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

Rhamnogalacturonase was purified from culture filtrate ofAspergillus 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 λ cDNA expression library. The clonedrhgA 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 forN-glycosylation. Limited homology withA. niger polygalacturonase amino acid sequences is found. A genomic clone ofrhgA was isolated from a recombinant phage λ 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 purifiedA. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence ofrhgA.A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either theA. niger pyrA gene or theA. aculeatus pyr A gene as selection marker. For expression of rhamnogalacturonase inA. awamori theA. 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.