Enzymatic Conversion of Biomass for Fuels Production - American

Chapter 23

Role of Acetyl Esterase in Biomass Conversion

Downloaded by NORTH CAROLINA STATE UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch023

James C. Linden, Stephen R. Decker, and Meropi Samara Departments of Microbiology and of Agricultural and Chemical Engineering, Colorado State University, Fort Collins, C O 80523

Acetyl esterases that deacetylate native xylan occur in cellulolytic systems of several fungi. The biochemical and kinetic properties of acetyl xylan esterases (AXE) that have been purified are reviewed. One of the major resistances of lignocellulosic materials to hydrolysis is the high degree of acetylation of the hemicellulose fraction. This is especially true for hardwood biomass. Enzymatic deacetylation may be a prerequisite for the technological utilization of biomass feedstocks for ethanol production.

Currently, the main conversion of plant biomass to fuels is from cellulose to ethanol. This process is incomplete as it does not utilize the entire sugar content of the plant biomass. In addition to cellulose, two other carbohydrate polymers are abundant in plant biomass. These other polymers are hemicellulose and pectin. Utilization of these carbohydrates, in addition to cellulose, may greatly improve the productivity and yield of fuels from biomass. The main problem with the utilization of hemicellulose is the high degree of substitution found on the hemicellulose backbone. These substituents interfere with the enzymatic degradation of the hemicellulose to monomeric sugars through steric hindrance. Removal of these side-chain groups would greatly facilitate the conversion of hemicellulose sugars to ethanol. Hemicellulose i n P l a n t C e l l W a l l Structure The most abundant source of carbohydrates in the world is plant biomass, which represents the lignocellulosic materials that comprise the cell walls of all plant cells. Plant cell walls are divided into two sections, the primary and the secondary cell walls. The primary cell wall, which functions to provide structure for expanding cells (and hence changes as the cell grows) is composed of three major polysaccharides, and one group of glycoproteins. The main polysaccharide, the most abundant source of carbohydrates, is cellulose. The second most important sugar source is

0097-6156/94/0566-0452$08.00/0 © 1994 American Chemical Society

In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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hemicellulose and the third polysaccharide is pectin. The compounds contained i n the strongly basic glycoprotein group are referred to as extensins. The secondary cell wall, which is produced after the cell has completed growing, also contains polysaccharides and is strengthened by lignin covalentiy cross-linked to the carbohydrate polymers, cellulose and hemicellulose. Cellulose is a linear p-(l-»4)-D-glucan and comprises 20 to 30 percent of the primary cell wall. Pectins are "chelating-agent-extractable polysaccharide plus chemically similiar inextractable polysaccharides"^,). Pectins are rich i n Dgalacturonic acid and also contain, in decreasing order, arabinose, galactose and rhamnose. Hemicellulose is a general term used to refer to cell wall polysaccharides that are not celluloses or pectins. Hemicelluloses include a variety of compounds including xylans, xyloglucans, arabinoxylans, mannans, and others. Hemicelluloses are always branched with a wide spectrum of substituents along the backbone polysaccharide. Hemicellulose is hydrogen bonded to cellulose as well as to itself, and can be isolated by extraction with alkali or dimethyl sulfoxide. The hydrogen bonding is thought to help stabilize the cell wall matrix and render the matrix insoluble i n water. Hemicellulose is also bonded to lignin through ferulic acid residues which covalentiy link lignin to the arabinosyl and 4-O-methyl glucuronyl residues of hemicellulose, further enhancing the stability of the cell wall. Xylan Structure Hemicelluloses that are based on a xylose backbone, regardless of source, are termed xylans. Xylans occur widely and may be present in all higher land plants; they have been isolated from grasses, legumes, ferns, mosses, softwoods and hardwoods (2). X y l a n is a linear P-(l—>4)-D-xylopyranose polymer with substituents attached at the number 2 and 3 hydroxyl. These substituents include a-L-arabinose and a-Dglucuronic acid residues, the 4 - 0 - methyl ester derivative of a-D-glucuronic acid, cinnamate-based esters, some short oligosaccharides, and acetyl esters (Figure 1). Examination of xylans isolated from leaves and stems of several grasses and cereals show that these xylans carry low proportions of L-arabinofuranose units on the 0-3 position i n the main chain, and the same or lower proportions of D-glucuronic acid and/or its 4-O-methyl ester on the 0-2 position in the main chain. Xylans from Zea mays include in their backbone D- and more rarely L-galactose units. Some xylans isolated from a few grasses carry high proportions of arabinose units in their backbone. The xylans from immature grasses are also esterified with phenolic acids (3). Coumaroyl and feruloyl moieties are thought to anchor the xylan chains to the lignin matrix by crosslinking the two polymers (2,4). The substituent chemical groups found on the xylan chain may be attached to the main chain as single units or occasionally as complex branches (5). The role of these groups is not clear. They may play a major role in the resistance to hydrolyzing agents, such as enzymes. The degree of substitution of carbohydrate side chains varies greatly and has a major influence on the solubility and functionality of the xylan. The more substituted xylans cannot self-hydrogen bond, and become more water soluble. Xylans substituted to a low degree hydrogen bond not only to themselves, rendering them insoluble, but also to cellulose, screening it from degradation. Highly substituted xylans do not hydrogen bond to cellulose very well and may have only a limited protective effect against cellulose degradation. A high degree of xylan acetylation also increases the solubility of xylan by preventing In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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interchain hydrogen bonding (6). According to Poutanen, et al. (7) acetate esters impacted xylan hydrolysis in two ways; acetyl substituents increase the solubility of polymeric xylan but also hinder complete enzymatic hydrolysis of oligosaccharides to xylose and xylobiose. B y removing acetyl groups and allowing interchain hydrogen bonding, A X E opens up binding sites for endoxylanase. Too much deacetylation without concurrent fragmentation of the polymer by endoxylanase, on the other hand, causes precipitation of the xylan, rendering it unavailable to the xylanases. Acetylated X y l a n . Acetylated xylan is mainly a component of higher land plants e.g. trees (8, 3, 9) and grasses (3,10) etc. The high content of acetic acid found i n many wood and cereal hydrolysates suggests that xylan is generally acetylated. The acetyl contents of some hardwoods are shown in Table I. The degree of acetylation is different for xylans of different origin. Usually, when isolated under milder conditions than alkali extraction, such as using D M S O , seven out of ten xylose units are found to be acetylated (6,11). However, lower and higher degrees of substitution have been reported. In hardwoods and mature grasses every second xylose residue is acetylated (12), while in mature beech leaves, almost all xylose residues carry acetyl groups (10). In a study of a hardwood hemicellulose preparation, the acetyl substituents were located at the hydroxyl groups of carbons 2 or 3 or both positions (2:3:2+3) of the xylopyranose ring i n the ratio 24:22:10 (13). It is assumed by analogy that the acetyl groups found in other xylans can be assigned to similar positions (10). It should be noted, however, that during extraction of xylan, transesterification can occur readily so these reported ratios may not reflect the true situation i n native xylan. E n z y m e s Relevant to X y l a n Digestion Xylanase. Xylanase is a general term given to the enzymatic system that degrades xylan. The system is based on two core enzymes; endoxylanase and P-xylosidase. Endoxylanase acts on the long chain, l o w substituted xylan polymers, cutting the long chains into short oligomers. A high degree of substitution on the chain prevents efficient hydrolysis, probably due to steric hindrance induced by the side chains. The short oligomers produced are degraded by P-xylosidase, yielding monomeric xylose and preventing end-product inhibition of the endoxylanase. P-xylosidase is most active on xylobiose, with decreasing activity on increasing chain length (14, 15). p-xylosidase is also hindered by substituents on the xylan oligomer. C o u m a r o y l and F e r u l o y l Esterases. In addition to the two core depolymerizing enzymes, other accessory enzymes are present in the xylanase system. T w o of these are coumaroyl esterase and feruloyl esterase, which remove coumaric and ferulic acid esters (respectively) from the xylan chain. Coumaric acid and ferulic acid are cinnamate-based acids that occur in an ester linkage to a-L-arabinofuranose units attached to the xylan chain. They function to protect the xylan chain from degradation by preventing xylanase access to the polymer. They also form cross-links to the lignin matrix, giving structural support to the cell wall (16). Ferulic acid has also been shown to form diferulic acid bridges between different xylan chains, giving


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additional strength through cross-linking of the xylan chains. Coumaroyl and feruloyl esterase removes these moieties, opening up the cell wall structure and allowing xylanase enzymes easier access to the xylan polymer. Although most reports indicate that there is a separate enzyme for removing each acid, a couple of studies have presented evidence of a single enzyme capable of removing both coumaroyl and feruloyl acid from the xylan chain (16, 17). a-L-arabinofuranosidase a n d a-D-glucuronidase. Three other substituents are common on xylan; a-L-arabinofuranose, cc-D-glucuronic acid, and 4-O-methyl glucuronic acid. These also need to be removed to prevent steric hindrance of the xylanase enzymes. The arabinose residues act as attachment points for ferulic and coumaric acid, as well as other sugars and are removed by a-L-arabinofuranosidase. a-D-glucuronic acid and its 4-0-methyl ester are removed by a-D-glucuronidase. A c e t y l Esterase. Acetyl esterase is a general term for an enzyme that cleaves an esterified acetic acid residue from a compound. In this paper, acetyl esterase refers to the enzyme that cleaves acetic acid from a short acetylated xylan oligomer and is inactive against acetylated long-chain xylan polymers. This enzyme acts on the short acetylated end-products of xylan degradation, removing any remaining acetyl groups and allowing P-xylosidase access to the xylose oligomer. A c e t y l esterases are distinguished from acetyl xylan esterases by the specificity of acetyl esterase for acetylated xylan oligomers and certain chromophoric substrates which mimic these short oligomers. Acetyl esterase is not active against intact longchain acetylated xylan. The activities of acetyl esterases on these chromophoric substrates indicates that the enzymes are probably active against small degradation products such as acetylated xylose, xylobiose, and xylotriose. Acetyl esterases have also been shown to be restricted to deacetylating xylobiose only at the C-3 position (18). A c e t y l xylan esterase ( A X E ) . In contrast to acetyl esterase, A X E is active against long-chain intact acetylated xylan. B y removing acetyl groups from the long chain xylose polymer, A X E opens up binding sites for endoxylanase, allowing production of short xylan fragments. Acetyl esterases active on acetylated xylan have been detected i n enzyme systems from fungi, streptomycetes, plants and animals. Acetyl xylan esterases from fungal systems have been found to exhibit higher activities against acetylated xylan than any acetyl esterase of non-fungal origin (28). Assay o f X y l a n a s e , A c e t y l X y l a n Esterase, a n d A c e t y l Esterases X y l a n a s e . Most studies on xylan digestion have employed deacetylated xylan as the test substrate. Native acetylated xylan is difficult to obtain. Xylans i n native cell walls are insoluble i n all known unreactive solvents, because of their covalent crosslinking to lignin, which prevents their isolation in an unmodified state (19). The procedures most commonly used for xylan isolation are harsh and usually utilize strong solutions (4-15%) of sodium or potassium hydroxide (9, 12). Treatment with alkaline solutions saponifies most or all ester groups and frees ester crosslinks (20). Therefore, these xylan preparations are extensively modified. This xylan preparation


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PRODUCTION


is useful for studying the action of endoxylanase or p-xylosidase, but it is not appropriate for the study of other xylanolytic accessory enzymes or for the study of the entire xylan-degrading system. A c e t y l x y l a n esterases. One of the largest drawbacks to studying the activity of acetyl xylan esterases directly is the lack of a readily available substrate. There are two general types of substrates currently used to assay for A X E . One type contains an acetyl group in an ester linkage with some easily detectable molecule. When the acetate is cleaved off, the indicator is freed and can be easily detected. Examples of this type include p-nitrophenyl acetate, a-napthyl acetate, and 4-methylumbelliferyl acetate. These substrates have the advantage of being readily available and quickly detected by simple means, such as colorimetric or fluorescent assays. The disadvantages include instability and nonspecificity. M a n y organisms produce a multitude of acetyl esterase enzymes, many of which w i l l cleave these simple molecules. Because of this, activity of an enzyme against these molecules is not a reliable indication of acetyl xylan esterase activity. The other type of substrate is acetylated xylan, either naturally derived or chemically acetylated. Isolation of "native" xylan is accomplished i n two ways, either by steam extraction or by thermomechanical extraction. In steam extraction, wood fibers are steamed at 200°C and the water soluble fraction is dialyzed against water, providing "native" hemicellulose (21). The thermomechanical method involves treating wood fibers i n an aqueous phase thermomechanical treatment loop for 103 s at 220°C with a shear value of 27 M P a . This treatment solubilizes about 60% of the starting hemicellulose. After centrifugation and dialysis, the hemicellulose is freeze dried for storage (22). Alternatively, D M S O can be used to extract intact xylan from delignified biomass (23). In order to obtain a substrate comparable to the native acetyl xylan, methods like chemical acetylation of commercially available xylans have been used in recent studies of xylan digestibility (24, 25). X y l a n purified from wood fibers is acetylated by mixing with D M S O at room temperature, then heating slowly to 55°C over a twenty minute period to solublize the xylan. Potassium borate is added to a final concentration of 0.8% (w/v) and stirred for 10 minutes at 55°C. Acetic anhydride (200 m L , 60°C) is added over five minutes. The mixture is then dialyzed for about five days against running tap water and then 24 hours against distilled water, after which it is lyophilized (25). It is difficult to determine what type of xylan best imitates native xylan. In a study comparing the action of different A X E s on different types of acetylated xylan, K a h n et al. examined the A X E activities of a number of Aspergillus species (22). Nine different strains of Aspergillus were studied, including five strains of A. niger and four other species. T w o different xylans were studied. Native xylan in this study was obtained by thermomechanical pulping of aspen wood. Analysis of this product showed that it was about 70% sugar and 16% acetic acid, with about 85% of the sugar being xylan, the rest being mannose, glucose and cellobiose. The second xylan was a chemically acetylated larchwood xylan. T w o thirds of the xylose residues i n the native hemicellulose were acetylated, compared to every xylose residue being acetylated i n the synthetic acetyl xylan. Results of the study showed that most of the species were able to deacetylate native hemicellulose more efficiently than synthetic


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acetyl xylan, indicating that A X E acts differently on chemically acetylated xylan than on native xylan, possibly due to different acetylation patterns. A c e t y l Esterases. A c e t y l esterases are easily assayed by using the previously mentioned colorimetric and fluorescent substrates. Although some A X E s may also cleave these substrates, the inactivity of acetyl esterase on intact acetylated xylan allows n o n - A X E acetyl esterase to be readily distinguished from A X E .


Specific E x a m p l e s of Investigated E n z y m e s (History) Acetyl esterases were documented i n plant cell wall degradation a few decades ago. In 1963, Frohwein et al. (26) demonstrated the existence of acetyl esterases in unpurified enzyme preparations. W i t h these preparations various acetylated substrates, such as acetyl-mannose, acetyl-glucose, acetyl-maltose and acetyl-cellobiose, were hydrolyzed. Almost twenty years later i n 1981, the role of acetylated polysaccharides was clarified when W i l l i a m s and Withers reported the presence of acetyl esterases again (27). These workers isolated more than 100 bacterial cultures from bovine rumen contents based on their ability to degrade plant cell wall structural polysaccharides. Thirty of the more active xylan degrading isolates, which were used to study the production and activity of the principal enzymes involved i n the hydrolysis of plant cell wall polysaccharides, demonstrated esterase activity. Esterases proved to act on acetylated xylan were not described until 1985. A c e t y l xylan esterase [ E . C . 3.1.1.6] was first reported as a new, distinguishable enzyme by B i e l y et al. (28). The presence of A X E was demonstrated using a nondialyzable fraction of birch hemicellulose as a substrate and the release of acetic acid as an indication of enzyme activity. The reaction was proven to be enzymatic by using solutions of different protein content. Esterases of fungal, animal and plant systems were compared; fungal A X E showed the highest activity (Table II). The purified A X E from A. niger studied by Kormelink, et al. (29) was tested against acetylated pectins and was shown to have no activity against them, suggesting the presence of another class of acetyl esterases specific for acetylated pectin. S y n e r g i s m Studies A X E a n d X y l a n a s e . Acetyl ester groups protect the xylan backbone from enzymatic attack and contribute to the resistance of plant cell wall to degradation. U n t i l recently, the effect of acetylation on the digestibility of native xylans has been overlooked. Acetylation greatly impedes enzymatic hydrolysis of xylan. From studies of the digestibility of dried grasses carried out i n ruminants, (30, 31) the presence of acetylated xylan was shown to be an important factor influencing digestibility of the plant cell wall. Higher degrees of the acetylation were related to lower degrees of digestion (79). Further research on the acetylated xylan itself showed that the xylan was more digestible by esterase-free xylanase preparations after it had been chemically deacetylated (24). The same xylan was found to be more susceptible to enzymatic hydrolysis when the xylanase was supplemented with acetyl xylan esterase enzymes (32). Puis et al. (13) showed that acetyl-4-O-methylglucuronoxylan from beechwood, treated first with acetyl xylan esterase and then xylanase, showed a high


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T a b l e I. A c e t y l content of various hardwoods*


Hardwood

A c e t y l content (% w/w)

3.4 4.2 4.2 3.9 3.3 3.1 4.4 3.9 1.8 3.8 3.2 1.5 2.8 3.3 3.8

Aspen Balsa Bass wood Beech Y e l l o w Birch White B i r c h Paper B i r c h American E l m Shellbark Hickory Red Maple Sugar Maple Mesquite Overcup Oak Southern Red Oak Tan Oak Data from ref. 46.

T a b l e II. A X E activity i n fungal a n d bacterial preparations*

Organism

Trichoderma reesei F. oxysporum Streptomyces olivochromogenes Aspergillus awamori Bacillus subtilis

Acetyl xylan esterase (nkat/mL) 366 53 28 3 1

*Data from ref. 40.



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degree of hydrolysis. M u c h higher amounts of xylose, xylobiose and xylotriose were produced i f A X E was added prior to the xylanase, than when xylanase was used alone or with xylanase followed by acetyl xylan esterase. Another study of an A X E from A. niger by Kormelink et al. showed that deacetylation of acetyl xylan was required before xylanases from A. awamori could depolymerize the xylan (29). In a study of A X E from Trichoderma reesei, xylanases and esterases were shown to have synergistic action i n the breakdown of glycosidic bonds (28). This cooperative effect of xylanases on the deesterification of acetyl xylan by esterases was attributed to a preference of esterases for deacetylation of shorter fragments of the substrate. The results of this study were later supported by Poutanen et al. (33). Complete degradation of hemicellulose requires a combination of enzymes (Figure 1). It was also demonstrated by Bachmann and McCarthy that A X E from T. fusca B D 2 1 operated i n a cooperative fashion with other enzymes of the xylanolytic system (34). Liberation of acetic acid from acetyl xylan by A X E increased approximately two-fold when endoxylanase was present, compared to use of A X E alone. Acetylation of xylan can greatly impede enzymatic hydrolysis by microorganisms, and may even be an important factor influencing digestibility of lignocellulose i n the rumen. A s a consequence, the enzymatic deacetylation of xylan may be a prerequisite for its breakdown. Presumably, deacetylation enhances the ability of the microorganisms to digest hemicellulose. M c D e r m i d et al. studied an A X E from Fibrobacter succinogenes, which colonizes digestion resistant plant material such as vascular tissue i n the rumen (35). The extracellular A X E produced by this organism was shown to deacetylate intact hemicellulose without the presence of xylanases, suggesting that the A X E helps prepare the hemicellulose for degradation. T w o acetyl xylan esterases were produced by this organism; one working on intact xylan, the other working on fragmented xylan. The latter enzyme was lost i n the purification step that removed most of the xylanase activity. This finding is supported by other studies. B i e l y et al. isolated a T. reesei A X E that worked very well on birchwood acetylated xylan, yet Poutanen and Sundberg isolated an A X E from the same species that deacetylated acetyl xylan only i n the presence of xylanase, working only on short oligomers of acetylated xylan (6, 15, 32, 36). Synergism between the xylanase enzymes and the acetyl xylan esterase enzyme was demonstrated i n both studies. B i e l y et al. showed that the xylanase enzymes were much more efficient i n degrading xylan after it had been deacetylated. Poutanen and Sundberg, however, showed that the esterase was much more effective in deacetylation after the xylan had been partially degraded. B i e l y first demonstrated synergism among these enzymes in 1986 (32). These and other studies have suggested that the action of xylanases, acetyl esterases and A X E are all required to get high efficiency of xylan degradation. Ester groups play an important role in the mechanisms of plant cell wall resistance to enzyme hydrolysis. Mitchell et al. found i n both aspen wood and wheat straw that the degree of acetylation of xylan was inversely related to the rate of enzymatic hydrolysis of the hemicellulose (24). Aqueous hydroxylamine solutions were used under very specific conditions to deacetylate the two plant materials to values up to 90 percent deacetylation. W i t h increasing degrees of deacetylation, the xylan fraction became five to seven times more digestible. The cellulose fraction also became more accessible and two to three times more digestible. The effect leveled



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A) a-L-arabinofuranosidase B) Acetyl xylan esterase C) a-D-glucuronidase

"

O

'

^

^

^

^

O

H

+

Acetic Acid

Figure 1. The structure and enzymology of native xylan degradation.



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off at approximately 75 percent deacetylation, and other mechanisms such as lignin shielding came into play for providing resistance to enzyme hydrolysis. Overend and Johnson found that lignin-carbohydrate complexes from Populus deltroides contained O-acetyl residues (37). Studies were conducted to characterize three distinct lignin-carbohydrate complexes ( L C C ) isolated from steam exploded poplar. There was no detectable release of reducing groups when these were treated with xylanase i n the absence of A X E . However, A X E isolated from Schizopyllum commune i n addition to xylanase was sufficient for removal of monosaccharides from the L C C fragments. Due to the high degree of acetylation i n native xylan, xylanases have only limited access to the polymer backbone i n the absence of esterases. Poutanen et al. showed that accessory enzymes, such as A X E , a feruloyl esterase and an acetyl esterase, were necessary enzymes involved i n the hydrolysis of arabinoxylans from wheat (15). A. oryzae was reported as a good source of these esterase enzymes. In another study, A X E purified from T. reesei was used separately, simultaneously and sequentially with an A. oryzae xylanase to quantitate the effects of acetate esters on the yield and nature of the products of hydrolysis (7). Acting alone, endoxylanases were able to depolymerize acetyl-4-O-methyl glucuronoxylan from beechwood. Because of steric hinderance of acetyl groups, only small amounts of xylose, xylobiose and xylotriose were recovered. Simultaneous hydrolysis with xylanase and A X E produced xylobiose and xylotriose as the main hydrolysis products. Xylanase followed by A X E produced mainly xylotriose; the opposite sequence, A X E followed by xylanase, produced mainly xylose and xylobiose. Removal of acetyl groups prior to xylanase action resulted in smaller degradation products than any other mode of degradation. K o n g et al. found that aspen wood substrates with varying degrees of deacetylation yielded proportional degrees of enzymatic hydrolysis (38). Deacetylation was controlled i n a stoichiometric manner by the ratio of alkali to wood that was used i n the preparation of the hemicellulose. The greater the degree of deacetylation, the greater the yield of sugars produced after enzymatic hydrolysis. The conclusion reached from this work was that for aspen wood, both acetyl groups and lignin were important barriers to enzyme hydrolysis, whereas the xylan backbone itself was not. Xylanase/Cellulase Synergism. In enzymatic hydrolysis of plant cell walls, cellulose digestion is highly dependent on hemicellulose digestion. Although other factors such as crystallinity and lignin content have been suggested as barriers to the enzymatic attack on lignocellulose (38j, the key to increasing lignocellulose digestibility depends on the increase of the cellulose surface that is accessible to the enzymes. Hemicellulose surrounds the cellulose fibrils, protecting them from any biological attack. This makes it a necessity to hydrolyze hemicellulose first. Digestion of hemicellulose loosens the rigid, complex structure of the cell w a l l and exposes the cellulose surface to cellulase attack.


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Physicochemical Studies Comparison of A X E from intra- and extracellular fractions of T. fusca by S D S - P A G E indicated that the intracellular A X E was an 80 k D a dimer. W h e n released from the cell, the dimer separated into two 40 k D a subunits which were the predominant extracellular form of the enzyme (34). The A X E from A . niger studied by Kormelink, et al. had a p i of 3.0-3.2, an optimal p H of 5.5-6.0 with a stability range of 3.0-8.0 and an optimal temperature of 50°C; activity was rapidly lost above this temperature (29). Using T. reesei R U T C-30 as a source, Sundberg and Poutanen isolated two different A X E enzymes (39). The two enzymes had exactly the same mobility on S D S - P A G E gels and were distinguished by differing isoelectric points. Some properties of these and other acetyl xylan esterases are summarized in Table HI. Enzyme Production Poutanen et al. compared the xylanolytic enzymes produced by fungal and bacterial strains (40). Trichoderma reesei produced the most active xylanolytic enzymes (Table 2). This study was valuable in that it also compared different substrates used to grow the microorganisms. O f all the actinomycetes studied, only two mesophilic species have been the objects of close A X E study, Streptomyces flavogriseus and S. lividans (25, 41). These streptomycetes are generally well suited to production of A X E . Johnson et al. (25,41) studied the response of different Streptomyces strains to different lignocellulosic materials. Some of the results are shown i n Table I V . Conditions have been optimized for production of A X E from A. niger A T C C 10864. Using 5 L fermentation vessels A X E (assayed by deacetylation of chemically acetylated oat spelt xylan) volumetric productivity is greatest at 33°C, 1.5 v v m aeration and 400 rpm agitation with no p H control (Linden, J . C , et al. Appl. Biochem. Biotechnol., in press.). A number of other studies have been carried out on production of A X E s from the fungal and bacterial sources listed in Table I E , with the studies consisting basically of comparisons between different microorganisms, different strains of the same species, various substrates and different assay conditions (21,22,35,36,42-44). Bachmann and McCarthy isolated the individual xylanolytic enzymes of the thermophilic actinomycete Thermomonospora fusca B D 2 1 (34). The growth yield was not affected to any great extent using different carbon sources but xylanase production was impacted. Xylanase production was 100 times greater when xylan was used as the main carbon source, compared to the use of glucose or arabinose and 5 times greater than levels obtained when induced by xylose alone. Extracellular acetyl esterase activity was very low (

Enzymatic Conversion of Biomass for Fuels Production - American

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