MOLECULAR-CLONING AND HETEROLOGOUS EXPRESSION OF THE ISOPULLULANASE GENE FROM ASPERGILLUS-NIGER ATCC-9642

Isopullulanase (IPU) from Aspergillus niger A.T.C.C. (American Type Cu lture Collection) 9642 hydrolyses pullulan to isopanose. IPU is import ant for the production of isopanose and is used in the structural anal ysis of oligosaccharides with alpha-1,4 and alpha-1,6 glucosidic linka ges. We have isolated the ipuA gene encoding IPU from the filamentous fungi A. niger A.T.C.C. 9642. The ipuA gene encodes an open reading fr ame of 1695 bp (564 amino acids). IPU contained a signal sequence of 1 9 amino acids, and the molecular mass of the mature form was calculate d to be 59 kDa. IPU has no amino-acid-sequence similarity with the oth er pullulan-hydrolysing enzymes, which are pullulanase, neopullulanase and glucoamylase. However, IPU showed a high amino-acid-sequence simi larity with dextranases from Penicillium minioluteum (61%) and Arthrob acter sp. (56%). When the ipuA gene was expressed in Aspergillus oryza e, the expressed protein (recombinant IPU) had IPU activity and was im munologically reactive with antibodies raised against native IPU. The substrate specificity, thermostability and pH profile of recombinant I PU were identical with those of the native enzyme, but recombinant IPU (90 kDa) was larger than the native enzyme (69-71 kDa). After deglyco sylation with peptide-N-glycosidase F, the deglycosylated recombinant IPU had the same molecular mass as deglycosylated native enzyme (59 kD a). This result suggests that the carbohydrate chain of recombinant IP U differed from that of the native enzyme.