Enzymes in Biomass Conversion - American Chemical Society

Chapter 33

Accessory Enzymes Involved in the Hydrolysis of Xylans 1

2

3

3

K. Poutanen , M. Tenkanen , H. Korte , and J. Puls 1

2

VTT, Food Research Laboratory and Biotechnical Laboratory, Box 202, SF-02151 Espoo, Finland BFH, Institute of Wood Chemistry, Leuschnerstrasse 91, D-2050 Hamburg 80, Germany

Downloaded by CORNELL UNIV on June 8, 2014 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch033

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Xylans are heteropolysaccharides which are depolymerized by β-1,4-D-endoxylanases. Due to the abundance and variety of substituents in native xylans, different accessory enzymes are also needed for the total hydrolysis of xylan. The knowledge of α-glucuronidase, α-arabinosidase, and acetyl xylan- and feruloyl esterases has increased considerably in recent years. In addition to acting in synergism with endoxylanases and β-xylosidase for the complete hydrolysis of xylan, some of these accessory enzymes are also capable of changing the structure of polymeric xylans.

Xylans are the major hemicelluloses of many plant materials, where they often contribute to the rigidity of plant cell walls. Most xylans are heteropoly saccharides with a homopolymeric backbone chain of 1,4-linked j3-D-xylopyranose units. The degree and type of substitution of the backbone is dependent on the plant origin of a xylan. In addition to xylose, xylans may contain L-arabinose, D-glucuronic acid or its 4-O-methyl ether, and acetic, p-coumaric, and ferulic acids. Due to the extreme variety of xylan structures, it is obvious that many kinds of enzymes are needed for their complete hydrolysis in nature. Xylanases (EC 3.2.1.8.) are the polysaccharide hydrolases responsible for the attack of the polymer backbone itself. The total hydrolysis or modification of heteroxylans requires in addition several different exo-glycosidases and esterases. The present knowledge of these enzymes is reviewed in this paper.

0097-6156/91/0460-0426$06.00/0 © 1991 American Chemical Society

In Enzymes in Biomass Conversion; Leatham, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Structure of xvlans Wood xylans are either O-acetyl-4-O-methylglucuronoxylans (in hardwoods) or arabino-4-O-methylglucuronoxylans (in softwoods). The degree of polymer ization of hardwood xylans (150-200) is higher than that of softwoods (70130) (1, 2). Wood xylans have very few branching points, but their degree of substitution is high. 4-O-Methylglucuronic acid and L-arabinofuranoside units are linked to the backbone by a-1,2- and a-l,3-glycosidic bonds, respectively. The average ratio of sugar units in softwood xylans is 8:1.6:1 (xylose:4-0-methylglucuronic acid:arabinose) (2). The average molar ratio of xylose, 4-O-methylglucuronic acid and acetic acid in hardwood xylan is 10:1:7 (3). Both D-glucuronic acid and/or its 4-O-methyl ether and arabinose are also present in cereal xylans (4). Endospermic arabinoxylans of annual plants, often referred to as pentosans, are because of their branched structures more soluble in water and dilute alkali than xylans of lignocellulosic materials. They also have at least one, or even two, substituents per xylose residue (5). The presence of feruloyl and p-coumaroyl acids linked via L-arabinose residues has been verified in several studies of xylan (6-8). The amounts of these components, however, are rather small. Every 15th arabinose unit in barley straw arabinoxylan is estimated to be esterified with ferulic acid, and every 31st with p-coumaric acid (7). These hydroxycinnamic acids are bound to C-5 of the arabinose residue (7, 8). It has been suggested that oxidative dimerization of ferulic acid residues crosslinks the arabinoxylan chains and renders them insoluble as a result of the diferuloyl bridges (9). Hardwood xylans and xylans of annual plants may contain up to 7% O-bound acetyl groups. Seven out of ten xylose residues in native hardwood xylan are acetylated on C-2 and/or C-3 (10). Because of the possible migration of O-acetyl groups between 2- and 3-positions during and after isolation of hemicellulose components, it is difficult to determine their original distribution in nature (11). The ratios reported for 2-, 3-, and 2,3-positions of acetyl groups in birch xylan have been 2:4:1 (3) and 2:2:1 (10) and in bracatinga xylan 3:3:1 (12). The existence of covalent bonds between lignin and hemicellulose, perhaps through xylan substituents in many cases, has been indicated in several studies. Evidence has been shown for the existence of an ether linkage between arabinose and lignin (13) and an ester linkage between glucuronic acid and lignin (14). Feruloyl groups may also crosslink xylan and lignin (15). g-Xylosidases Exo-l,4-/3-D-xylosidases (EC 3.2.1.37) hydrolyze xylooligosaccharides and xylobiose to xylose by removing successive D-xylose residues from the nonreducing termini. /3-Xylosidase is part of most microbial xylanolytic systems, but the highest extracellular production levels have been reported for fungi.



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ENZYMES IN BIOMASS CONVERSION

0-Xylosidases are rather large enzymes, with molecular weights exceeding 100 kDa, and are often reported to consist of two or more subunits (16-20). Most purified β-xylosidases show highest activity towards xylobiose and no activity towards xylan. The activity towards xyloologosaccharides generally decreases rapidly with increasing chain length (17,21). In addition to formation of xylose, many j8-xylosidases produce transfer products with higher molecular weights than that of the substrate (17, 22). Some 0-xylosidases have also been reported to possess j8-glucosidase activity (17, 19). An important characteristic of 0-xylosidases is their susceptibility to inhibition by xylose, which may significantly affect the yield under process conditions (17, 20, 23). jS-Xylosidase is the key enzyme for production of monomeric xylose from solubilized xylan fragments, such as those obtained from a steaming process (20). They have been shown to act in synergism with the substituent-cleaving enzymes in the hydrolysis of substituted xylooligosaccharides (24, 25). The β-xylosidase of Trichoderma reesei was not able to hydrolyze xylobiose bearing an acetyl substituent at the non-reducing end without the presence of acetyl xylan esterase (Poutanen, K.; Sundberg, M.; Korte, H.; Puis, /. Appl Microbiol Biotechnol in press). q-Arabinosidases a-I^Arabinofuranosidases (EC 3.2.1.55) hydrolyze non-reducing a-L-arabinofuranosyl groups of arabinans, arabinoxylans, and arabinogalactans, as reviewed by Kaji (26). The production of α-arabinosidases in microorganisms is often associated with the production of pectinolytic or hemicellulolytic enzymes, e.g. in Corticium rolfsii (27), Sclerotina fructigena (28), Γ. reesei (29, 30), and different Streptomyces species (31-33). Some reported molecular characteristics of α-arabinosidases are presented in Table I.

Table I. a-Arabinosidases

Microorganism

Aspergillus niger Trichoderma reesei Streptomyces spp. Streptomyces purpurascens Ruminococcus albus

MW (kDa)

pi

1}

53 53 92 495 , 62 305 , 75 2>

2)

7>

7)

2)

2)

3.6 7.5 4.4 3.9 6.8

Reference

34, 35 30 31 36 37

1) Gel chromatography 2) SDS-PAGE



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The purified α-arabinosidase of Aspergillus niger (38), as well as that partially purified from a commercial pectinase preparation (39), was able to release L-arabinose from wheat L-arabino-D-xylan. As the reaction proceeded an amorphous precipitate, consisting mainly of D-xylan with only traces of arabinose, was formed. Adrewartha et al (40) prepared a series of arabinoxylans from purified wheat-flour arabinoxylan by partial removal of arabinosyl side branches using an α-L-arabinosidase. They suggested that the solubilizing effect of the arabinosylsubstituents was not a result of increased hydration, but due to their ability to prevent intermolecular aggregation of unsubtituted xylose residues. Cereal endospermic arabinoxylans especially are known to form viscous solutions and gels. It is obvious that suitable α-arabinosidases could be used to control the degree of substitution and hence the water-binding capacity of these pentosans. In a similar way α-galactosidases have been used in adjusting the degree of α-galactosyl substitution and hence the gelling properties of galactomannans (41, 42). α-Glucuronidases α-D-Glucuronidases are required for hydrolysis of the o>l,2-glycosidic linkage between xylose and D-glucuronic acid or its 4-O-methyl ether. The presence of acidic oligosaccharides in xylan hydrolyzates produced by hemicellulolytic enzyme preparations indicates the absence or inadequacy of this enzyme (29, 43). 4-O-Methylglucuronic acid wasfirstdetected in the enzymatic hydrolyzates of glucuronoxylan by Sinner et al 1972 (44). The presence of an uronic acidliberating enzyme was together with β-xylosidase claimed to increase the xylose yield in the enzymatic hydrolysis of hardwood xylan (45). The presence of α-glucuronidase in the hemicellulolytic system of 7! reesei was demonstrated in 1983 (23). The production of α-glucuronidase by many fungi and bacteria (Table Π) has recently been reported. Only a few α-glucuronidases have been totally or even partially purified and characterized. The α-glucuronidase isolated from a culture filtrate of Agaricus bisporus by gel chromatography is a very large protein (450 kDa) (24). The enzyme had a very low isoelectric point and a pH-optimum of about pH 3.3. Of a series of 4-0-methylglucuronosubstituted xylooligosaccharides up to DP 5 tested as substrates, it showed highest activity against 4-O-methylglucuronoxylobiose (52). The α-glucuronidase of A. bisporus had no activity towards polymeric xylan. The α-glucuronidase of T. reesei also had an acidic isoelectric point (25). It had a molecular weight of about 70 kDa as estimated by gel chromatography and a pH-optimum at pH 6 with 4-O-methylgucurono-xylobiose as substrate. The very recently characterized α-glucuronidase of the thermophilic fungus Thermoascus aurantiacus (51) was a single polypeptide chain with a M W of


430



Table IL Microbial producers of α-glucuronidases

Microorganism

Reference

Agaricus bisporus Pleurotus ostreatus Trichoderma reesei Trichoderma spp. Schizophyllum commune Aspergillus awamori Aspergillus niger Tyromyces palustris Streptomyces flavogriseus Streptomyces olivochromogenes Streptomyces spp. Thermobacter aurantiacus

24, 46, 47 46, 47 25, 46 47 48 46 48 47 49 50 33 51

118 kDa. The enzyme had a pH optimum at pH 4.5 and it hydrolyzed 443-methylglucurono-substituted xylooligomers from X to X at rates com parable with that of xylan. 7

7

Esterases In addition to enzymes hydrolyzing the glycosidic linkages in xylans, the requirement of esterases to remove esterified acids from xylans has recently been discovered. Acetyl xylan esterases remove O-acetyl groups from the C-2 and C-3 positions of xylose residues in both xylan and xylooligomers. Feruloyl esterases liberate ferulic acid from arabinoxylans of monocotyledons. Esterases are classified according to their substrate specificity (53). This classification is not, however, very clear due to the rather wide substrate specificities of many esterases. Acetyl esterases (EC 3,1.1.6), a group of enzy mes having highest activity against esters of acetic acid, are widely distributed in nature. They are produced by many animals, plants and microorganisms (54). The occurrence of microbial esterases acting on various synthetic acetyl derivatives of mono- and disaccharides was demonstrated in several studies long ago (55-58), but the role of esterases in the hydrolysis of native acetylated xylans was emphasized only rather recently (59, 60). Biely et al. (59) first reported the presence of acetyl xylan esterases in fungal cellulolytic and hemicellulolytic systems: Trichoderma reesei, Aspergillus niger, Schizophyllum commune and Aureobasidium pullulans. As compared with plant and animal esterases, these fungal esterases exhibited high specific activities



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towards acetylated glucuronoxylan and were therefore named acetyl xylan esterases. During recent years the production of esterases deacetylating xyl an by a number of hemicellulolytic microorganisms has been reported (Table III). Hitherto, only the acetyl xylan esterases of T. reesei have been purified and characterized (67, Sundberg, M.; Poutanen, K., Biotechnol. Appl Biochem., in press). The enzymes both had neutral isoelectric points, but differed in their native molecular weights as assayed by gel chromatography. Both enzymes showed optimal activity at pH values between 5 and 6. One enzyme released only a little acetic acid from acetylated xylooligomers, but showed high activity towards acetyl xylobiose. The other occurred as multiple isoenzymes and showed high activity towards acetylated xylan fragments and polymeric acetyl4-O-methylglucuronoxylan (Poutanen, K.; Sundberg, M.; Korte, H.; Puis, J., Appl Microbiol Biotechnol, in press). Enzymatic deacetylation of beechwood xylan caused precipitation of the polymer. This was shown to be due to molecular aggregation analogous to the behavior of arabinoxylan after a-arabinosidase treatment. Feruloyl esterase activity was first detected in culture filtrates of Strepto myces olivochromogenes (49), and has thereafter also been reported for some hemicellulolytic fungi (Table III). A partially purified feruloyl esterase from S. commune liberated hardly any ferulic acid without the presence of xylanase (65). Very recently a feruloyl esterase was purified from Aspergillus oryzae (Tenkanen, M.; Schuseil, J.; Puis, J.; Poutanen, K., /. Biotechnol, in press). The enzyme is an acidic monomeric protein having an isoelectric point of 3.6 and a molecular weight of 30 kDa. It has wide substrate specificity, liberating ferulic, p-coumaric, and acetic acids from steam-extracted wheat straw arabino xylan. The late discovery of acetyl xylan and feruloyl esterases has been partly due to the lack of suitable substrates. Xylans are often isolated by alkaline extraction, in which ester groups are saponified. Treatment of plant materials under mildly acidic conditions, as in steaming or aqueous-phase thermomechanical treatment, leaves most of the ester groups intact. These methods, however, partly hydrolyze xylan to shorter fragments (63, 69). Polymeric acetylated xylan can be isolated from delignified materials by dimethyl sulfoxide extraction (70). The choice of substrate is especially important in studies of esterases for deacetylation of xylans. The use of small chromophoric substrates (p-nitrophenyl acetate, α-naphthyl acetate, and methylumbelliferyl acetate) analogously to the assays of disaccharidases may lead to the monitoring of esterases unable to deacetylate xylan (33, 63, 64). The substituent-cleaving enzymes in the hydrolysis of xylans It is obvious that some of the accessory enzymes are capable of releasing substituents from polymeric xylan, and consequently they could be used to



+ + + + +, one +, three different +, two different types purified

+ + +, three different +, five different +

Acetyl xylan esterase

-

+, three different + nd nd + nd +, one purified nd +, two different nd

-

nd

Feruloyl esterase

Esterase activity^

1) + = activity detected, - = no activity detected, nd = activity not determined 2) in press, see text for the reference

Butyrivibrio fibrisolvens Streptomyces flavogriseas Streptomyces olivochromogenes Streptomyces mbiginosus Aspergillus awamori Aspergillus japonicus Aspergillus niger Aspergillus versicolor Aspergillus oryzae Fusarium oxysporum Schizophyllum commune Trichoderma viride Trichoderma reesei

Microorganism

Table III. Occurence of hemicellulolytic esterases


2)

2

61 33, 49 29, 33, 49 62 29, 63 63 48, 59, 63 63 64, Tenkanen et al 29 48, 59, 65, 66 59 59, 66, 67, 68, Sundberg and Poutanen ^

Reference


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change the viscosity and solubility of xylans (40, 51, Poutanen, K.; Sundberg, M.; Korte, H.; Puis J., Appl Microbiol Biotechnol, in press). On the other hand, some substituent-cleaving enzymes have been reported to prefer short oligosaccharides as substrates (24, 30, 65). In nature, however, all these enzymes usually occur in mixtures with other biomass-degrading enzymes and act synergistically with them to break down plant cell walls. Synergism between α-arabinosidase, xylanase and jS-xylosidase has been demonstrated in the hydrolysis of wheat straw arabinoxylan with purified enzymes of T. reesei (71). When only xylanase and j3-xylosidase were used in the hydrolysis, the xylose yield was only 66% of that produced by the whole culture filtrate at the same activity levels of these two enzymes, and no arabinose was produced. Addition of α-arabinosidase increased the yields of both xylose and arabinose. Enhanced hydrolytic action of hemicellulolytic or pectinolytic enzymes in the hydrolysis of alfalfa cell wall polymers by addition of Ruminococcus albus α-arabinosidase has also been reported (37). The synergistic action of depolymerizing and side-group cleaving enzymes has most clearly been demonstrated using acetylated xylans as substrates. Due to the high degree of acetylation, xylanases have only limited access to the polymer backbone in the absence of esterases (71). Deacetylation by acetyl xylan esterase prior to the action of xylanases, however, resulted in a lower yield than that obtained by the simultaneous action of xylanase, 0-xylosidase and esterase (Poutanen, K.; Sundberg, M.; Korte, H.; Puis, J., Appl Microbiol Biotechnol, in press). The sequence of enzyme application not only influenced the extent of hydrolysis but also the nature of the oligomeric end products.

Acknowledgments Maija Tenkanen has a personal grant from the Neste Oy Foundation.

Literature cited 1. 2. 3. 4. 5. 6. 7.

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