Pectinases of Aspergillus niger : a molecular and biochemical characterisation

The major topics of this thesis are the microfilamentous fungus <em>Aspergillus niger</em> and the pectinases a group of extracellular enzymes. Many 'products' of this species hold the GRAS (Generally Regarded As Safe) status and thus pectinases find a broad range of applications in food, feed and beverage industries.Pectinases are enzymes which degrade pectin, a heteropolysaccharide found in the middle lamella and the primary cell wall of higher plants. The number of pectinase-encoding genes identified in this fungus reflects the chemical and structural complexity of this polymer. Amongst more than twenty pectinase genes from <em>A. niger</em> isolated in our research group up to the present day, two gene families encoding pectin lyases (PELs) and endopolygalacturonases (PGs) are found.The first part of this study focuses on the identification and characterisation of new members of the endopolygalacturonase-encoding ( <em>pga</em> ) gene family from <em>A. niger</em> . It includes isolation of four new genes, gene expression studies, overproduction and purification of enzymes and their biochemical characterisation. The study of the pectinase gene regulation is extended in Chapter 5. In the second part of this study one of the pectinase gene, <em>viz.</em> pectin lyase A ( <em>pel</em> A), was used as a probe in a restriction fragment length polymorphism (RFLP) analysis to develop a quick and reliable method for the identification of an unknown black <em>Aspergillus</em> isolate. The establishment of the taxonomic position of a new strain is a necessary prerequisite for its application in biotechnological processes.PGs are enzymes, which hydrolyse theα-[1→4] glycosidic bond between two adjacent<font size="1">D</font>-galacturonic acid residues in the main backbone of pectin called homogalacturonan. One of our questions in the beginning of the pectinase studies was, why a simple eukaryotic micro-organism such as <em>A. niger</em> contains multiple genes-encoding one specific pectinase activity. The molecular and biochemical characterisation of four new <em>A. niger</em> PGs (PGA, PGB, PGD and PGE) presented here together with the data published on three other PGs (PGI, PGII and PGC) during the course of this study now confirm that each of these enzymes can have a specific <em>in vivo</em> role for the fungus.Except for <em>pga</em> D all the <em>pga</em> genes encode enzymes with a high mutual amino acid similarity (66.7 % - 85.0 %). They are closely related to PG-encoding sequences of other aspergilli present in the gene and protein Databases. Expression studies revealed that <em>pga</em> A, <em>pga</em> B and <em>pga</em> E are constitutively expressed. The <em>pga</em> I mRNA was detected in the mycelium grown on pectin whereas the inducing conditions for <em>pga</em> II, <em>pga</em> C and <em>pga</em> D could not be established. This study thus showed that the members of the PG-encoding gene family are differentially expressed. The individual overproduction of these enzymes was made possible by using a strong <em>pki</em> A (pyruvate kinase) glycolytic promoter, which drives the expression of the particular <em>pga</em> gene under growth conditions, which represses the expression of the majority of other pectinases. The isolation of PGA, PGB, PGD and PGE now allowed comparing the biochemical properties of all the members of the family.All enzymes are active in the acidic pH range (3.8 to 5.0), and they differ in their kinetic parameters ( <em>K</em>_m,app and <em>V</em>_max,app ), when using polygalacturonate as a substrate. For PGA and PGB, the constitutively expressed PGs, the <em>V</em>_max,app values are 16.5μkat.mg <sup>-1</SUP>and 8.3μkat.mg <sup>-1</SUP>, respectively, which are of the same order as determined for PGI and PGII. However, both PGA and PGB prefer a partially methylesterified pectin (pectin with a degree of esterification of 22 %) as a substrate. PGB is the only <em>A. niger</em> PG, which shows higher activity on polygalacturonate with increasing ionic strength. PGE shows much lower specific activity on polygalacturonate (0.5μkat.mg <sup>-1</SUP>) and is comparable to PGC in this respect. It may therefore preferentially hydrolyse an as yet unidentified part of the pectin structure. For PGD it was tentatively concluded that it represents an oligogalacturonase. The analysis of the product progression using polygalacturonate as a substrate together with the kinetic studies on oligogalacturonates with defined length identified PGA and PGD as processive enzymes, whereas the PGB and PGE product progression profiles were typical for endolytic non-processive enzymes. All these enzymes attack the oligomeric substrates from the reducing end.The study on the regulation of the expression of several pectinase genes encoding enzymes active on the homogalacturonan and rhamnogalacturonan parts of pectin revealed three typical induction profiles (type I-III). The type I and II induction is triggered by the presence of D-galacturonic acid or possibly an intermediate metabolite (type I) and was typically found for the genes encoding enzymes active on homogalacturonan. The type III induction profile, which is characterised by a high expression of genes on pectin later during growth, has been found for <em>rgh</em> A and also in the case of <em>rgl</em> A expression. Both <em>rgh</em> A and <em>rgl</em> A encode enzymes active on the rhamnogalacturonan part of pectin. All the selected pectinase genes studied were repressed in the presence of sucrose. Thus, in the process of the pectin degradation by <em>A. niger</em> , the constitutively expressed PGA and PGB and later the cell wall attached oligogalacturonase, PGD, generate D-galacturonic acid, which is the inducer of a set of other pectinase genes. The products of these genes can at a later stage of the pectin degradation release another inducer molecule, which might activate the type III regulatory system. It may be that other factors play a role in this induction process.In the next step we addressed the isolation of <em>A. niger</em> mutants affected in the expression of the exopolygalacturonase ( <em>pgx</em> ) and pectin lyase A ( <em>pel</em> A) genes. We exploited a methodology based on the <em>pyr</em> A gene as a reporter, which was successfully applied in the regulation study of the <em>A. niger</em> xylanolytic system and led to the isolation of XLNR, the positively acting regulator. This approach was not successful here, however it led to some partial achievements, which help to better understand the <em>A. niger</em> pectinase regulation. For <em>pel</em> A and <em>pgx</em> , the representatives of the type I and II induction profiles respectively, shorter promoter fragments were identified, which are responsible for the high induction of the genes in the presence of D-galacturonic acid. In the case of <em>pel</em> A this promoter fragment was also responsible for induction of expression in the presence of other sugars like L-rhamnose, D-xylose and D-glucose, although to a lower extent. Furthermore, in the course of this study evidence was found that pectin or its degradation products induce the D-galacturonic acid transport system.Molecular techniques such as DNA sequencing, RLFPs and RAPDs are reliable tools to establish the taxonomical origin of industrial strains, which very often underwent a number of morphological changes in the process of 'domestication'. These changes render classic morphology criteria inapplicable. In our studies we developed a RFLP based strain identification method, which based on the combination of the restriction enzymes <em>Sma</em> I, <em>Pst</em> I/ <em>Sal</em> I and <em>Kpn</em> I/ <em>Xho</em> I and the 28S, <em>pki</em> A and <em>pel</em> A probes provides the diagnostic RFLP patterns for each species. This method now clearly identifies nine species amongst the black aspergilli, i.e. <em>A. niger</em> , <em>A.</em><em>tubingensis</em> , <em>A. foetidus</em> var., <em>A. brasiliensis</em> , <em>A. carbonarius</em> , <em>A. heteromorphus</em> , <em>A. ellipticus</em> , <em>A. aculeatus</em> and <em>A. japonicus</em> .