MOLECULAR-CLONING AND CHARACTERIZATION OF A RHAMNOGALACTURONAN ACETYLESTERASE FROM ASPERGILLUS-ACULEATUS - SYNERGISM BETWEEN RHAMNOGALACTURONAN DEGRADING ENZYMES

A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-termin al amino acid sequence was determined, Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymer ase chain reaction-generated product as a probe, The 936-base pair rha 1 cDNA encodes a 25O-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide, The rha1 cDNA was overexpressed in As pergillus oryzae, a filamentous fungus that does not possess RGAE acti vity, and the recombinant enzyme was purified and characterized, Mass spectrometry of the native and recombinant RGAE revealed that the enzy mes are heterogeneously glycosylated, In addition, the observed differ ences in their molecular masses, lectin binding patterns, and monosacc haride compositions indicate that the glycan moieties on the two enzym es are structurally different, The RGAE was shown to act in synergy wi th rhamnogalacturonase A as well as rhamnogalacturonase B from A, acul eatus in the degradation of apple pectin rhamnogalacturonan. RNA gel b lot analyses indicate that the expression of rhamnogalacturonan degrad ing enzymes by A, aculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.