CLONING AND CHARACTERIZATION OF 2 RHAMNOGALACTURONAN HYDROLASE GENES FROM ASPERGILLUS-NIGER

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes o f Aspergillus niger. The corresponding proteins, rhamnogalacturonan hy drolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme, In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expressio n of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently in duced after 3 h of growth on apple pectin, The rhamnogalacturonan hydr olase B gene was not induced by apple pectin, but the rhgB gene was de repressed after 18 h of growth on either apple pectin or sucrose, Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain hi gh-producing A. awamori transformants which were then used for the pur ification of the two A. niger rhamnogalacturonan hydrolases. High-perf ormance anion-exchange chromatography of oligomeric degradation produc ts showed that optimal degradation of an isolated highly branched pect in fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogal acturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeat us rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5), Co mpared to the A enzymes, the A. niger B enzyme appears to have a diffe rent substrate specificity, since additional oligomers are formed.