1999). The active site cavity is confined by a lid, similar

to that of lipases, and by a loop that confers plasticity to the substrate binding

site. The lid presents a high ratio of polar residues, which, in addition to a unique

N -glycosylation site, stabilizes the lid in an open conformation conferring the

esterase character to this enzyme (Hermoso et al., 2004). The structure and the

sequence homology of AnFaeA are different from that reported for the cellulosomal

enzymes XynY and XynZ from C. thermocellum, although the catalytic triads can be

superimposed allowing direct extrapolation of the position of the oxyanion pocket.

Co-crystallization studies of the inactive forms of XynY and AnFaeA with ferulic

acid (Prates et al., 2001; McAuley et al., 2004) or XynZ and AnFaeA with feruloyl

oligosaccharides (Schubot et al., 2001; Faulds et al., 2005) were conducted in order

to identify the residues involved in substrate binding and reveal the hydrolytic

mechanism of FAEs. Furthermore, the structures of XynY FAE Ser-Ala mutant

complexes with syringate, sinapinate and vanillate methyl esters were reported by

Tarbouriech et al. (2005) indicating the importance of the meta-methyl group of

the ferulic ring for binding. Faulds et al. (2005) solved the crystal structure of