Accessory enzymes from Aspergillus involved in xylan and pectin degradation

The xylanolytic and pectinolytic enzyme systems from Aspergillus have been the subject of study for many years. Although the main chain cleaving enzymes and their encoding genes have been studied in detail, little information is available about most of the accessory enzymes and their corresponding genes. This thesis describes the purification and characterisation of two accessory enzymes from Aspergillus , feruloyl esterase A (FaeA) andα-glucuronidase A (AguA), and the activities of these enzymes on polymeric substrates in relation to other accessory enzymes. Furthermore, the characterisation and regulation of the FaeA and AguA encoding genes ( fae A and agu A), and some genes encoding other accessory enzymes is studied. FaeA is the major feruloyl esterase produced when Aspergillus niger is grown on xylan or crude substrates such as wheat bran or sugar beet pulp. Addition of ferulic acid, the product of FaeA, to media containing xylan increases the production of this enzyme. FaeA is able to release ferulic acid from xylan and pectin oligosaccharides, as well as from synthetic substrates such as methylferulate. The fae A gene was cloned from A. niger and Aspergillus tubingensis . A blast of the deduced amino acid sequence of FaeA revealed no significant homology to other proteins, except for a small region of FaeA which was highly similar to the active site of lipases. Based on this homology, a 3-dimensional model for FaeA was proposed by Pickersgill et al. Although only 16 amino acid differences were observed between FaeA from A. niger and A. tubingensis , the latter enzyme was found to be much more sensitive to proteolytic degradation. AguA from A. tubingensis was able to release (4-O-methyl-) glucuronic acid from xylan derived oligosaccharides, but had very little activity towards the intact polysaccharide. The agu A gene was cloned, and database analysis of the deduced amino acid sequence revealed homology to theα-glucuronidases from Trichoderma reesei and Thermotoga maritima . Regulation of fae A and agu A expression was studied in A. niger and compared to other xylanolytic genes. Both genes were found to be under the control of the xylanolytic transcriptional activator protein XlnR, which also regulates endoxylanase,β-xylosidase, acetylxylan esterase, arabinoxylan arabinofuranohydrolase, and endoglucanase gene expression. In a XlnR negative mutant no expression of fae A and agu A was observed on xylose or xylan. Expression of fae A in this mutant was observed in the presence of ferulic acid, indicating the presence of a second system for the induction of this gene. This system seems to be specific for fae A, since no expression of agu A or other xylanolytic genes was observed under these conditions. In a wild-type A. niger strain, expression levels of fae A were higher on a combination of xylose and ferulic acid than the sum of the expression levels on xylose and ferulic acid alone, suggesting a synergistic effect of these two inducing systems. The carbon catabolite repressor protein CreA is involved in the repression of xylanolytic gene expression in the presence of easy metabolisable carbon sources, such as glucose or fructose. Expression of agu A and fae A on xylose and xylan, as well as expression of fae A on ferulic acid was repressed in the presence of glucose. Depending on the concentration of xylose present in the medium, this sugar also triggers CreA mediated repression of xylanolytic gene expression. Using a concentration range from 1 to 100 mM, it was shown that expression levels of fae A, agu A, and genes encoding endoxylanase B andβ-xylosidase decreased with increasing xylose concentrations in an A. niger wild type strain. In a CreA derepressed mutant constant levels of XlnR induced gene expression were observed indicating that the xylose concentration has a modulating effect via CreA. A gene ( agl B) encoding anα-galactosidase, which was produced when A. niger was grown on crude wheat arabinoxylan, was cloned and the expression of this gene was compared with the expression of two otherα-galactosidase encoding genes ( agl A and agl C) and aβ-galactosidase encoding gene ( lac A) from A. niger . All four genes had specific expression profiles with respect to monomeric sugars, galacto-oligosaccharides and polymeric substrates. High expression on xylan was only observed for agl B and lac A, suggesting that these genes may be part of the xylanolytic system from A. niger . This was confirmed using a XlnR negative mutant, which showed no ( lac A) or reduced ( agl B) expression of these genes on xylose. Synergy was studied between the accessory enzymes from Aspergillus involved in xylan degradation and two main chain cleaving enzymes, endoxylanase A (XlnA) andβ-xylosidase (XlnD). Except forα-L-arabinofuranosidase B (AbfB), the activity of all accessory enzymes on xylan was increased in the presence of XlnA. Similarly, the presence of accessory enzymes increased the activity of XlnA on xylan, indicating synergy between these enzymes. Synergy was also observed between the accessory enzymes, resulting in more efficient degradation of xylan. These results confirm that the gene products of XlnR regulated genes are in fact all part of the xylanolytic enzyme system of Aspergillus . Similarly, the synergy of enzymes involved in the degradation of the hairy regions in sugar beet pectin was studied. Degradation of the pectin backbone did not influence the activity of the arabinose releasing enzymes, AbfB and endoarabinase (AbnA), but had a strong effect on the release of ferulic acid by FaeA and the release of galactose by endogalactanase (GalA) andβ-galactosidase (LacA). Synergy was also observed between galactose- and ferulic acid- releasing enzymes. The accessory enzymes from Aspergillus involved in the degradation of xylan and pectin form a diverse group of enzymes which actively co-operate in polysaccharide degradation. Common factors have been identified in the regulation of the genes encoding these enzymes, but the expression patterns of the different genes also indicate the presence of other factors influencing specific genes. This most likely enables Aspergillus to modulate the production of these enzymes to obtain an efficient mixture for the degradation of the variety of substrates it encounters.