An extracellular exoinulinase from the filamentous fungus Penicillium sp. strain TN-88 has a 14-fold higher specific activity of 743 U/mg toward inulin than its equivalent from the Aspergillus niger strain 12 and possesses an internal 157-amino-acid sequence whose corresponding region is absent in the A. niger enzyme. On the basis of sequence alignment, the internal region D' encoding the 157-amino-acid sequence in the Penicillium exoinulinase gene inuD cDNA was inserted into the site between the nucleotides 897 and 898 of the A. niger exoinulinase gene inuE cDNA. The resultant inuE::D' fusion was expressed in the methylotrophic yeast Pichia pastoris. The K(m) value of the secreted hybrid enzyme InuE::D' for inulin hydrolysis was about 1/15 that of the A. niger InuE, whereas its k(cat) value did not differ greatly from that of the InuE. These observations indicate that the Penicillium exoinulinase has evolved by the horizontal transfer and integration of a relevant DNA segment and that the internal sequence D' functions as an additional noncatalytic inulin-affinity region.