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

Suykerbuyk, M.E.G., Schaap, P.J., Stam, H., Musters, W., Visser, J.
Applied microbiology and biotechnology 1995 v.43 no.5 pp. 861-870
Aspergillus aculeatus, amino acid sequences, amino acids, antibodies, antiserum, apples, bacteriophages, carbon, complementary DNA, culture filtrates, culture media, gene overexpression, genes, genomic libraries, high performance liquid chromatography, introns, liquefaction, mice, molecular weight, open reading frames, peptides, polygalacturonase, pulp, sugar beet
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 gamma 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 45962 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 gamma 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.