Biotechnolo

ELSEVI ER Journal of Biotechnology 57 (1997) 151-166

Endo-P - 1,4-xylanase families: differences in catalytic properties’

P. Biely a,*, M. VrSanskh ‘, M. Tenkanen b, D. Kluepfel ’

aInstitute of Chemistry, Slovak Academy of Sciences, DlibravskLi cesta 9, 84238 Bratislava, Slovakia
b VTT Bioiechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
c Institute Armand-Frappier, Universire da Quebec, We de Laval, Quebec, Canada H7N 423

Received 25 August 1996; received in revised form 19 March 1997; accepted 21 March 1997

Abstract

Microbial endo-b-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 (formerly F) and 11 (formerly
G) differ in their action on 4-O-methyl-D-glucurono-D-xylan and rhodymenan, a a-l,3-/I-l,4-xylan. Two high
molecular mass EXs (family IO), the Cryptococcus albidus EX and XlnA of Streptomyces liuiduns, liberate from
glucuronoxylan aldotetrauronic acid as the shortest acidic fragment, and from rhodymenan an isomeric xylotriose of
the structure Xylpl-3Xyl/?l-4Xyl as the shortest fragment containing a ,/?-1,3-linkage. Low molecular mass EXs
(family 1 I), such as the Trichoderma reesei enzymes and XlnB and XlnC of S. lividans, liberate from glucuronoxylan
an aldopentauronic acid as the shortest fragment, and from rhodymenan an isomeric xylotetraose as the shortest
fragment containing a fi-1,3-linkage. The structure of the oligosaccharides was established by: NMR spectroscopy,
mass spectrometry of per-O-methylated compounds and enzymic hydrolysis by /?-xylosidase and EX, followed by
analysis of products by chromatography. The structures of the fragments define in the polysaccharides the linkages
attacked and non-attacked by the enzymes. EXs of family 10 require a lower number of unsubstituted consecutive
/3-1,Cxylopyranosyl units in the main chain and a lower number of consecutive /!-1,4-xylopyranosyl linkages in
rhodymenan than EXs of family 11. These results, together with a greater catalytic versatility of EXs of family 10,
suggest that EXs of family 10 have substrate binding sites smaller than those of EXs of family 11. This suggestion
is in agreement with the finding that EXs of family 10 show higher affinity for shorter linear /I-1,4-xylooligosaccha-
rides than EXs of family 11. The results are discussed with relevant literature data to understand better the
structure-function relationship in this group of glycanases. 0 1997 Elsevier Science B.V.

Keywords: Endo-/?- 1,4-xylanase; Enzyme family; Mode of action; Catalytic properties

1. Introduction
* Corresponding author. Tel.: + 42 7 3782295; fax: + 42 7
375565; e-mail: chempbsa@savba.sk
’ Dedicated to Prof. Roger W. Jeanloz on the occasion of One of the questions of microbial utilization of
his 80th birthday. heteroxylans to be answered is why microorgan-

016%1656/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved

PII SO 168-l 656(97)00096-5
152 P. Biely et al. 1Journal of Biotechnolo,sg 57 (1997) I51 -166

isms produce more than one type of endo-a-1,4- In spite of limited knowledge on the differences
xylanase (EX, EC 3.2.1.8) the crucial enzyme for in catalytic properties of EXs in the two families,
substrate depolymerization. The synthesis of mul- it is certain that the enzymes belonging to family
tiple EXs as distinct gene products by one mi- 10 exhibit greater catalytic versatility or lower
croorganism was revealed on the basis of their substrate specificity than enzymes of family 11. As
different physicochemical properties, such as a rule, the EXs of family 10 catalyze hydrolysis of
molecular mass and isoelectric point, rather than cellulase substrates, aryl p -D-cellobiosides (van
on the basis of different catalytic properties. A Tilbeurgh and Claeyssens, 1985) at the agluconic
careful comparison of EXs by Wong et al. (1988) linkage (Grepinet et al., 1988; Gilbert et al., 1988;
indicated that these enzymes can be divided into Ltithi et al., 1990; Shareck et al., 1991; Hass et al.,
two groups, one consisting of high-molecular- 1992). EX of Dichomitus squulens (Rouau and
mass enzymes with low pI values, and the other Odier, 1986) and EXII of Schizophyllum commune
consisting of low-molecular-mass enzymes with (Biely, 1987) which catalyze the same reaction, are
high p1 values. As usual, there are exceptions. apparently members of the family 10. The low-
Interestingly, the grouping of EXs in the work by molecular-mass EXs do not possess this catalytic
Wong et al. (1988) was found to be in consonance property. A common feature of both enzyme
with the classification of glycanases on the basis families are their endocharacter demonstrated vis-
of hydrophobic cluster analysis and sequence sim- cosimetrically and the double displacement mech-
ilarities (Gilkes et al., 1991a,b; Shareck et al., anism of the hydrolysis of glycosidic bond, which
1991; Yaguchi et al., 1992; Henrissat and Bairoch. means that both types of enzymes are retaining
1993; Tiirriinen et al., 1993). The hydrophobic glycanases (Withers et al., 1986; Gebler et al.,
cluster analysis is a comparative method designed 1992; Biely et al., 1994).
to predict protein folding based on hydrophobic/ The need to elucidate structureefunction rela-
hydrophilic patterns and is used to correlate mem- tionship among EXs is obvious. Progress in this
bers of a protein group of similar catalytic direction is somewhat hindered by the fact that
functions (Gaboriaud et al., 1987). Acidic high- representatives of the two EX families produced
molecular-mass EXs ( > 30 kDa) were found to by a single microorganism, have been purified and
belong to one family, assigned as glycanase family sequenced in two cases only. During growth on
10 (Henrissat and Bairoch, 1993; formerly family xylan, Streptomyces lividuns produces three genet-
F), and basic low-molecular-mass EXs, were ically distinct EXs designated, XlnA, XlnB and
found to belong to another family, designated as XlnC (Shareck et al., 1991). XlnA belongs to
glycanase family 11 (formerly family G). As far as family 10 of glycanases, while XlnB and XlnC are
the protein structure of the members of the two members of family 11 (Shareck et al.. 1991). An
families is concerned, the most conserved region analogous situation occurs with three EXs of
within a family appears to be the catalytic do- Aspergillus kuwachii (Ito et al., 1992). Although
main. The last published list of identified mem- only the EX of family 11 of Schizophyllum com-
bers of the two EX families (Hazlewood and mune has been characterized (Yaguchi et al.,
Gilbert, 1993; Coughlan and Hazlewood, 1993) 1992), there is indication for the existence of EX
includes 10 species of family 10 and 17 species of of family 10 in this microorganism (Biely et al.,
family 11 (Henrissat and Bairoch, 1993). Family 1986a). The enzyme differs from the 11 type EX
10 can be updated by EXs from Thermoanarr- in that it degrades acetylxylan to a much higher
ohacterium .scxcharolyticunz B6A-RI (Lee et al., degree. However, because an analogous difference
1993) and from an alkalophilic Bucillus strain in the pattern of hydrolysis of acetylxylan has
(Tabernero et al., 1995) and family 11 by two EXs been observed with the two EXs of T. rersei (Biely
of T. rresei (Tiirronen et al., 1992) and XynA of et al., 1993b), both belonging to glycanase family
Aureobasidium pulluluns (Li and Ljungdahl, 1994). 11, but significantly different (Torriinen et al.,
The latest data-base information on glycanase 1992) several substrates are required to differenti-
families shows 23 EXs in family 10 and 17 in ate between catalytic properties of EXs of families
family 11 (Henrissat and Bairoch, 1996). 10 and 11.
 P. Biely el al. /Journal of t?iolechnology 57 (1997) 151- I66 153

In this work, the action of two members of merit xylotriose, Xyl,& 1-3XylB 1-4Xyl-/?-O-Me
family 10 and four members of family 11 on (Kovac and Hirsch, 1980), was a gift from Di J.
various polysaccharide and oligosaccharide sub- Hirsch (Institute of Chemistry, Slovak Academy
strates was investigated with the aim of contribut- of Sciences, Bratislava). Aldotriuronic acid
ing to further understanding of the (MeGlcAcr I-2Xylp l-4Xyl) and aldotetrauronic
structure-function relationship among EXs and acid (MeGlcAcr 1-2Xylp 1-4Xyla 1-4Xyl) were
to elucidate the reasons of their multiplicity. from Megazyme (Australia).

2.3. Enzyme-substrate mixtures

2. Materials and methods
Solutions (2% (w/v)) of polysaccharides in 0.05
2.1. Enzymes M pyridine-acetic acid buffer (pH 5.0 for fungal
enzymes and pH 6.0 for Streptomyces enzymes)
Family 10 EXs: Cryptococcus EX (48 kDa, p1 were incubated with appropriately diluted enzyme
5.0) and XlnA of S. lividans (47 kDa, p1 5.2) were solutions at 40°C and the products of hydrolysis
purified as described (Biely and VrSanska, 1986; were analyzed by TLC on microcrystalline
Morosoli et al., 1986). cellulose (Merck) in the solvent system ethyl ac-
Family 11 EXs: XlnB (31 kDa, p1 8.4) and etate/acetic acid/water (3:2:2, by vol.). Two-di-
XlnC (22 kDa, p1 10.25) of S. Zividans were mensional TLC under the same conditions, with
purified from culture filtrates of genetically engi-
deacetylations between the runs, was used for
neered clones harbouring the plasmids with the
analysis of acetylated xylooligosaccharides gener-
corresponding EX genes as described in previous
ated from acetylxylan (Biely et al., 1985). Reduc-
papers (Kluepfel et al., 1990, 1992); EXI (19 kDa,
ing sugars were visualized by the aniline-hydrogen
p1 4.8-5.5) and EXII (20-21 kDa, p1 8.5-9.0) of
phthalate reagent. The enzymes were tested also
T. reesei were purified by ion-exchange chro-
with synthetic substrates such as 4-nitrophenyl-p-
matography and size-exclusion chromatograhy
D-xylopyranoside, -cellobioside and isomeric xy-
(Tenkanen et al., 1992).
lotrioside, Xyla l-3Xylp 1-4Xyl-P-O-Me. Liber-
ation of the aryl aglycon was followed by TLC
2.2. Saccharides
followed by visualization under UV-light and the
4-O-methyl-D-glucurono-D-xylan formation of reducing sugars as given above. Sim-
Beechwood
containing Xyl and 4-O-methyl-D-glucuronic acid ilar large-scale incubations of polysaccharides in
(MeGlcA) in the ratio 7:l was isolated from saw- water containing 0.01% sodium azide were run to
dust (Biely et al., 1986b). Beechwood acetyl4-O- isolate the selected products of hydrolysis.
methyl-D-glucurono-D-xylan was obtained by
DMSO extraction of holocellulose (Bouveng et 2.4. Isolation and identification of products of
al., 1960), and rhodymenan from the seaweed hydrolysis
Rhodymenia stenogona, a linear homoxylan hav-
ing /3 - 1,4- and p - 1,3-linkages in the ratio 2: 1, was Products of hydrolysis of polysaccharides by
a gift from Prof. A.I. Usov (Russian Academy of various EXs were isolated from lyophilized and
Sciences, Moscow). Xylooligosaccharides, Xyl, concentrated reaction mixture by preparative
through Xyl,, were purchased from Megazyme chromatography on water-washed Whatman No.
(Australia). Linear oligosaccharides containing 3MM paper in the solvent sytem ethyl acetate/
one ,&-1,3-linkage, XylB 1-3Xylj3 l-4Xyl and acetic acid/water (18:7:8, by vol.). The acidic na-
Xylp l -4Xyla 1-3Xylp I-4Xy1, were prepared from ture of some products of 4-O-methyl-D-
phenyl j?-D-xylopyranoside by glycosyl transfer glucurono-D-xylan hydrolysis was established by
reactions catalyzed by EX of C. albidus (Biely and their adsorption on Dowex 1 (acetate form) or by
VrSanska, 1983). Methyl P-glycoside of the iso- TLC on cellulose in the solvent system ethyl
154 P. Biely et al. /Journal of Biotrchnology 57 (1997) I.51 -166

Table I
TLC mobility of shortest acidic products liberated from 4.O-methyl-o-glucurono-[I-xylan by EXs of family IO (F) and I I (G)

Standard compounds Products liberated

Enzyme Family Relative chromatographic mobility (Rx,,)

XYlz 0.72
XYl, 0.47
XYL 0.28
XYl, 0.1I
MeGlcAXyl, 0.53
C. albidus EX 10 (F) 0.55
S. lividans XlnA 10 (F) 0.55
T. rersei EXI 11 (G) 0.36
T. reesei EXII 11 (G) 0.37
S. liaidans XlnB 11 (G) 0.39
S. lividam XlnC 11 (G) 0.39

acetate/pyridine/water (5:3:2, by vol.). Aldouronic ucts liberated by EXs of family 10 and 11 con-
acids show extremely slow mobility under these cerned the length of the products containing
conditions in comparison with neutral oligosac- 4-O-methyl-D-glucuronic acid. The enzymes of
charides. Three runs in the system are required to family 10, i.e. EXs of C. ulbidus and XlnA of S.
achieve a good separation of aldouronic acids. lividans, liberated aldouronic acid having chro-
Rhodymenan hydrolysates were also analyzed by matographic mobility of aldotetrauronic acid
HPLC on an Aminex HPX-42A column (Biorad; (MeGlcAXyl,) as the shortest acidic oligosaccha-
Kluepfel et al., 1990). The methods used to eluci- ride (Table 1). This aldouronic acid was never
date the structure of products of hydrolysis in- found in the hydrolysates of the EXs of family 11.
cluded NMR-spectroscopy, mass spectrometry XlnB and XlnC of S. lividuns and EXs of T. reesei
and electrospray mass spectrometry of per-O- liberated acidic oligosaccharide larger than those
methylated oligosaccharides, periodate oxidation, produced with the enzymes of family 10, the
Smith degradation and enzymic hydrolysis by As-
shortest fragment being presumably an aldopen-
pergillus niger p-xylosidase and C. ulbidus EX, as
tauronic acid.
described earlier (Biely and VrSanska, 1983).
The shortest acidic oligosaccharides from the
reaction mixtures of both EXs families were iso-
lated by preparative paper chromatography and
3. Results
subjected to structural analysis. The structure of
the aldouronic acid liberated by EXs of family 10
3.1. Products of 4-O-methyl-D-glucurono-D-xylan
were in both cases established as 2”-O-a-(4-0-
hydrolysis
methyl-x-D-glucuronosyl)-xylotriose (MeGlcAx l-
The polysaccharide was incubated with the en- 2-Xylp 1-4Xylp l-4Xy1, MeGlcAXyl,) based on
zymes for several days to assure the maximum the following criteria. The compound showed
extent of its hydrolysis. The mixtures were ana- chromatographic mobility identical to that of the
lyzed by TLC to compare the products of hydrol- standard aldotetrauronic acid. The electrospray
ysis. The tested enzymes differed considerably in mass spectrometry of the per-0-methylated and
the amont of liberated monosaccharide in relation esterihed oligosaccharide gave molecular ions
to Xyl, and Xyl,, however, these differences did compatible with an aldotetrauronic acid. The
not reflect the grouping of the enzymes into two compound was resistant to P-xylosidase which
families. The main difference between the prod- means it contains at the non-reducing end a xy-
 P. Bidy et al. /Journal oj’ &otechnofogy 57 (1997) 151- 166 155

xylpl-4xylpl-4xylpl-4xyl Xyl + xy1p 1-4xy1p 1-4Xyl

2 B-xylosidase 2
I > I
Ctl EX CL1
MeGlcA MeGlcA
Scheme I.

lopyranosyl residue substituted with MeGlcA. The 13C-NMR spectra of the shortest acidic
The linkage of MeGlcA to the non-reducing D-xy- fragments liberated by EXs of family 10 and 11
lopyranosyl residue of Xyl, was also confirmed by were fully consistent with the deduced structures.
the ‘H-NMR spectrum in which only one doublet The assignment of the most important signals
signal corresponding to the anomeric proton of (Fig. 1, Table 2) was done on the basis of pub-
MeGlcA at 5.2 ppm was observed. Substitution of lished data for analogous compounds (Cavagna et
the first or the second xylopyranosyl residue from al., 1984; Excoffier et al., 1986; Debeire et al.,
1990).
the reducing end results in two doublets corre-
sponding to the anomeric proton of MeGlcA, as a
3.2. Products of rhodymenun hydrolysis
consequence of the a,/?-anomeric equilibrium of
the reducing terminal xylopyranosyl residue (De-
It has been mentioned earlier that rhodymenan
beire et al., 1990).
is hydrolyzed by both EXs of both families, how-
The structure of the shortest acidic fragment ever, to a higher degree by EXs of family 10
liberated from 4-O-methyl-D-glucurono-D-xylan (Biely et al., 1993a). To illustrate this fact, Fig. 2
by EXs of family 11 was established as aldo- shows the resolution of products of rhodymenan
pentauronic acid 2”-0-sr-(4-o-methyl-cr-D-glu- hydrolysis by XlnA and XlnB of S. Iividuns. XlnA
curonosyl)-xylotetraose (Xyl-p - 1,4-[MeGlcA-l- was capable of almost complete depolymerization
1,2-]Xyl-p - 1,4-Xyl-p - 1,4-Xyl, MeGlcAXyl,) on of the polysaccharide to oligosaccharides. XlnB,
the basis of the following criteria. The compound as a representative of the family 11, left a consid-
showed slower chromatographic mobility than al- erable part of this polysaccharide undegraded as a
dotetrauronic acid. The electrospray mass spec- high-molecular-mass fraction. The rhodymenan
trometry of the per-O-methylated and esterified hydrolysate by XlnB does not contain an isomeric
derivative afforded molecular ions compatible xylotriose (isoxyl,), which was also confirmed by
with aldopentauronic acid. The substitution of an TLC analysis. The liberation of the isoXyl,, which
internal xylopyranosyl residue was suggested by is typical also for EX of C. albidus (Biely and
periodate oxidation and Smith degradation pre- VrSanska, 1983), does not occur in the case of all
four EXs of family 11 tested. The shortest iso-
serving one D-xylose residue. The ‘H-NMR spec-
merit xylooligosaccharide liberated from rhody-
tra pointed out that MeGlcA is not linked to any
menan by EXs of family 11 is a xylotetraose,
of the first two xylopyranosyl residues from the
designated as isoXy1, (Table 3).
reducing end. The anomeric proton of MeGlcA
The isomeric trisaccharide liberated from
appeared as one doublet at - 5.2 ppm, unaffected
rhodymenan by EXs of family 10 was isolated by
by the cr,a-equilibrium of the reducing terminal preparative paper chromatography and subjected
(Debeire et al., 1990). The substitution of the to structural analysis. On thin-layers of cellulose,
second xylopyranosyl residue from the non-reduc- the trisaccharide showed chromatographic mobil-
ing end by MeGlcA was unambiguously demon- ity between Xyl, and Xyl,, which is an indication
strated by its conversion to aldotetrauronic acid that it contains one linkage other than p-1,4.
and xylose upon incubation with A. niger a-xy- Mass spectrometry of the fully methylated
losidase or C. albidus EX (Scheme 1). product revealed that the isomeric trisaccharide
156 P. Biely et al. /Journal qf Biotechnology 57 (1997) 151~ 166

PPfl

I I I 1 - I
105 100 95 so 85

Fig. I. Anomeric regions of the “C-NMR spectra of aldouronic acids liberated from 4-O-methyl-D-glucurono-u-xylan by EXs of
family 10 (part A) and family II (part B). The signal around 83.5 ppm corresponds to C-4 of MeGlcA residue.

contains one p - 1,3-linkage and one /I - 1,4-linkage. p-1,4-linkage is attacked much faster than ,8-l ,3-
The 13C-NMR spectrum of the trisaccharide was linkage, D-xylose was found as the only product
consistent with the structure 3-0-/I-D-xylopyra- of hydrolysis by ,5’-xylosidase. Xyl, as the second
nosyl-4-0-/I-D-xylopyranosyl-D-xylose (XYW l- initial product of hydrolysis was cleaved as soon
3XylBl-4Xyl) (Kovac et al., 1980). This structure as it had been formed. The cleavage at the p - 1,3-
was supported by the action of p-xylosidase. The linkage to give D-XylOSe and Xyl, was obtained by
isomeric trisaccharide was hydrolyzed by the en- the action of C. albidus EX (Biely and VrSanska,
zyme only slowly in comparison with the rate of 1983). The action of the two enzymes on the
hydrolysis of Xyl, or Xyl,. Due to the fact, that isomeric xylotriose is shown in Scheme 2.
 P. Siely et al. /Journal of Biotechnology 57 (1997) 151~ 166 157

Table 2 isolated. It is anticipated that it contains a ,8-1,3-

“C-NMR data for anomeric carbons in aldotetrauronic acid linkage in the middle.
and aldopentauronic acid liberated from 4-O-methyl+glu-
curono-r)-xylan by EXs of family 10 (F) and I I (G), respectively
3.3. Products of acetylxylan hydrolysis
Carbon Xyl/I I -4XylB I-4Xyl XylB I -4Xylp 1-4Xyl/~’ I-4Xyl
2 2 Beechwood acetylxylan was found to be a sub-
strate which does not allow differentiation of EXs
11 rl
belonging to family 10 and 11. Two-dimensional
MeGlcA MeGlcA
TLC analysis of acetylated products generated by
(ppm) (ppm) EXs of family 10 pointed out that these enzymes
tolerate well the small substitutents on the main
C-lx 93.09 C-ID! 93.12 xylan chain and liberate acetylated Xyl, and Xyl,
c-1g 97.58 c-1p 97.59
in significant quantities. No acetylated Xyl, was
C-l’ 102.71 C-l’ 102.46
C-l” 102.71 C-l” 102.75 found in the hydrolysate by XlnB and XlnC of S.
C-I”’ (acid) 98.68 C-l”’ 103.06 lividans and EXII of T. reesei. Similar results were
C-l”” (acid) 98.67 obtained earlier with EXI of Schizophyllum com-
mune, an EX of family 11 (Biely et al., 1987).
However, EXI of T. reesei, as a member of family
The isomeric tetrasaccharides liberated from
11, showed the pattern of acetyl xylan hydrolysis
rhodymenan by EXs of family 11 has not been as did EXs of family 10. They both released
considerable amounts of acetylated dimer and
trimer.

3.4. Action on urtiJicia1substrates

We have already mentioned that EXs of family

10 in contrast to EXs of family 11, tolerate the
replacement of xylopyranosyl units in aryl xylo-
biosides by glucopyranosyl units. EXs of family
10 hydrolyze aryl cellobiosides at the agluconic
linkage similarly as xylobiosides. In this work we
confirm that the two tested representatives of
family 10 liberated 4-nitrophenol and 4-methy-
lumbelliferone and cellobiose from the corre-
sponding cellobiosides. The substrates were

j$Y&_
J, , ,
resistant to the action of all four tested EXs of
family 11.
X,“e Additional synthetic substrate for differentia-
tion of EXs of the two families is 4-nitrophenyl
p-D-xylopyranoside (NPh-Xyl). The glycoside is
practically not attacked by EXs of family 11, but
0 5 10 15 20 serves as a substrate for EXs of family 10. The
Retention time (mln) reaction rate with both enzymes of family 10
showed a sigmoidal dependence on substrate con-
Fig. 2. HPLC of products of a long-term hydrolysis for centration. At higher substrate concentrations the
rhodymenan by XlnA and XlnB of S. ficidans. Symbols: Xyl,,
enzymes catalyze the degradation of the substrate
/i-l ,4-xylooligosaccharides; isoXyl,, xylooligosaccharides con-
taining /&1,4-xylosidic linkages and at least one p-1,3-xylo- by a complex reaction pathway which involves a
sidic linkage. series of glycosyl transfer reactions followed by
158 P. Biely et al. /Journal of’ Biotechnology 57 (1997) 151-166

Table 3
TLC mobility of the shortest P-1,3-linkage-containing products liberated from rhodymenan by EXs of family IO (F) and I I (G)

Standard compounds Products liberated

Enzyme Family Relative chromatographic mobility (R,,,)

XYh 0.72
XYh 0.47
XYl, 0.28
XYl, 0.11
isoXyl, (Xyl/I 1-3Xylb 1-4Xyl) 0.62
isoXy1, (XyljI 1-4Xylp 1-3Xylp I -4Xyl) 0.38
C. alhidus EX 10 (F) 0.61
S. licidans XlnA 10 (F) 0.62
T. reesei EXI 11 (G) 0.40
T. veesei EXII 11 (G) 0.43
S. licidans XlnB 11 (G) 0.44
S. liridans XlnC 11 (G) 0.44

hydrolysis. The reaction mixture contains signifi- 4. Discussion

cant amounts of lower xylooligosaccharides and
4-nitrophenyl P-D-xylobioside and -trioside, Comparison of the mode of action on various
which function as much better xylosyl donors substrates of EXs representing members of family
than NPh-Xyl. They also represent the primary 10 (formerly family F) and family 11 (formerly
precursors of generated xylooligosaccharides. The family G), under the same conditions, may afford
critical, rate-limiting step in this reaction pathway data to elucidate the structureefunction relation-
is the formation of NPh-Xyl, and NPh-Xyl,. ship in this group of glycanases. The literature
Therefore, the liberation of NPh-OH can be accel- data on catalytic properties of variety of EXs, e.g.
erated by addition of /J’- 1,4-xylooligosaccharides see the early review by Dekker and Richards
(1976), are vast. However, they are not very help-
serving as glycosyl donors for the formation of
ful because the information on the enzyme family
4-nitrophenyl glycosides of oligosaccharides. Slow
is absent. Earlier data were accumulated before
and faster reactions of aryl P-D-xylopyranoside
the existence of the various glycanase families had
degradation by EXs of family 10 are outlined in
been recognized. For this reason the results ob-
Scheme 3. For simplicity, the hydrolytic cleavages
tained in the present study are compared only
of xylooligosaccharides larger than xylobiose are
with such data from sources where the enzyme
not presented. family can be deduced either on the basis of its
The last substrate which was found to be useful physico-chemical properties or on characteristic
for differentiation of EXs of family 10 and 11 is catalytic properties.
methyl j?-D-glycoside of the isomeric xylotriose
which is identical with that liberated from rhody- 4. I. Action on heteroxyluns and rhodymenan
menan by EXs of family 10, Xyla l -3Xylp 1-4Xyl-
Me. The compound serves as substrate for EXs of It can be concluded that EXs belonging to
family 10 only. At low substrate concentrations, glycanase family 10 hydrolyze heteroxylans and
0.552 mM, the glycoside is cleaved almost exclu- rhodymenan to a higher degree than EXs of fam-
sively to give trisaccharide and methanol (Scheme ily 11. The EXs of family 10 show better capabil-
4). Under similar conditions, the glycoside is resis- ity of cleaving glycosidic linkages in the xylan
tant to EXs of family 11. main chain closer to the substituents, such as
 P. Biely et al. /Journal of Biotechnology 57 (1997) 151-166 159

XYl
t B-xylosidase
EX
xylpl-3xylpl-4xyl ________) Xyl + xy1p1-4xy1

Scheme 2.

Slow reactions:
NPh-Xyl + Xyl + NPh-OH dd
NPh-Xyl + NPh-Xyl -+ NPh-Xyl, + NPh-OH
NPh-Xyl,! + NPh-Xyl + NPh-Xyl, + NPh-OH

Faster reactions:

NPh-Xyl, + Xyl, + NPh-OH

NPh-Xy13 -+ Xy13 + NPh-OH
NPh-Xyl + Xy13 -+ Xylz + NPh-Xy12

Scheme 3.

XylPl-3Xylpl-4XyLMe +Xyl~1-3Xyl~1-4Xyl + MeOH

Scheme 4.

MeGlcA and acetic acid. The disturbance of the EXs of family 10, in contrast to EXs of family 11,
xylan main chain by replacing /? -1,4-linkages by are capable of attacking the glycosidic linkage next
/?- 1,3-linkages, like it is in rhodymenan, represents to the branch and towards the non-reducing end.
a more serious steric barrier for EXs of family 11 While EXs of family 10 require two unsubstituted
than for EXs of family 10. In consonance with xylopyranosyl residues between the branches, EXs
these considerations, EXs of family 10 liberate of family 11 require three unsubstituted consecu-
from 4-0-methyl-D-glucurono-D-xylan, rhody- tive xylopyranosyl residues (Scheme 5).
menan and, with some exceptions, also from A similar scheme can be drawn for the mode of
acetylxylan, smaller products than those formed action on rhodymenan. EXs of family 10 attack
with EXs of family 11. It has been clearly demon- the polysaccharide at /?-1,4-linkages which do not
strated in this study and elsewhere (Biely et al., follow a /?-1,3-linkage towards the reducing end.
1993a) that an EXs of family 10 can further Since the isomeric xylotriose, Xyla l-3Xylp l-4Xy1,
hydrolyze the shortest branched or isomeric xy- does not occur among the products of rhody-
looligosaccharides which are released from the menan hydrolysis by EXs of family 11, these
corresponding polysaccharides by EXs of family enzymes obviously attack fi - 1,4-linkages which are
11. more distant to the /2-1,3-linkage. Although the
The identification of the shortest acidic frag- structure of the tetrasaccharide released from
ments released from 4-O-methyl-D-glucurono-D- rhodymenan by the family 11 enzymes has not
xylan allows to propose the linkages which are been established, two different modes of attack
accessible to hydrolysis by the two types of EXs. can be proposed (Scheme 6).
160 P. Biely et al. /Journal of Biotechnology 57 (1997) 151.. 166

EXs 104 -1 -1 J -1
-4xylpl-4xylpl-4xylpl-4xylpl-4xylpl-4xylpl-4xylpl-4xylpl-4xylpl-4xylp1-
EXs 11 ?- 2 ? 2 2
I I I
al al al
MeGlcA MeGlcA MeGlcA
Scheme 5

EXsF L & 1 L
-3xylp1-4xylpl~4xylpl-4xylpl-3xylpl-4xylpl-~l~l-4xyl~l-3xyl~l-4xyl~l-
EXs G (mode 1)
? T (mode 2)

Scheme 6.

The situation with rhodymenan is, however, Sporotrichum dimorphosporum (Comtat and Jose-
complicated by the fact that some EXs, at least leau, 1981) and Polyporus tulipzjkue (Brillouet,
the EXs of family 10, are capable of cleaving also 1987) was documented by isolation of oligosac-
p-1,3-linkages (Biely and VrSanska, 1983; Chen et charides of the structures MeGlcAx l -2-Xylb l-
al., 1986). 4Xylb l-4Xyl and L-Araa 1-3Xy1/3 l-4Xyl. In both
The above schemes resemble the degradation of oligosaccharides the substituent is attached to the
a cereal t_-arabino-D-xylan with two EXs of As- non-reducing D-xylopyranosyl unit. There are sev-
pergillus awamori, which, unfortunately, have not eral exceptions from the above mode of action.
been classified by hydrophobic cluster analysis. For instance, the structure of the most abundant
Nevertheless, based on physico-chemical proper- substituted oligosaccharides obtained from glu-
ties the enzymes appear to be representatives of
curonoxylan (MeGlcAa l-2-Xylp I-4Xylp 1-4Xyl)
the two families: EXI (39 kDa, p1 5.776.7), most
and arabinoxylan (4-0-b-D-xylopyranosyl-[3-0-
probably a member of family 10, and EXIII (26
x-L-arabinofuranosyll-4-0-/?-D-xylopyranosyl-~-
kDa, p1 3.333.5), most probably a member of
xylopyranose) upon the action of EXs of Strepto-
family 11 (Kormelink et al., 1993b). As shown on
myces E86 (Yoshida et al., 1990; Kusakabe et al.,
the Scheme 7, modified from Kormelink et al.
1983) and A. oryzae EXII (28 kDa, p1 6.9; Tele-
(1993a), EXI and EXIII differ in the number of
man et al., 1996) suggests that the ability of such
cleavage sites of a putative L-arabinosyl-D-xylan
EXs to cleave glycosidic linkage adjacent to the
in a way as do EXs of family 10 and 11 on
4-O-methyl-D-glucurono-D-xylan. The L-ara- substituent on the main chain may depend on the
binosyl substituents represent a more serious position of the substituent. The substitution at
steric hindrance for the formation of productive position 3 could create a more serious steric ob-
complexes of the polysaccharide with EXIII than struction for an enzyme to attack the glycosidic
with EXI. Only the larger enzyme is capable of oxygen than the substitution at position 2.
attacking the linkages between substituted and Action pattern on acetylxylan may become an
unsubstituted xylopyranosyl residues. The cleav- additional criterium for a finer classification of
age of the glycosidic linkage in the vicinity of both EXs. Mono-acetylated Xyl, is a characteristic
4-O-methyl-D-glucuronosyl or L-arabinofuranosyl fragment of hydrolysis of acetylxylan by EXs of
branches in arabinoglucuronoxylan by EXs of family 10. Of the 4 EXs of family 11 investigated
 P. Sk/y et al. /Journal of Biofeehnology 57 (1997) 151~ 166 161

LAra
CXl
I
EXI$ & L J 2 J
-4xy1p 1-4xy1p 1-4xy1p l -4xy1p 1-4xy19 1-4xylpl-4xyl-xylpl-4xylp1-4xylp1-
EXIII ‘? 3 f 3 3
I I I
al al al
LAra LAra LAra
Scheme 7.

in the present study, only the EXI of T. reesei increase of their polymerization degree, starting
degraded acetylxylan to products analogous to with xylotriose, is much greater with EXs of
those generated by family 10 EXs. This observa- family 11 than with EXs of family 10. These data
tion is in a consonance with differences in the speak for the fact that, in spite of a higher molec-
three-dimensional structures of EXI and EXII of ular mass, EXs of family 10 have smaller sub-
T. reesei (Tbrriinen and Rouvinen, 1995) and strate binding sites than EXs of family 11. Four
suggests that there are further differences in cata- subsites may be proposed for EXs of family 10
lytic properties among EXs belonging to family (Biely and VrSanska, 1986) however, more than
11. One can expect the existence of subfamilies four subsites may be anticipated in substrate bind-
within the family 11 reflecting the wide range of ing sites of EXs of family 11 (Biely et al., 1983;
the sequence identity data of the enzymes of Meagher et al., 1988; Bray and Clarke, 1992). A
family 11, ranging from 42.5 to 99.5% proposal for a three subsite substrate binding site
(Wakarchuk et al., 1992). The enzymes of family of EXI of T. reesei (Tiirriinen and Rouvinen,
11 can be classified into three groups (Yaguchi et 1995) does not find support in kinetic data with
al., 1992). oligosaccharides.

4.2. Action on oligosaccharides 4.3. Relation to tertiary structures

Although both types of EXs show preference to The greater catalytic versatility of EXs of fam-
attack internal glycosidic linkages in xylans, the ily 10 in comparison with that of family 11, can
structure of the final products of hydrolysis of be ascribed to differences in their tertiary struc-
heteroxylans suggests that EXs of family 10 pos- tures which has been established by crystallogra-
sess several catalytic activities which are compat- phy (Fig. 3). EXs of family 11 appear to be
ible with p-xylosidases. The EXs of family 10 smaller and well-packed molecules, formed mainly
liberate terminal xylopyranosyl residues attached of a-sheets (Katsube et al., 1989; Campbell et al.,
to a substituted xylopyranosyl residue, but they 1993; Wakarchuk et al., 1994; T&-r&en et al.,
also exhibit aryl P-D-xylosidase activity. Detailed 1994). These enzymes appear very small in native
studies with linear xylooligosaccharides carried state (Dean and Anderson, 1991; Grabski and
out with two EXs of family 10 and several species Jeffries, 1991). The catalytic groups are present in
of family 11 pointed to a relatively higher cata- the cleft that accommodates a chain of from five
lytic efficiency of degradation of shorter xy- to seven xylopyranosyl residues.
looligosaccharides of EXs in family 10 (Biely and The crystal structure of the 32 kDa catalytic
VrSanska, 1986; Biely et al., 1993a). Increase in domain of XlnA of S. lioiduns points to significant
the rates of oligosaccharide hydrolysis with the differences between construction of EXs of family
162 P. Biely et al. /Journal of Biotechnolog~~ 57 (1997) 15L 166

EX of family 10 EX of family 11
Fig. 3. Ribbon representation of the main fold of the catalytic domains of EXs of family 10 and 1 I P-strands are shown in gray
and cc-helices in black. Courtesy of Dr B. Henrissat (Davies and Henrissat, 1995).

10 and 11 (Fig. 3). The tertiary fold of XlnA is a points to the tolerance of the two subsites of the
typical S-fold E/B barrel (~18)~ resulting in a substrate binding site of these enzymes to the
‘salad bowl’ shape of the molecule (Derewenda et replacement of one or two consecutive D-xylopy-
al., 1994; Prade, 1995). The substrate binds to the ranosyl residues by D-glucopyranosyl residues in
shallow groove on the bottom of the ‘bowl’. The substrates. Kitaoka et al. (1993) demonstrated
(E/B)~ barrel appears to be the structure of two that some high molecular-mass EXs hydrolyze
other EXs of family 10, EX A from Pseudomonas agluconic bond in substrates containing more
fluorescens (Harris et al., 1994) and XynZ from than two consecutive glucopyranosyl residues,
Clostridium thermocellum (Dominguez et al., namely in 4-nitrophenyl b-~glycosides of larger
1995) however, the binding sites of these two cellooligosaccharides. Regarding this property it
enzymes were not considered to be as flat as in the could be expected that EXs of family 10 will also
case of XlnA of S. litliduns. Anyway, the substrate attack the aglyconic bond of glycosides of b-1.4-
binding sites of the family 10 EXs are apparently linked oligosaccharides composed of D-glucose
not such deep clefts as the substrate binding sites and D-xylose (Scheme 8).
of family 11 EXs. This fact together with a possi- Aryl glycosides of 4-0-j?-n-glucopyranosyl-D-
ble greater conformational flexibility of the larger xylopyranoside have already been synthesized
enzymes than of the smaller enzymes, may ac- (Kitaoka et al., 1993; Christakopoulos et al.,
count for a lower substrate specificity of family 10 1995). Three EXs hydrolyzing aglyconic bonds in
EXs. 4-nitrophenyl /J-D-glycosides of homo- and het-
erodisaccharides, showed much lower affinity for
4.4. Action on urtiJiciu1 substrates cellobioside than for xylobioside and ~-O-/J-D-
glucopyranosyl-b -D-xylopyranoside (Kitaoka et
All so far tested EXs were capable to hydrolyze al., 1993). Relevant kinetic data are also available
aryl p-glycosides of xylobiose and xylotriose at for EXs of Cellulornonas fimi, designated Cex, a
the aglyconic bond (Biely et al., 1992; Kitaoka et member of family 10 (Bedarkar et al., 1992). The
al., 1993; Christakopoulos et al., 1995), therefore catalytic efficiency (k,.,/K,,,) of the enzyme for
these two synthetic substrates do not seem to be hydrolysis of 4-nitrophenyl B-D-xylobioside was
suitable for a ‘yes and no’ enzyme differentiation. approximately 50-fold higher than for hydrolysis
The opposite is true with aryl p-D-cellobiosides. of the cellobiose analogue. The information that
Hydrolysis of aryl P-D-cellobiosides at the aglu- one of two low-molecular-mass EXs of Fusarium
conic bond, exhibited by EXs of family 10 only, oxysporum liberates 4-methylumbelliferone from
 P. Biely et al. /Journul of Biotechnology 57 (1997) 151-166 163

EXs 10 -1 J -1 &
Xylp l-4XylP-R GlcP 1-4GlcP-R Glcf3 1-4Xyl@R Xylp 1-4GlcfSR
EXs 11 ? ( ,I-?>
Scheme 8.

EXslOd L J -1 L -4
-4Xyl~l-4Xyl~l-4Glc~l-4Xyl~l-4Xyl~l~~l-4Glc~l-4Glc~l-4Xyl~l~Xyl~l-
EXsll? T T
Scheme 9.

4-methylumbelliferyl-/? -D-glucopyranosyl-P -D-xy- Acknowledgements

lopyranoside (Scheme 7, arrow in brackets) but
not from the corresponding cellobioside, suggests The authors are indepted to Mrs M. Cziszrirova
that the glycosides of mixed-type oligosaccharides and Mrs N. Gaigneault for excellent technical
may become useful tools for further differentia- assistance. The authors thank Dr J. Alfiildi for
tion of EXs of family 11 (Christakopoulos et al., NMR measurements, Dr R.D. Plattner (USDA,
1995). Aryl glycosides of Xyl/3 l-4Glcb which ARS, Peoria, Illinois, USA) for electrospray mass
have not been synthesized yet, may be expected to spectrometry and Dr B. Henrissat (CERMAV,
be resistant to all enzymes that do not cleave Grenoble, France) for providing the picture of
cellobiosides, but could be useful for further clas- three-dimensional structures of endoxylanases.
sification of EXs of family 10. This work was supported by a grant 2/1095 from
Thus, the ability to cleave the /?-1,6glucosidic the Slovak Grant Agency for Science (VEGA).
linkage to D-xylopyranosyl residue is one of the
major differences between EXs of family 10 and
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