Cloning, sequence and expression of the gene coding for rhamnogalacturonase of Aspergillus aculeatus; a novel pectinolytic enzyme

Abstract

Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da. The protein contains a potential signal peptidase cleavage site behind Gly-18 and three potential sites for N-glycosylation. Limited homology with A. niger polygalacturonase amino acid sequences is found. A genomic clone of rhgA was isolated from a recombinant phage lambda genomic library. Comparison of the genomic and cDNA sequences revealed that the coding region of the gene is interrupted by three introns. Furthermore, amino acid sequences of four different peptides, derived from purified A. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence of rhgA. A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either the A. niger pyrA gene or the A. aculeatus pyrA gene as selection marker. For expression of rhamnogalacturonase in A. awamori the A. awamori pyrA gene was used as selection marker. Degradation patterns of modified hairy regions, determined by HPLC, show the recombinant rhamnogalacturonase to be active, and the enzyme was found to have a positive effect in the apple hot-mash liquefaction process.

The Value of Genome Sequences in the Rapid Identification of Novel Genes Encoding Specific Plant Cell Wall Degrading Enzymes

“…8) and Blast analysis showed that this enzyme is also the highest homologue of EAA63357 ( Table 9). EAA61967 is in the same group as the rhamnogalacturonan hydrolases from A. aculeatus [77,78] and A. niger [79] and is the orthologue of A. niger RhgA [79] ( Table 9). …”

Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis

“…leatus rhamnogalacturonase A and B (Kofod et al, 1994;Suykerbuyk et al, 1995) were included in the alignment. Rhamnogalacturoiiases are novel enzymes, first described by Schols et al (1990a), which hydrolyze the backbone of the hairy regions of pectin.…”

“…When rhamnogalacturonase A is included, the sequence identity decreases even more. In the sequence NXD, only the aspartate residue seems to be conserved; DD is replaced by DE, a conservative change and a HG sequence is found, but is located at a different position (Suykerbuyk et al, 1995) and of RXK only the lysine residue is conserved. Rhamnogalacturonase A is more closely related to A. tubingensis exopolygalacturonase than to the known endopolygalacturonases.…”

Cloning, targeted disruption and heterologous expression of theKluyveromyces marxianus endopolygalacturonase gene (EPG1 )

“…PG is widely used as a plant cell wall-degrading enzyme. A number of EPG-encoding genes from plants, prokaryotes and fungi, including Aspergillus niger (Bussink et al, 1990;Ruttkowski et al, 1990), Cochliobolus carbonum (Walton et al, 1990), Aspergillus flavum , A. oryzae (Kitamoto et al, 1993), A. parasiticus , Sclerotinia sclerotiorum (Reymond et al, 1994), Fusarium moniliforme (Caprari et al, 1993), A. aculeatus (Suykerbuyk et al, 1995), Colletotrichum lindemuthianum (Centis et al, 1997) and Cryphonectria parasitica (Gao et al, 1996), have recently been cloned and characterized. On the other hand, the pectolytic properties of yeast have also been reported but, in the case of EPG, mainly microbiological and biochemical aspects have been described (Luh et al, 1951;McKay et al, 1990;Gainvors et al, 1994;Schwan et al, 1997).…”