Xylanolytic Enzymes from Fungi and Bacteria

Critical Reviews in Biotechnology, 17(1):39-67 (1997)

Xylanolytic Enzymes from Fungi and Bacteria A. Sunna and G. Antranikian* Technical University Hamburg-Harburg, Institute of Biotechnology, Department of Technical Microbiology, Denickestrasse 15, 21071 Hamburg, Germany

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*

Address all correspondence to G. Antranikian, Technical University Hamburg-Harburg, Institute of Biotechnology, Department of Technical Microbiology, Denickestrasse 15, D-21071 Hamburg, Germany; Phone: +4940-77 18-3117; Fax: +4940-77 18-2909; E-mail: antranikian @tu-harburg.d400.de

ABSTRACT: The development of new analytical techniques and the commercial availability of new substrates have led to the purification and characterization of a large number of xylan-degrading enzymes. Furthermore, the introduction of recombinant DNA technology has resulted in the selection of xylanolytic enzymes that are more suitable for industrial applications. For a successful integration of xylanases in industrial processes, a detailed understanding of the mechanism of enzyme action is, however, required. This review gives an overview of various xylanolytic enzyme systems from bacteria and fungi that have been described recently in more detail.

KEY WORDS: xylan, endoxylanase, P-xylosidase, a-arabinofuranosidase, a-glucuronidase, esterases, synergism.

1. INTRODUCTION Xylan is the major component of the plant cell wall and the most abundant renewable hemicellulose (Whistler and Richards, 1970). Xylans are structurally of mixed composition. Thus, the enzymatic degradation of the substrate to its monomer, xylose, is a complex process involving a battery of enzymes (Biely, 1985; Zimmermann, 1992). Endoxylanase and P-xylosidase have the most important activities among the xylanolytic enzymes involved in xylan hydrolysis. Side-chain cleaving enzymes like a-arabinofuranosidase, a-glucuronidase, and acetylxylan esterase play important roles in the removal of side substituents of heteroxylans. Substituent groups in xylan are a limiting factor in achieving the efficient hydrolysis of the substrate (Debeire et al., 1990). These groups mainly comprise sterics obstacles

to the enzyme-substrate complex formation (Gorbacheva and Rodionova, 1977). The interest in xylanases has increased greatly in the last decade due to their potential biotechnological applications, especially in the paper industry (Biely, 1985; Viikari et al., 1991, 1994; Yang et al., 1992; Daneault et al., 1994). Due to this growing interest, intensive investigations have been undertaken, mainly with xylanolytic enzymes derived from mesophilic fungi and bacteria.

II. STRUCTURE AND OCCURRENCE OF XYLAN Hemicelluloses are noncellulosic polysaccharides that are found in plant tissues (Woodward, 1984). In the cell walls of land plants, xylan is the most common hemicellulosic

0738-855 1/97/$.50 0 1997 by CRC Press LLC

39


polysaccharide, representing more than 30% of the dry weight (Joseleau et al., 1992). Xylans are composed of 1,4-1inked P-D-xylopyranosyl residues (Whistler and Richards, 1970). Most xylans occur as heteropolysaccharides, containing different substituent groups in the backbone chain and in the side chain (Biely, 1985; Puls and Poutanen, 1989). The common substituents found on the backbone of xylan are acetyl, arabinosyl, and glucuronosyl residues (Whistler and Richards, 1970). Homoxylans, on the other hand, consist exclusively of xylosyl residues. This type of xylan is not widespread in nature and has been isolated from esparto grass (Chanda et al., 1950), tobacco stalks (Eda et al., 1976), and guar seed husk (Montgomery et al., 1956).

a higher 4-0-methylglucuronic acid content than do hardwood xylans. The 4-0-methylglucuronic acid residues are attached to the C-2 position. Softwood xylans are not acetylated, and instead of acetyl groups they have a-L-arabinofuranose units linked by a-1,3-glycosidic bonds to the C-3 position of the xylose (Puls and Schuseil, 1993). The arabinosyl substituents occur on almost 12% of the xylosyl residues (Wong et al., 1988). The ratio of p-Dxylopyranose, 4-~-methyl-a-~-glucuonic acid, and L-arabinofuranose is 100:20:13 (Puls and Schuseil, 1993). Softwood xylans are shorter than hardwood xylans, with a DP between 70 and 130. They are also less branched (Zimbo and Timell, 1967).

A. Hemicellulose from Hardwood

111. THE XYLANOLYTIC ENZYME SYSTEM

The xylan of hardwoods is 0-acetyl-4-0methylglucuronoxylan (Figure 1). This polysaccharide consists of at least 70 P-xylopyranose residues (average degree of polymerization [DP] between 150 and 200), linked by P- 1,4-glycosidic bonds (Timell, 1964). Every tenth xylose residue carries a 4-0-methylglucuronic acid attached to the 2 position of xylose (Woodward, 1984). Hardwood xylans are highly acetylated (e.g., birch xylan contains more than 1 mol of acetic acid per 2 mol of xylose [Dekker, 19891). Acetylation occurs more usually at the C-3 than at the C-2 position, however, acetylation at both positions has been reported (Bouveng, 1961; Lindberg et al., 1973). The presence of these acetyl groups is responsible for the partial solubility of xylan in water (Whistler and Richards, 1970). These acetyl groups are readily removed when xylan is subjected to alkali extraction (Dekker, 1989).

Due to the heterogeneity of xylan, its hydrolysis requires the action of a complex enzyme system (Figure 3). This is usually composed of p-1,4-endoxylanase, P-xylosidase, a-L-arabinofuranosidase,a-glucuronidase, acetylxylan esterase, and phenolic acid esterases. All these enzymes act cooperatively to convert xylan to its constituent sugar. The presence of such multifunctional xylanolytic enzyme systems is quite widespread among bacteria and fungi (Woodward, 1984; Wong et al., 1988; Coughlan and Hazlewood, 1993). Heteroxylans contain different substituent groups in the backbone and side chain. Thus, the degradation of such a complex polysaccharide may involve synergistic action between the different components of the xylanolytic enzyme system (Biely et al., 1986; Bachmann andMcCarthy, 1991; Smith andForsberg, 1991; Kleupfel et al., 1992).

B. Hemicellulose from Softwood

A. Occurrence of Xylanases

Softwoods are composed of arabino-4-0methylglucuronoxylans (Figure 2). They have

Xylanases are widespread in nature and they have been reported to be present in marine

40

FIGURE 1. Composition of Oacetyl-4-Omethylglucuronoxylan (hardwood xylan). Numbers indicate the carbon atoms at which substitutions take place. Ac: Acetyl group; a-4-OMe-GlcA: a-4-Omethylglucuronic acid.

OH


P N

HO-C

a-Araf

OH

a-Araf

OH

d HOH2C

a91

a4O-Me-GlcA

FIGURE 2. Composition of arabino-4-Omethylglucuronoxylan(softwood xylan). Numbers indicate the carbon atoms at which substitutions take place. a-Araf: aArabinofuranose; a-4-OMe-GlcA: a-4-Omethylglucuronic acid.

a4O-Me-G kA



x

c

..

9

..

A”

I

43


and terrestrial bacteria, rumen and ruminant bacteria, fungi, marine algae, protozoa, snails, crustaceans, insects, and seeds of terrestrial plants. Interestingly, until now xylanolytic enzymes have not been reported in hyperthermophilic Archaea, which grow optimally between 75 and 100°C (unpublished results). Among the different functions of xylanases are biodegradation in order to provide a source of metabolizable energy, degradation of cell-wall components in interaction with other polysaccharide-degrading enzymes, degradation of xylans during germination of barley, and digestion of dietary vegetation (for a detailed review see Dekker and Richards, 1976). B. Endoxylanase

p- 1,4-Endoxylanase

(1,4-p-o-xylan xylohydrolase; EC 3.2.1.8) cleaves the internal glycosidic linkages of the heteroxylan backbone, resulting in a decreased DP of the substrate. The attack of the substrate is not random, and the bonds to be hydrolyzed depend on the nature of the substrate (e.g., length and degree of branching of the substrate or the presence of substituents [Reilly, 19811). During the early course of hydrolysis of xylan, the main products formed are xylooligosaccharides. As hydrolysis proceeds, these oligosaccharides will be further hydrolyzed to xylotriose, xylobiose, and xylose (Dekker and Richards, 1976; Wong et al., 1988; Debeire-Gosselin et al., 1992a, b). Endo-acting xylanases have been differentiated according to the end products released from the hydrolysis of xylan (e.g., xylose, xylobiose and xylotriose, and/or arabinose). Thus, xylanases may be classified as nondebranching (arabinose nonliberating) or debranching (arabinoseliberating) enzymes (Dekker and Richards, 1976; Reilly, 1981). Arabinose-cleaving endoxylanases have been purified from Aspergillus niger (Takenishi and Tsujisaka, 1975), Neurospora crassa (Mishra et al., 1984), Streptomyces roseiscleroticus (Grabski and Jeffries, 199 l), Talaromyces byssochlamydoides (Yoshioka et al., 1981), and Tricho44

derma koningii (Wood and McCrae, 1986). Furthermore, many organisms are able to produce both debranching and nondebranching xylanases, resulting in the maximum efficiency of xylan hydrolysis (Takenishi and Tsujisaka, 1975; Wong et al., 1988). An interesting physicochemical property of fungal and bacterial endoxylanases seems to be the strong relationship between their molecular weight (MW) and isoelectric point (PI) values. Thus, Wong et al. (1988) suggested that endoxylanases could be grouped into those that are basic proteins with MW below 30,000 and those that are acidic with MW above 30,000. In Table 1, the physicochemical properties of purified endoxylanases are presented, Almost 70% of the acidic endoxylanases have MW values above 30,000. Nearly the same percentage of basic endoxylanases show MW values lower than 30,000. These values seem to be in agreement with the observations of Wong et al. (1988). However, it should be mentioned that there are several exceptions to this general pattern and endoxylanases with low PI and low MW values have been reported, and vice versa. For example, the endoxylanase from Myrothecium verrucaria has a PI value of 4.3 and a MW of 15,900 (Filho et al., 1993), and the endoxylanase 2 from Fibrobacter succinogenes shows a pI of 8.0 and a MW of 66,000 (Matte and Forsberg, 1992). Most characterized endoxylanases are optimally active at temperature ranges between 45 and 75"C, and only a small number of purified bacterial and fungal endoxylanases show maximal activities at temperatures above 80°C. The purified endoxylanases from various species belonging to the genus Thermotoga are optimally active at temperatures between 80 and 105°C (Simpson et al., 1991; Sunna et al., 1996; Winterhalter and Liebl, 1995). C. p-Xylosidase P-D-Xylosidases (p-D-xyloside xylohydrolase; EC 3.2.1.37) are exoglycosidases that hydrolyze short xylooligosaccharides and

4.5

55 (-)d

x22 x34

37

30

28

28

28

20

Aspergillus nidulans CECT 2544

Aspergillus oryzae D5

Aureobasidium SP. NRRL-Y-2311-1

Aureobasidium pullulans Y-2311-1

Bipolaris sorokinianu H83

flaws IF00407

Cryptococcus

5.5

70 (5)

A B C

30

11

54 (15)

45 (-)d

55 (3)

60 (30) 55 (30) 50 (30) 62 (10) 56 (10)

4.8

4.5

5.0

5.5 4.5 2.0 5.5 6.0

5.0

Aspergillus kawachii IF0 4308

55 (30)

28

55 (60) 5.5-6.0 50 (60) 5.0 45-50 (60) 4.0

Aspergillus jlavipes

II Iu

24 I

Growth Xylanase Optimal Optimal temperature temperature pH ["CI ( I= ["CI

Aspergillus awarnori CMI 142717

Fungi:

Organism

TABLE 1. Physicochemical properties of purified B-1.4-xylanases

10.0

9.5

9.4

8.5

3.6

6.7 4.4 3.5 6.4 3.4

N.DC

5.7-6.7 3.7 3.3-3.5

pi

Oatspelt

Source ofxylan

x2 x3

Xi,XzX3 X2XnA

Hydrolysis products

endo endo endo

CMXj

Oatspelt

N.D

N.D

Xi&&&

x2xn&&

Xl,XzXn& Xi,XzXn& Xi,Xzxn&

25.0 N.Mm

30.0

X2X3,&&

N.D

25.0 Birchwood x1.X~

20.0

46.5

26.0 29.0 22.0 34.0

35.0 Oatspelt

endo

endo

endo

endo

endo

endo endo endo endo endo

3.10

N.D

7.20

N.D

2.60

N.D N.D N.D 4.20 4.15

N.D

1.00 0.33 0.09

+

N.D

N.D

N.D

N.D

Hg2+, NBS. SDS

N.D

N.D

2 . 6 5 ~ 1 0 ~-

N.D

N.D

N.D

w+, Hg2+

N.D N.D N.D hk2+, EDTA

N.D

w+

w+, Hg2+ w+

N.D

N.D

N.D L N.D 7 . 1 0 ~ 1 0 ~1 . 5 9 ~ 1 0 ~-

N.D

N.D

l.oOx104 N.D 3 . 3 3 ~ 1 0 ~N.D 4 . 5 5 ~ 1 0 ~N.D

Mode Km vma~ Cellulase Inhibitors' of activityh action [mghl] [u/mgl

45.0 Larchwood XLX~,XI,X~ endo

39.0 23.0 26.0

h4Wb [ma]


120

76, 126

98

94

52

47, 46

63

145

86

Ref.

P

QI

22

45

40

30 39

28

25

Gloeophyllum trabeum BAM Ebw 109

Humicola grisea var. thennoidea

Humicola grisea var. thennoidea

Myrothecium verrucaria CMI 45541

Neocallimastix frontalis MCH3

Neurospora crassa 870

Penicillium chrysogenum

28 30 30 30 25

Penicillium purpurogenum

Pichia stipitis CBS 5775

Robillarda sp. Y-20

Schizophyllum commune

Schizophillum radiatum CMI 90341

4176

26

Fusarium oxysporum f . sp. melonis

TABLE 1. (continued)

60 (-)d

A

rI

I

B

55 (30)e

50 (10)

50 (15) 50 (15)

30 (-)d

50 (-)d

4.Y

5.0

4.5-6.0

4.5-6.0

5.0

5.0

6.0

N.D

4.5

9.5 3.5

N.D

5.9

4.2

4.8

4.8

50 (30)

11 40 (15)

4.5

4.8

endo

XI

27.7 Oat spelt

X Z X ~ & X ~endo

21.0 Larchwood Xi,X2

endo endo endo

N.D

N.D 43.0

17.6 Larchwood Xi,XzX3 59.0 Xi,X2X3

N.D

endo N.D

Xi,X2

X 1 , X z X ~ x l endo XsA Xi,XzX3& endo X sA

endo

endo

X,

X3

23.0

35.0 Oat spelt

30.0

33.0 N.Mm

70.0

N.D

15.9 Wheat straw

XI

25.5 Larchwood

endo

x i , x z X 3 & endo xl,xzXn& endo

endo

endo

95.0 Oatspelt 13.0

45.0 Oatspelt

50 (30)

5.5

55 (10)

11

N.D

39.0 Birchwood X2X3

80.0 Oat spelt

N.D

4.3

N.D

N.D N.D

5.0

N.D

I

6.0

5.5

5.5

6.0-7.0 7.0

55 (10)

45 (30)

70 (15)

60 G)d

4.0

5.0

I

2

I II

80 (30)

50 (-)d


N.D

9.5

12.50 2.00 8.37

N.D

N.D

4.10

5.00

4.00

2.50

1.22

6.60

3.30

4-

+

0.37"

225.70" 8.10" 0.44"

N.D

N.D

N.D

N.D

N.D

N.D

*,&,a2+

N.D N.D

SIB

19

69

87

122

N.D

€+?

57 45

Hg2+

N.D

48

113

160

135

1

+ N B S

8 . 8 0 ~ 1 0 ~+

1 . 4 4 ~ 1 0 ~+

P4.D

N.D

8.7d

NBS,PHMB

N.D N.D

+ -

+

notidentified

-

pb2+,Hg2+

W+,Hg2+

N.D

2.29~10~-

2 . d 1.80~10' 3.3$ 200x10'

N.D

1.00" N.D

25 29 30 29

Trichodenna koningii IMI73022

Trichoderma ligrwrum

Trichoderma reesei Rut C30

Trichodem reesei VTT-D-80133

A

37 48-50

Bacillus sp. (2-125

Bacillus sp. NCIM 59

II

I

N

6.0-10.0 6.0-7.0

55-60 (30) 6.0 5030) 6.0

70(10) 70(10)

N.D N.D 30-37 (30) 6.8

V

Iv

30

Aeromonas caviae ME-1

7.0 5.5 5.0

4.0-5.0 4.0-5.0

4.0-4.5 5.0-5.5

3.5 6.5

4.9-5.8 4.9-5.5

55 (10) 45 (10) 50 (10)

N.D N.D N.D N.D

45 (-)d 45 (-)d

50 (60)

60 (60)

5.0 50(10) 45-50 (10) 4.5-5.0 60 (10) 5.0

1 2 3

30

n

I

PI 5.5 PI 9.0

B

A

1 2

20 kD 22 kDa 29 kDa

I1

Aeromonas caviae W-61

Bacteria:

25

Trichoderma harzianwn E58

60-65 (30) 4.0

45

5.1

4.2 3.5

80(30)

78(15) 67(15)

45

m

II

Thennoascus aurantiacus C436 Thielavia terrestris 255B

5.5 4.5 5.0

45

75 (60) 70 (60) 70 (60)

Talaromyces emersonii CBS 814.70

X-a X-b-I X-b-11

50

Talaromyces byssochlamydoides YH-50


N.D N.D 4.0 8.0

N.D 5.4

9.2 11.5 2.5

4.1-4.2 6.4-6.5

5.5 9.0

7.2 7.3 5.1 8.7

9.4 8.5 9.5

6.1

7.1

5.3 4.2

4.3 3.8 4.0

x1.x2

Xi,Xz&A X1,X2

xz x,

XzX, N.D

N.D

XzX,

endo endo endo

N.D

endo

endo endo

endo endo endo

1.60 N.D 0.66

N.D

N.D N.D 1.70

N.D N.D N.D

Oatspelt

X2

x4

endo endo endo endo

exo exo

Xi.XzX3X4 endo Xz&.&X, endo &rg endo

43.0 N.Mm XzX3,&& 16.0 %&&XI 35.0 Larchwood XzX3&X, 15.8 xzxn&&

41.0 46.0

22.0 Oat spelt 41.0 58.0

N.D 2.00 N.D N.D 1.58 3.50

N.D N.D N.D

29.0 Oat straw XI,Xz X3 A endo 1.40 18.0 X z X ~ x l l endo 4.20 21.o Oat spelt XI,Xz X3 X, endo N.D 20.0 XI,Xz X3 X, endo N.D 19.0 Birchwood XI,Xz X3 X, endo 22.30 20.0 XI, xz x3 X, endo 3.80 32.0 Birchwood Xl,XzX, endo N.D 23.0 Xi,X2& endo N.D

20.0 Aspen22.0 wood 29.0

25.7

32.0 Oat spelt

74.8 Beech XzX3&X, 54.2 "Lenzing" XzX>&&

76.0 Broad54.0 leaved 45.0 trees


N.D

N.D

L

N.D N.D N.D

N.D N.D

-

-I-

N.D N.D N.D N.D 1 . 7 0 ~ 1 0 ~7.42~10' -

N.D 1.82x 102

N.D N.D N.D

N.D N.D

3 . 0 7 ~ 1 0 ~1 . 4 1 ~ 1 0 ~-

N.D N.D N.D N.D

5 . 8 0 ~ 1 0 ~L N.D 1.alx102 L

N.D

N.D N.D 6.10~10'

N.D N.D

m

NBS

NBS

N.D N.D

N.D N.D

N.D N.D N.D

N.D N.D N.D N.D

@+,I@+

w+,Hg2+

N.D N.D

not identified N.D not identified

a2+,

w+

w2+

N.D N.D

W2+? %2+. Kh4fo4

Hg2+,KMno4

38

62

89 88

168 42

93

159

64

179

156, 157, 177

49

155

163

i 82

30 55 60

30 37

37 37

65

Bacillus pumilus P O

Bacillus srearothemophilus 21

Bacillus stearothemphilus T-6

Bacillus subzilis P A P 1 15

Cellulomonas fimi

Clostridium sp. SAIV

Clostridium acetobutylicwn ATCC 824

Clostridium stercorarium

40 (30)

C

75 (-)d 75 (-)d

75 (-)d

B C

60 (-)d

B A

50 (-)d

A

50 (30)

40 (30)

B

A

45 (30)

50 (10)

75 (5)

60 (30)

A

T-6

x61

x2z

6.0-7.0 4.0-6.0 6.0-7.0 6.5

6.0

9.0

5.5-7.0

5.5-7.0

5.5-7.0

5.5-6.0

5.0

5.5-6.5

5.5-6.5

6.0

5.0

5.0

6.5

7 .O

45-60 (10) 6.5

45 (40) 50-62 (10) 55 (10) 50(10)

x34E

x34c

30

polymyxa CECT 153

Bacillus

75 (30)

55

50(10)

Bacillus sp. XE

J

37

Bacillus sp. 41 M-1


4.3

4.4

4.5

8.5

4.4

N.D

4.5

8.0

8.5

N.D

7.0

4.8

N.D

>9.3 9.0 4.7

9.3

7.8

5.5

Oat spelt

Oat spelt X1,Xz

x2x3

3.70

Xi.XZ Xs & endo 62.0

72.0

2.90

6.70

XI, x2 Xn & endo

endo

3.20

xz x 3

44.0 Larchwood x i . X z X ~ x 4 endo

29.0

6.00

7.00

1.25

N.D N .D

N.D

1.68

N.D N.D

%*+

-

PHMB &+, Hg2+,w, PHMB 4.00~10~ -

w, a2+ H$+, ,

3.50~103 -

PHMB

8

97

I18

m+.a+,w,

C@+, Hg2+,pb2+

@+,Hg2+,Pam

Q2+

m+, @+,*+,

Hs2'

@+,@+,@+,

i-

5.50~10~ -

223x10'

224x10'

-

N.D

N.D

3.60~10'

N.D

N.D

79

N.D

N.D

@+,H$+,NEM 1.72

9

7.00~10~ 1.60

80

A P ,NJ3s N.D N.D

N.D

%+,

w+, Ni+,Hgz+,

a?+, &2+,

121

N.D 2 8 8 ~ 1 0 ~L

N.D

125

N.D

1.63

3.80

N.D

115, 116

N.D N.D N.D N.D

1.60~10' N.D 6.20~10' L 1.50~10' N.D 1.12~10~ -

0.65 6.30 0.32 17.70

N.D

29

N.D

119

N.D

Hs2+.NBs

0.60

N.D N.D

N.D

endo

endo

endo

65.0 Larchwood XzXd&X5 endo

30.0 Larchwood

150.0

22.0

13.2 Larchwood

32.0 Larchwood xl,x2X3

43.0

39.5

24.0 Larchwood XzX%&&

endo

endo endo endo endo

34.0 Birchwood xl.xzX3 34.0 Xl,xzX3 22.0 xzX3x4 61.0 oat spelt

xzx?,,%

endo

endo

22.0 Birchwood Xzx?,,%

36.0 Larchwood X2Xn&


60 65

37

50 36

37

37 45

37

Clostridium stercorarium HX-1

Clostridium thermolncticum TC 21

Fibrobacter succinogenes S 85

Streptomyces sp. T7

Streptomyces sp. 3137

Streptomyces sp. EClO

Streptomyces sp. A45 1

Streptomyces q.B12-2

Streptomyces cyaneus MT 8 13


55 (-)d

2

72 (10) 65 (10)

60 (-)d 60 (-)d 60 (-)d

2 3 4

I LI

55 (-)d

60 (-)d

la

50 (-)d 50 (-)d

Ib

I I1

8.0 6.5

7.0 7.0 6.0

6.0

6.0

5.6 5.4

7.0-8.0 7.0-8.0 7.0-8.0

60-65 (10) 5.0-6.0

X-II-B 60(30) 60(30) 60(30)

60-65 (10) 5.0-6.0

X-II-A

XIA XIB Xu

60-65 (10) 5.5-6.5

4.5-5.5

6.3

7.0

6.5 6.5 6.5

6.5

X-I

60 (30)

39 (-)d

80 (20) 80 (20) 80 (20)

75 ( - ) d

1

B C

A

D

5.1 5.2

7.5 8.3 5.4 5.0 4.8

8.6 8.9

6.8 8.9 5.2

10.2

10.0

7.1

7.8

8.0

8.9

4.4 4.5 4.6

4.5

Xzx3 endo

XI. XZ Xn endo &%A x l . X z x n & endo

37.5 34.0

26.4 23.8 36.2 36.2 40.5

22.8 33.1 Oatspelt

Oatspelt

32.0 Oat spelt 22.0 21.o

25.0

25.0

endo endo

XI.x2

xi.x2

21.8 Larchwood Xi. X2 Xn endo &X5 50.0 "Sanpearl" Xi,X2 endo

66.0

53.7 Oat spelt

39.0 Larchwood X L X ~ X ~endo 55.0 xi.Xzx3 endo 65.0 x1.xZX3 endo

53.0 Oat spelt


N.D N.D

5.80 3.40 3.OO 1.20 0.80

N.D N.D

3.00 3.40 5.00

N.D

N.D

N.D

10.00

1.30

2.60

0.40 0.53 0.48

1.40

+

-

-

-

N.D N.D

N.D N.D N.D N.D 1 . 6 2 ~ 1 0 ~4 . 7 0 ~ 1 0 ~210x10* 3.38~10~2 4 3 ~ 1 0 ~-

N.D N.D N.D

N.D

N.D

N.D

7.60~10~-

1 . 1 8 ~ 1 0 ~+

3.30~10'

3 . 3 0 ~lo3 N.D 3 . 1 4 ~ 1 0 ~N.D 3 . 9 5 ~ 1 0 ~N.D

2200103 N.D

110

30

13s

N.D N.D

Hg2+,NES

Hg2"m.Y

N.D N.D Hg2+,NBs Hg2+,NES,PHMB Hg2+.NES

N.D N.D N.D

SDS,NES

&2+, Hg2+,F&,

SDS,NBS

171

44

59

102

Hg2+,NBS,PHh4B? 77 HNBB C$+,Hg2+,F&, 106 SDS, NBS C@+,Hg2+,w,

al2+,Hg2+

C@+, Hg2+,EJYIX EcirA

N.D N.D N.D

N .D

50

60

Streptomyces themviolaceus OPC-520

Thermoanaerobacterium sp.

0.24

0.36

N.D N.D N.D

1.9510’

1.18

3.74~10’ 4.760103

-

-

-

1 . 2 6 ~ 1 0 ~N.D

N.D N.D N.D

N.D

@+, Hg2+,Nl2+

C@+,w+

w+

N.D

NBS

SDS; dodecylsulfate . Na-salt, NEM; N-maleiamide, EGTA; ethylene glycol-bis (Barninoethyl ether). N, N, N , “-tetraacetic acid, HNBB; 2-hydroxy-5-nitrobenzyl bromide.

N.M; source of xylan was not mentioned in the reference Activity in U.

Xn; xylooligosaccharides (depending on the reference, n is usually larger than 5). A; arabinose. h +; positive, -;negative, L; low activity.

*

Activity in U / d .

f Size exclusion chromatography. g X I ; xylose, X2; xylobiose, X3; xylotriose, X4;xylotetraose, X5;xylopentaose,

k Concentration in m ~ .

j CMX; c d x y m e t h y l xy~an.

p- chloromercuribenzoate, EDTA; ethylene diamine tetraacetic acid . N a p a l t ,

152

175

137, 146

149

142

162

N.D; not determined. Assay period was not mentioned in the reference. Measured in crude extract.

NBS;N-bromosuccinimide, PHMB; p-hydroxymercuribenzoate. PCMB;

endo

endo

1.10 0.29

0.07

2.50 1.40 2.00

N.D

3.00

-I-

+

SDS-PAGE.

a Assay period in min.

35.0

XZ

N.D

90-100 (30) 7.0

(150)b

266.6Birchwood XZ%XS& (105)b

N.D

6.0

80 (30)

77

Thennotoga thermarum

endo endo

XzX3 XZ x3

120.0 Oat spelt 40.0

N.D 5.6

6.2 5.4

92 (5) 105 (5)

80

Thennotoga maritima MSB8

endo

XzX3

Oatspelt

31.0

N.D

5.3

105 (20)

80

endo

Xn&Xs,&

15.6

8.4

~3X4.16 endo

endo

N.D

19.6

Themtoga sp. Fj SS3-B.l

N.D

(24Ib 3 6 . 6 Birchwood XzX&

(1

350.6 Oat spelt

7.1

7.8 7.2 6.8

75 (20)e 75 (20)e 75 (20)e

55

4.2

4.37

6.2

80 (5)

33.0

N.D

endo

8.O

7 .O

60 (10)

N.D

N.D

N.D

N.D

N.D

N.D

endo

4.2

7.0

70 (10) x1,X2X3

2.50

endo

&

N.D

N.D

7.0

55 (-)d

16.60

exo

N.D

N.D

5.5

50 (-)d

Xl,&

& 12.50

Oat spelt

endo

N.D

N.D

5.5

55 (-)d

Themmonospora curvata

JWISL-YS485

30

Streptornyces exfoliutus MC1




xylobiose from the nonreducing end to liberate xylose (Wong et al., 1988). True P-xylosidases are able to cleave artificial substrates like p-nitrophenyl P-D-xyloside (Coughlan and Hazlewood, 1993). P-Xylosidases have been reported in bacteria and fungi. These are larger enzymes with molecular weights between 60and 360 kDa, and they may be mono- or dimeric proteins (Table 2). P-Xylosidases appear to be mainly cell associated in bacteria and yeast (Biely, 1985). However, extracellular P-xylosidase activity has also been reported (Deleyn et al., 1978; Zimmermann et al., 1988; Dobberstein and Emeis, 1991). In the yeast Cryptococcus albidus, xylobiose and xylotriose enter the cells through a P-xyloside permease transport system and are converted by P-xylosidase to xylose (Biely, 1985). Generally, purified P-xylosidases are unable to hydrolyze xylan. However, there are some reports of P-xylosidases that are able to attack xylan slowly to produce xylose (Dekker and Richards, 1976). Among the xylooligomers, xylobiose is usually the best substrate. The affinity of the enzyme toward xylooligosaccharides decreases with increasing DP (Van Doorslaer et al., 1985). The P-xylosidase of A . awamori is not able to release significant amounts of xylose from xylan. whereas incubation with xylobiose, xylotriose, and xylotetraose is accompanied by the formation of xylose as a major product (Kormelink et al., 1992). A number of P-xylosidases have been reported to possesses a-arabinosidase activity (e.g., the enzymes from A . niger [Rodionova et al., 19831, T. reesei [Poutanen and Puls, 19881, T. ethanolicus [Shao and Wiegel, 19921, and Penicillium wortmanni [Deleyn et al., 19781).Most of the P-xylosidases studied so far are completely inhibited by their hydrolysis product xylose (Poutanen and Puls, 1988; Dobberstein and Emeis, 1991; Buttner and Bode, 1992). An important role of P-xylosidase seems to be relieving the end product inhibition of endoxylanase. Thermomonospora fusca produces a cell-associated P-xylosidase that was found to enhance the saccharification of xylan (Bachmann and

McCarthy, 199 1). Many 0-xylosidases have transxylosidation (transferase) activity, especially at high substrate concentrations, resulting in products of higher molecular weight than that of the substrate (Conrad and Noethen, 1984). Transfer reaction may result in the formation of both P-1,3 and P-1,4 bonds (Claeyssens et al., 1966, 1971).

D. a-L-Arabinofuranosidases Despite the important role played by arabinosidases in the hydrolysis of xylan, only a few enzymes have been isolated and characterized. There are two types of arabinases, the exo-acting a-L-arabinofuranosidase (EC 3.2.1.53, which is active againstp-nitrophenyla-L-arabinofuranosides and on branched arabinans, and the endo- 1,5-a-~-arabinase(EC 3.2.1.99), which is active only toward linear arabinans (Van der Veen et al., 1992). Endoarabinases have been reported in Bacillus subtilis (Kaji and Saheki, 1975), Clostridium felsineurn (Kaji, 1984), and various fungi (Kaji and Tagawa, 1970; Poutanen, 1988; Bachmann and McCarthy, 1991; Kaneko et al., 1993). These enzymes hydrolyze 1,5-a-~-arabinans, but are not able to hydrolyze the chromogenic substrate phenyl-a-L-arabinofuranosideor gum arabic. Most of the arabinan-degrading enzymes studied so far are of the exo-acting type (Dekker and Richards, 1976). The size of native arabinofuranosidases may reach up to 495 kDa and are found in mono-, di-, tetra-, hexa-, and octameric forms (Table 3). The production of arabinofuranosidase in several actinomycetes seems to be induced, among others, by arabinan, xylan, and wheat bran (Mackenzie et al., 1987; Zimmermann et al., 1988). Similarly, several rumen bacterial isolates investigated by Williams and Whiters (1982) showed high levels of arabinofuranosidase activity when grown in the presence of arabinose or arabinose-containing polysaccharides. On the other hand, only low levels of arabinofuranosidase were detected when the isolates were grown with glucose or

51

TABLE 2 Properties of Purified Fungal and Bacterial p-Xylosidases Organism


Fungi Aspergillus a wamori A. niger A. oryzae A. pulverulentus Aureobasidium pullulans Chaetomium trilaterale Emericella nidulans Neurospora crassa Penicillium wortmanni Pichia stipitis Talaromyces emersonii Trichoderma reesei T. viride Bacteria Bacillus pumilus 6.stearothermophilus Clostridium acetobutylicum Thermoanaerobacter ethanolicus Thermotoga sp. FjSS3-B.l a

MW Wa)

Subunit (kW

Form

PI

Ref.

110.0 78.0 168.0 180.0 190.0 240.0

82.0 65.0 100.0 121.o

Monomeric Monomeric Dimeric Trimeric Dimeric Dimeric

4.2 N.D.” 4.1 4.7 3.5 ~3.0

86 65 52 150

240.0

118.0

Dimeric

4.8

164

240.0 83.0 100.0

116.0

Dimeric Monomeric Monomeric

3.2

4.3 5.0

109 37 35

Monomeric Dimeric

N.D.” 8.9

122 163

Monomeric Monomeric

4.7 4.4

129 108

Dimeric Dimeric Dimeric

4.4 4.2 5.8

124 121 95

34.0 181.0

97.5

100.0 101.0 130.0 150.0 224.0

70.0 75.0 85.0

63.0 165.0

85.0

Dimeric

4.6

140

174.0

92.0

Dimeric

4.1

137

N.D.: not determined.

cellobiose as substrate. a-L-Arabinofuranosidases capable of hydrolyzing both 1,3- and 1,5-a-~-arabinofuranosyllinkages in arabinoxylan have been reported in A . niger (Kaji and Tagawa, 1970), B. subtilis (Kaji, 1984), and S.purpurascens (Komae et al., 1982). The A . niger enzyme first attacks the a-~-l,3-linked arabinofuranosyl residues in arabinan to an extent of 30% and then proceeds with the slow attack of the a-L1,5-arabinan, which will then be completely converted to arabinose (Kaji and Tagawa, 1970). A novel arabinosidase from A . awamori was recently purified (Kormelink et al., 1991a). This I ,4-P-~-arabinoxylan arabinofuranohydrolase is highly specific for

52

39

arabinoxylans and is able to release only arabinose from arabinoxylan. During the arabinose release, the xylan backbone is not degraded and there is no production of xylooligosaccharides. The enzyme is not active toward a - ~ - 1 , 3 -or a-l,5-linked arabinose from arabinans, arabinogalactans, or p-nitrophenyla-L-arabinofuranoside (Kormelink et al., 199la, b). Similar to xylanases, multiple forms of a-I.arabinofuranosidases have been detected in streptomycetes (Komae et al., 1982; Zimmermann, 1989). Synergism between a-L-arabinofuranosidases and xylanases has been reported. An increase in xylose, xylobiose, and arabinose production was observed when both en-

TABLE 3 Occurrence and Properties of a-Arabinofuranosidases MW

Organism


Fungi Aspergillus niger A. niger 5-16 Phanerochaete chrysosporium Talaromyces emersonii Trichoderma reesei Bacteria Bacillus polymyxa

9. subtilis 9 . stearothermophilus Bacteroides xylanolyticus Butyrivibrio fibrisolvens Clostridium acetobutylicurn Ruminococcus albus Streptornyces sp. 17-1 Streptomyces diastaticus S. purpurascens Therrnornonospora fusca a

(kD4

Subunit Wa)

53.0 67.0 55.0 210.0

105.0

53.0 166.0

65.0 33.0

Form

PI

Ref.

Monomeric Monomeric Monomeric

3.6 3.5 7.3

73 74 27

Dimeric

3.5

27

Monomeric

7.5

128

Dimeric

4.7

115

65.0 256.0 364.0

64.0 61.O

Monomeric 5.3 Tetrameric 6.5 Hexameric N.D.a

172 50 139

240.0

31.O

Octameric

6.0

61

Monomeric

8.2

96

94.0 310.0 92.0 38.0 60.0 495.0 92.0

75.0

62.0 46.0

Tetrameric 3.8 Monomeric 4.4 Monomeric 8.8 Monomeric 8.3 Octameric 3.9 Dimeric N.D.”

56 72 153 82 2

N.D.: not determined.

zymes were used simultaneously (Greve et al., 1984; Poutanen, 1988).

E. a-Glucuronidases a-I>-Glucuronidases(3.2.1 .-)hydrolyze the a-1,2 linkages between glucuronic acid and xylose residues in glucuronoxylan. Due to the lack of glucuronidase activity in many fungal hemicellulase preparations (Puls and Poutanen, 1989), this enzyme was not described until 1986 (Puls et al., 1986). Despite the role played by a-glucuronidases in the biodegradation of xylan, few enzymes have been reported so far (Table 4). The five enzymes that have been purified have molecular weights above 100 kDa.

The substrate specificities of a-glucuronidases differ according to the enzyme source. The enzymes from Agaricus bisporus and S . olivochromogenes require a low-molecularweight glucuronoxylan substrate (Puls et al., 1987; Johnson et al., 1989). They release 4-0-methylglucuronic acid from 4-0-methylglucuronose-substituted xylooligomers, but not from the polymer (Puls et al., 1987). Furthermore, the presence of acetyl groups next to the glucuronosyl substituents hinders the action of the a-glucuronidase produced by A. bisporus (Puls and Schuseil, 1993). The ruminal bacterium F . succinogenes produces an a-glucuronidase that is unable to release 4 - 0 methylglucuronic acid from intact xylan. This enzyme is active against low-molecular-weight

53

TABLE 4 Occurrence of Microbial a-Glucuronidases Ref.

Organism Fungi Agaricus bisporus Aspergillus niger Pleurotus osfreatus Schizophyllum commune Therrnoascus aurantiacus Trichoderrna reesei

1 32 67

132 67 78

32

Bacteria


Fibrobacter succinogenes Streptornyces flavogriseus S. olivochromogenes Therrnoanaerobacteriurnsp. JWISL-YS485

glucuronoxylan fragments that result from the hydrolysis of xylan with endoxylanase. The aglucuronidase is active only against substituted xylooligomers of DP greater than 2 (Smith and Forsberg, 1991). On the other hand, the aglucuronidases produced by Aspergillus niger and Schizophyllum commune are able to liberate 4-0-methylglucuronic acid from methylglucuronoxylan (Johnson et al., 1989).

F. Acetylxylan Esterases Acetylxylan esterases (EC 3.1.1.6) remove the 0-acetyl substituents at the C-2 and C-3 positions of xylose residues in acetylxylan. The production of acetylxylan esterase by fungi and bacteria was first reported in 1985 (Biely et al., 1985b). The main reason for the late discovery of this enzyme was due to the lack of suitable substrates. Although xylan is highly acetylated in its native state, most of the xylans used to study xylanolytic enzyme systems were deacetylated xylans obtained from alkali extraction (Tenkanen and Poutanen, 1992). Acetylxylan esterase activity has been recognized as being a part of the xylanolytic systems of many organisms such as Trichoderma reesei, T. viride, A . niger, S. commune (Biely et al., 1985b), Streptomyces sp. (Mackenzie et al., 1987), and F . succinogenes (McDermid et al.,

54

147 67 67 143

1990). The highest specific activity of acetylxylan esterase has been reported in A. niger (Tenkanen and Poutanen, 1992). Few acetylxylan esterases have been purified and characterized until now and little is known about their physicochemical properties. T. reesei produces two monomeric isoenzymes that are glycosylated and have a molecular weight of 34 kDa (Sundberg and Poutanen, 1991). The acetylxylan esterases from T. reesei liberate acetic acid from acetylated xylooligomers. The newly described acetylxylan esterases I and I1 from Thermoanaerobacterium sp. JWISL-YS485 deacetylates acetylated xylan. It has been proposed that acetylxylan esterase II removes the acetyl groups from xylan extracellularly, whereas acetylxylan esterase I acts on short acetylated xylooligosaccharides intracellularly (Shao and Wiegel, 1995). The enzyme from S. commune releases acetic acid from xylan. However, acetylated xylooligosaccharides are the preferred substrate (Biely et al., 1986, 1988). Acetyl groups present on the xylan backbone inhibit the action of xylanases by steric hindrance. Acetylxylan esterase from F . succinogenes may play an important role in relieving this inhibition by releasing acetic acid from xylan, thereby forming new unsubstituted sites on the polysaccharide backbone (McDermid et al., 1990). This, therefore,


increases the susceptibility of the polymeric xylan to endoxylanases (Biely, 1985).

ferulic acid when incubated with ferulic acid containing tri- and tetrasaccharides.

G. Ferulic and pCoumaric Acid Esterases

IV. STRUCTURE AND SYNERGISTIC EFFECT OF XYLANOLYTIC ENZYMES

Ferulic and p-coumaric acids are linked to xylan by ester bonds (Bacon et al., 1975). Ferulic acid esterase (EC 3.1.1.-) cleaves the ester linkages between arabinose side chains and ferulic acids in xylan. Similarly, p-coumaric acid esterase (EC 3.1.1 .-) cleaves the ester linkage between arabinose and p-coumaric acid (Miiller-Harvey et al., 1986; Mackenzie et al., 1987). These enzymes were first described by Mackenzie et al. (1987). Few p-coumaric and ferulic acid esterases have been purified and characterized (Christov and Prior, 1993). Higher concentrations of ferulic acid esterase were measured if S. commune was grown on Avicel cellulose instead of oat spelt xylan. The enzyme releases ferulic acid from wheat bran only in the presence of xylanase (Mackenzie and Bilous, 1988). The best substrate for the production of the ferulic acid esterases by Streptomycrs olivochromogenes is oat spelt xylan (Mackenzie et al., 1987). From this organism, three feruloyl esterases have been partially purified (Johnson et al., 1988). Similarly, a p-coumaric acid-liberating enzyme has been reported and partially purified from cultures of S . viridosporus (Deobald and Crawford, 1987; Donnelly and Crawford, 1988). Among the few purified and characterized phenolic acid esterases are those of the fungus Neocallimastix strain MC-2 (Borneman et al., 1993). This anaerobic fungus produces a p-coumaroyl and two feruloyl esterases. The purifiedp-coumaroyl esterase is a dimer with a molecular weight of 11 kDa. In contrast to thep-coumaroyl, the two feruloyl esterases are monomeric, with molecular weights of 68 and 24 kDa. The p-coumaroyl esterase is not able to release ester-linked acetyl groups from xylan. Furthermore, no reducing sugars are released from oat spelt xylan. The two feruloyl esterases release

A. The Xylanosome Concept Discrete multifunctional, multienzyme complexes found on the surface of several cellulolytic microorganisms are called “cellulosomes.” These complexes play an important role in the degradation of cellulose and hemicellulose (Bayer et al., 1983, 1994; Lamed et al., 1983b). The cellulosome is a cell-associated entity that mediates the adhesion of the bacterium to cellulose. Cellulosomes can also be found as an extracellular enzyme complex. In both forms, the cellulosome is responsible for the efficient degradation of the polymeric substrate (Lamed et al., 1987; Bayer et al., 1994). Recently, the presence of a structure analogous to the cellulosome, the xylanosome, was reported in Butyrivibriofibrisolvens H17c (Lin and Thomson, 199 1). The extracellular complex B (C,) from B. fibrisolvens exists as a multisubunit protein aggregate. The complex has a molecular weight higher than 669 kDa, and is composed of 11 protein bands with xylanase activity and 3 bands showing endoglucanase activity. Similarly, C. papyrosolvens C7 possesses a multicomplex cellulase-xylanase system that is responsible for the hydrolysis of cellulose and xylan (Pohlschroder et al., 1994). This multicomplex system consists of at least seven different protein complexes. The molecular weight of the complexes ranges from 500 to 660 kDa. A single noncatalytic glycoprotein (subunit S4), with a molecular weight of 125 kDa, is present in all of the enzyme complexes. This glycoprotein may have a function similar to the noncatalytic glycoprotein, called subunit S 1, found in C. thermocellum (Lamed et al., 1983a; Bayer et al., 1985), chiefly as a substrate binding and

55

scaffolding component. The presence of xylanosomes in the anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was reported by Zeikus et al. (1991). In this anaerobe, xylanase exists as a highmolecular-weight complex, and the enzyme activity is mainly cell-associated when the organism is grown on insoluble xylan.


B. Multiple Forms of Xylanases Due to the complex structure of heteroxylans, not all of the xylosidic linkages in the substrate are equally accessible to xylan-degrading enzymes. Thus, xylan hydrolysis requires the action of multiple xylanases with overlapping but different specificities (Wong et al., 1988). These enzymes can be grouped into families on the basis of conserved amino acid sequences in the catalytic domains and by hydrophobic cluster analysis. Thus, all highmolecular-weight xylanases belong to the F family of glycanases, whereas low-molecularweight xylanases belong to the G family (Gilkes et al., 1991; Henrissat, 1992). Multiplicity of xylanolytic enzymes has been reported for several organisms. Streptomyces lividans produces a xylanolytic enzyme system that is composed of three xylanases encoded by three different xln genes (Shareck et al., 1991). xlnA belongs to the F family of glycanases, which shows exoxylanase or exoglucanase activity. On the other hand, xlnB and xylC are members of the G family. Streptomyces sp. B-12-2 produces five endoxylanases when grown on oat spelt xylan (Elegir et al., 1994). Based on their physicochemical properties, they can be divided into low-molecular-weightbasic (group 1) and highmolecular-weight/acidic proteins (group 2). The two endoxylanases belonging to group 1 have low activity against xylotetraose, whereas the three endoxylanase from group 2 show higher activity toward xylotetraose. When the white rot fungus Phanerochaete chrysosporium was grown on Avicel, more than 30 different protein bands could be separated by analytical isoelectric focusing (Dobozi et al., 1992). All 56

these proteins showed xylanase activity and were classified into three groups: I, 11, and 111. All three groups also possessed arabinofuranosidase and p-xylosidase activities. Similarly, the culture filtrate of Aspergillus niger was composed of 15 (Biely et al., 1985a), B. fibrisolvens of 1 1 (Lin and Thomson, 1991), Trichoderrna viride of 13 (Biely et al., 1985a), and Talaromyces emersonii between 11 and 13 xylanases (Coughlan et al., 1993). In addition to endoxylanases, enzyme multiplicity has also been observed with other xylanolytic enzymes. Matsuo et al. (1987) reported on the presence of four P-xylosidases in the culture of Penicillium wortmanni, and investigations on Cryptococcus albidus indicated that this yeast produces two forms of p-xylosidases (Biely et al., 1980). Streptomyces diastaticus secretes two different a-arabinofuranosidases when grown on wheat bran or oat spelt xylan (Tajana et al., 1992). Multiple forms of a-L-arabinofuranosidases have also been detected in S.purpurascens (Komae et al., 1982) and S. diastatochromogenes (Zimmermann, 1989). Two acetylxylan esterases from Trichoderma reesei were purified and characterized. Both isoenzymes were monomeric glycoproteins with similar properties (Sundberg and Poutanen, 1991). Furthermore, three bands with acetylxylan esterase activity were detected in zymograms of Thermomonospora fusca grown on oat spelt xylan (Bachmann and McCarthy, 1991). In addition, the fungus Neocallimastix strain MC-2 produces a high- and a low-molecular-weight ferulic acid esterase when grown on cellulose as substrate (Bomeman et al., 1993). Several factors could be responsible for the multiplicity of xylanases. These include differential mRNA processing, postsecretional modification by proteolytic digestion, and posttranslational modification such as glycosylation and autoaggregation (Biely, 1985; Coughlan et al., 1993). Multiple xylanases can also be the product from different alleles of the same gene (Wong et al., 1988). However, some of the multiple xylanases are the result of independent genes (Hazlewood and Gilbert, 1993).


C. Synergism between XylanDegrading Enzymes Highly branched polysaccharides like heteroxylans may contain a large number of side-chain substituents. In xylan hydrolysis, cooperativity or synergism has been observed between enzymes acting on the 1,4-P-~-xylan backbone (P- 1,4-endoxylanase) and side chains cleaving enzymes (a-L-arabinofuranosidase, acetylxylan esterase and a-glucuronidase). The synergistic action between acetylxylan esterase and endoxylanases results in the efficient degradation of acetylated xylan. The release of acetic acid by acetylxylan esterase increases the accessibility of the polysaccharide backbone for endoxylanase attack. The endoxylanase creates shorter acetylated polymers, which are the preferred substrates for esterase activity (Biely, 1985; Biely et al., 1988). Synergism between the xylanases and a-arabinofuranosidases was described for the enzyme system of Taluromyces emersonii. The xylanases I1 and I11 from T. emersonii were unable to hydrolyze wheat straw xylan (arabinoxylan), unless the polymer had been previously treated with an a-arabinofuranosidase of the same organism (Tuohy et al., 1993). The thermophilic actinomycete Thermomonospora fusca possesses a complex multicomponent xylan-degrading enzyme system that consists of endoxylanase, P-xylosidase, a-arabinofuranosidase, and acetyl esterase activities (Bachmann and McCarthy, 1991). The cooperative action between these enzymes has been demonstrated. The hydrolysis of xylan by endoxylanase was enhanced by the addition of P-xylosidase. P-Xylosidase seems to be responsible for relieving the end-product inhibition of endoxylanases. Similarly, the addition of aarabinofuranosidase to endoxylanase enhances the saccharification of arabinoxylan. Similarly, the incubation of oat spelt xylan with endoxylanase and P-xylosidase from Bacillus stearothermophilus caused the complete conversion of this substrate to xylose (Nanmori et al., 1990).

The release of 4-0-methyl-a-~-glucuronic acid from birchwood xylan by the F . succinogenes a-glucuronidase was enhanced by treating the substrate with xylanase (Smith and Forsberg, 1991). The purified a-glucuronidase and P-xylosidase from the thermophilic anaerobic bacterium Thermoanaerobacterium sp. JW/ SL-YS485 are needed in order to achieve the complete hydrolysis of 4-0-methylglucuronosyl xylotetraose. The P-xylosidase hydrolyzes 4-0methylglucuronosyl xylotetraose and produces a mixture of 4-0-methylglucuronosyl xylobiose and 4-0-methylglucuronosyl xylotriose. The a-glucuronidase shows greater activity against these two compounds than against 4-0-methylglucuronosyl xylotetraose, resulting in the release of 4-0-methylglucuronic acid (Shao et al., 1995b). Synergism between phenolic acid esterases and xylanases has also been reported. The amount of released p-coumaroyl and feruloyl groups from coastal Bermuda grass increased when the p-coumaroyl and feruloyl esterases from Neocallirnastix strain MC-2 were incubated with xylanase. The polymer-degrading enzyme may provide free phenolic oligosaccharides, which act as substrate for the phenolic acid esterases (Borneman et al., 1993). Synergism also occurs between various endoxylanases. F . succinogenes S85 secretes two types of endoxylanases. Unlike endoxylanase 2, endoxylanase 1 possesses a debranching activity, liberating arabinose from xylan. It has been shown that the release of arabinose precedes the hydrolysis of the xylan backbone into xylooligosaccharides (Matte and Forsberg, 1992). This indicates that the removal of arabinose substituents, which act as hindrance, lets endoxylanase have access to the xylan backbone.

V. BIOTECHNOLOGICAL APPLICATIONS Xylan-degrading enzymes have considerable potential in several biotechnological applications. In some processes, the use of puri-

57


fied enzymes is required. However, in other applications, the presence of additional enzyme activities is desired. Commercial applications suggested for xylanases involve the conversion of xylan, which is present in wastes from agricultural and food industry, into xylose (Biely, 1985). Similarly, xylanases could be used for the clarification of juices, for the extraction of coffee, plant oils, and starch (Wong and Saddler, 1993), and for the production of fuel and chemical feedstocks (Link0 et al., 1989). Another application of xylanases is the use of this enzyme in poultry diets. Depression in weight gain and feed conversion efficiency in rye-fed broiler chicks has been associated with intestinal viscosity (Van Paridon et al., 1992). Bedford and Classen (1992) reported that incorporating xylanase from Trichoderma longibrachiatum into a rye-based diet of broiler chickens resulted in reduced intestinal viscosity, thus, improving both the weight gain of chicks and their feed conversion efficiency. The efficiency of xylanases in improving the quality of bread has also been demonstrated. The introduction of Aspergillus niger var. awamori xylanase into bread dough resulted in an increase in specific bread volume. This is further enhanced when amylase in combination with xylanase is used (Maat et al., 1992). Recently, the use of xylanolytic enzymes in pulp bleaching has been considered as one of the most important new biotechnological applications of these enzymes (Viikari et al., 1994). The environmental impact of wastewaters arising from the pulp and paper industry, especially the formation of toxic organic chlorines, has attracted public attention in the last few years (Viikari et al., 1991, 1994; Coughlan and Hazlewood, 1993). The most important enzyme that is used in enzyme-aided bleaching is endoxylanase (Kantelinen et al., 1988; Paice et al., 1988; Viikari et al., 1994). However, the use of other xylanolytic and hemicellulolytic enzymes in combination with endoxylanases has been shown to be effective in improving the enzymatic process (Kantelinen et al., 1988; Clark et al., 1990). The use of

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xylanases and side-cleaving enzymes in pulp bleaching helps mainly in reducing the kappa number and increasing the brightness of thc pulp (Viikari et al., 1991, 1994; Yang et al., 1992; Daneault et al., 1994). Most of the studies on the effect of xylanases in pulp prebleaching have been conducted with enzyme preparations from Trichoderma sp. The enzymatic pulp prebleaching resulted in a 20 to 30% reduction of chlorine requirements (Viikari et al., 1991, 1994).

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