Purification and properties of an extracellular exoinulinase from Penicillium sp. strain TN-88 and sequence analysis of the encoding gene.

An exoinulinase, P-I, was purified from the culture filtrate of Penicillium sp. strain TN-88 grown on inulin. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis with an apparent Mr of 81 kDa. The purified enzyme had extremely high specific activity, 743 U/mg, toward inulin. Inulinase activity was optimal at pH 4.0 and 55 degrees C. A genomic DNA and cDNAs encoding this protein were cloned and sequenced. The exoinulinase gene (inuD) was present as a single copy in the genome. An open reading frame of 2,106 bp was interrupted by a single intron of 56 bp, and encoded a 25-amino acid signal peptide and a 677-amino acid mature protein. The mature protein contained two Cys residues and eight potential N-linked glycosylation sites. The 5'-noncoding region had a putative CAAT box at position -239. Four distinct transcription start points were observed at positions -98 (A), -91 (A), -80 (A), and -76 (A) from the start codon. The exoinulinase gene inuD was located 860-bp upstream of the previously reported endoinulinase gene inuC in the opposite direction of transcription.