Xylan degradation by the anaerobic bacterium Bacteroides xylanolyticus

Plant cell walls are the major reservoir of fixed carbon in nature. The mineralization of the fiber material, the so called lignocellulosic complex, proceeds almost exclusively by microbial processes in both aerobic and anaerobic environments. In anaerobic microbial processes the energy of the plant polymers can be conserved in fermentation products. The valorization of agricultural waste plant materials can consist of low and high technological processes. These include the production of biogas, ethanol, solvents and enzymes.The first step in the anaerobic conversion of plant cell wall material is enzymatic degradation of the polysaccharides to soluble sugars. This is the rate limiting process and it is often incomplete. The hernicellulose xylan is one of the major constituents of plant cell walls. The structure of this polysaccharide is dependent on the source from which it has been isolated. The diversity of xylan is one of the reasons why the hydrolysis of this polysaccharide can be slow or incomplete. For a thorough comprehension of anaerobic digestion it is therefore important to obtain a better insight into the microbiology of xylan-degrading bacteria.The aim of this thesis was to examine the nature of the enzymes needed for a complete degradation of xylan and to study their regulation. In parallel a research was conducted by Steef Biesterveld to investigate the bacterial fermentation of xylose, the major constituent of xylan. The xylose uptake system as well as the xylose metabolic pathways of the model organism Bacteroides xylanolyticus X5-1 were investigated. The main topics of his research are summarized here. Xylose enters the cell through an active uptake system (Fig. IC and ID) and is converted via the pentose phosphate pathway followed by the glycolysis to acetate, ethanol, H 2 , C0 2 and formate as the main fermentation products. The first two enzymes in the xylose metabolism, xylose-isomerase and xylulose-kinase were inducible, whereas the xylose transport was constitutive. The regulation of product formation and the regulation of the formation of some key enzymes by H 2 were investigated. No diauxic growth, that is no preference for glucose over xylose or arabinose was observed. External electron acceptors could be used to shift the metabolic pathways. It was shown that it is possible to modulate the xylose metabolism by several methods and at different levels.The complete microbial degradation of branched xylan involves the action of several hydrolytic enzymes: endo-1,4-β-D-xylanases (EC 3.2.1.8) which hydrolyze the internal β-1,4- xylosic linkages of the xylan backbone, β-D-xylosidases (EC 3.2.1.37) which release xylose residues from small oligomeric substrates, and several enzymes capable of hydrolyzing substituents from the xylan backbone such as arabinofuranosidases and acetyl esterases.Bacteroides xylanolyticus X5-1 , a predominant strain isolated from fermenting cattle manure, grows efficiently on xylan. The organism produces at least five different enzymes to degrade this polymeric substrate. These enzyme activities include xylanase, β-D- xylosidase, acetyl-esterase and α-L-arabinofuranosidase activities. The enzymes are not secreted into the medium, but stay associated to the cell during exponential growth (Fig. IA and 1B). This enables this organism to efficiently use the degradation products of the different enzymes in a highly competitive environment. The production of the enzymes is tightly regulated. The mechanism of control of xylanolytic enzyme synthesis varies considerably among different microorganisms as is revealed by an analysis of literature data. Induction, catabolite repression, growth rate and other environmental factors can influence the activity of the xylanolytic enzymes. High molecular weight xylan can not enter the cells, and consequently can not directly induce the synthesis of xylanolytic enzymes. Low molecular weight sugars are often involved in either the induction and/or the repression of xylan degrading enzymes.In chapter 2 the regulation, purification and some properties of two endo-β-1,4-xylanases were presented. During growth on xylan, B . xylanolyticus X5-1 produces two different endo-β-1,4-xylanases. These enzymes were purified by column chromatography to apparent homogeneity. Both enzymes are monomeric with a molecular weight of 38000 (xylanase 1) and 63000 Da (xylanase 11), respectively. Xylanase I degraded xylan and xylo-oligomers with a polymerization degree of 4 and higher. Xylanase I of B. xylanolyticus released arabinose after prolonged incubation with xylan, and after incubation with arabinose containing xylo-oligomers. Since arabinose release coincided with xylose appearance in the assays, this side activity seems to be the result of unspecific cleavage. The final products of the enzymatic degradation of arabinoxylan by xylanase I are xylotriose, xylobiose, xylose and arabinose. Xylanase 11 degraded xylan to xylose and xylobiose. Small xylo-oligomers were degraded much slower than the polymeric substrate. Arabinose was not released from oat spelt arabinoxylan or smaller oligosaccharides by this enzyme.The regulation of the formation of the two xylanases was investigated. Little attention has so far been paid to the possibility that the formation of individual xylanases might be under different control. B. xylanolyticus has a differential regulation for the synthesis of the two xylanases. Xylanase I production did not seem to be induced by a direct product of xylan degradation, but was constitutively synthesized when no easily metabolizable sugars were present, and enough energy was available for the cells (Fig. 1 A). The formation of xylanase I was repressed by readily metabolizable sugars as well as by the non-metabolisable sugar D-arabinose. The uptake of D- arabinose and the other sugars must therefore be involved in the repression of xylanase synthesis or the sugars themselves are direct repressors (Fig. 1C). Short xylo-oligosaccharides were good inducers of xylanase I. In resting cell suspensions pyruvate induced high levels of this enzyme. Pyruvate, an intermediate of the sugar degradation, probably acted as a good inducer because it circumvented the catabolic repression normally occurring when easily metabolisable sugars are available to the cells. Under growing conditions with pyruvate as sole carbon source, B. xylanolyticus did not produce xylanase activity. B. xylanolyticus produced low levels of xylanase activity in the presence of low concentrations of pentoses, which are released during xylan degradation. Regulation of xylanase I can be interpreted as constitutive synthesis under catabolite control. The availability of enough energy was the main factor responsible for xylanase I formation, but sugars present in the medium repressed the xylanase I formation. The uptake of the sugars is likely to be involved in the xylanase repression (Fig. 1C).Xylanase II was only produced in significant amounts when the organism was grown on xylan. It was produced in higher amounts on birch- or larch-wood xylan, compared to oat spelt xylan. On monomeric sugars, as well as short xylo-oligomers, no xylanase II was produced. The formation of this enzyme was also catabolite repressed by easy metabolisable substrates. The exact nature of the inducer of this enzyme was not identified, but one could think of a xylooligomer containing specific side chains, found in wood-xylan more than in grass xylan (Fig. 1A). B. xylanolyticus X5-1 is able to fine time its xylanase synthesis according to demand. It is possible that the difference in substrate specificities of the two purified xylanases reflect their role in xylan degradation. Xylanase I has a broad specificity and is produced constitutively. Xylanase II may be better adapted to attack wood xylan, because it is preferentially produced under these conditions. The different regulation mechanism and the substrate specificities of the two xylanases allows a flexible response to changes in nutritional conditions.In chapter 3 the regulation, purification and properties of a β-xylosidase of B. xylanolyticus X5-1 was presented. The formation of β-xylosidase activity is induced by the pentoses D-xylose and L-arabinose and repressed by intermediates of the sugar metabolism and pyruvate (Fig. 1 B). A simultaneous induction and repression of the β-xylosidase synthesis by xylose was observed, which resulted in an optimal inducer concentration of about 20 mM. The repressive effect of glucose on the β-xylosidase induction by xylose was immediate and can be interpreted in terms of catabolite repression. A regulation by inducer exclusion, by which glucose prevents the entry of inducers, is not likely since both xylose and glucose were consumed simultaneously by cell suspensions.The β-xylosidase induced by xylose was purified by column chromatography. The purified enzyme had a very low thermostability. In vivo, the enzyme would probably be more stable when located in the cytosol. It had an apparent molecular weight of 165 kDa and was composed of two subunits of 85 kDa. The enzyme exhibited optimal activity at pH 6 and 40°C. The isoelectric point was 6.3. It hydrolyzed p-nitrophenyl-β-D-xyloside with a Km of 0. 125 mM. The activity was strongly inhibited by Hg 2+. The β-xylosidase of B. xylanolyticus hydrolyzed p-nitrophenyl-β-D-xylopyranoside and can be denoted as a typical β-D-xylosidase in the sense that it could cleave of single xylose units from short xylo-oligosaccharides. The activity for xylobiose and xylotriose was much higher than for the longer xylo-oligomers. Xylan and other p-nitrophenylglycosides were no substrates for the enzyme.In chapter 4 the purification of a cell-associated α-L-arabinofuranosidase of B. xylanolyticus X5-1 is described. The enzyme was purified 41 -fold to apparent homogeneity. The native enzyme had an apparent molecular mass of 364 kDa and was composed of six polypeptide subunits of 61 kDa. The enzyme was stable under the conditions found in the extracellular environment. The enzyme was not affected by divalent cations and was very sensitive to sulfhydryl inhibitors like mercury indicating the presence of essential thiol groups in the enzyme. The anaerobic growth environment of the organism probably ensures that the sulfhydryl groups remain reduced. The substrate specificity of the purified α-L-arabinofuranosidase was very narrow. It was only able to release the a-linked L-arabinose in the furanose form from the synthetic substrates tested. The K m and V max for p-nitrophenyl-α-L-arabinofuranoside were 0.5 mM and 155 U/mg of protein, respectively. Compared with the high activities found with the artificial substrate p-nitrophenyl-α-L-arabinofuranoside the activities with potential natural substrates were low. The enzyme was unable to release L- arabinose from arabinogalactan or oat spelt arabinoxylan, it could however cleave of arabinose residues from arabinose containing xylo-oligosaccharides with a polymerization degree of about 2 to 5. The enzyme belongs to the Streptomyces purpurascens -type of α-L- arabinofuranosidase.The synthesis of the multimeric α-L-arabinofuranosidase in B. xylanolyticus X5-1 was regulated. High activities were found after growth on L-arabinose and D-xylose, compared to growth on xylan (Fig 1B). This suggest that the synthesis of α-L-arabinofuranosidase is induced by these pentoses or by metabolites directly derived from these sugars. The enzyme was not produced when the organism was grown on glucose or cellobiose. These hexoses acted as catabolite repressor. The enzyme was mainly extracellularly attached to the cell when the organism was grown on xylan and was not released into the medium. In this way the enzyme can cooperate with the xylanases of this organism.Chapter 5 describes the purification and characterization of a xylose acetyl esterase of B. xylanolyticus X5-1. No acetyl-xylan esterase activity could be detected in cultures grown on xylan, but B. xylanolyticu s X5-1 produced high activities of an acetyl-xylose esterase. The synthesis of the acetyl-xylose esterase in B. xylanolyticus X5-1 was regulated. The acetyl xylose esterase was only produced in significant amounts when the organism was grown on xylan (Fig. 1A). The acetyl esterase was purified by column chromatography from cell extracts of B. xylanolyticus X5-1 grown on xylan. The enzyme had an apparent molecular mass of 245 kDa and was composed of 4 identical subunits of 62 kDa. No metal ions were required for activity. The enzyme was stable under the conditions found in the extracellular environment. The acetyl esterase from B. xylanolyticus X5-1 was not active on acetylated xylan, but hydrolyzed several low molecular weight acetyl esters, but not esters of fatty acids with a longer chain length. This enzyme could therefore be classified as an acetyl esterase (E.C. 3.1.1.6.). Compared to other acetyl esterases this enzyme had a high activity on β-D-xylose tetraacetate. Furthermore, this enzyme hydrolyzed all four acetyl groups from the β-D-xylose tetraacetate. The fact that the acetyl esterase was mainly synthesized when the organism was grown on xylan, makes it probable that it plays a role in the degradation of xylan. The cell associated acetyl esterase of B. xylanolyticus X5-1 could be involved in delivering unsubstituted xylose, xylobiose and xylotriose to the cell, thus facilitating the uptake of these sugars and the subsequent hydrolysis by β-xylosidase.B . xylanolyticus X5-1 has a set of enzymes enabling it to efficiently grow on xylan. All the xylanolytic enzymes studied in this research do not seem to be located in one regulon. The α-L-arabinofuranosidase and β-xylosidase could be located in one operon. By having different induction mechanisms for the synthesis of the xylanolytic enzymes, this organism is able to adapt specifically to the environmental conditions. In figure 1, the xylan degradation and enzyme regulation by B. xylanolyticus X5-1 is represented schematically.