Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424

Abstract

Cell wall inulinase (EC 3.2.1.7) was purified from Kluyveromyces marxianus var. marxianus (formerly K. fragilis) and its N-terminal 33-amino acid sequence was established. PCR amplification of cDNA with 2 sets of degenerate primers yielded a genomic probe which was then used to screen a genomic library established in the YEp351 yeast shuttle vector. One of the selected recombinant plasmids allowed an invertase-negative Saccharomyces cerevisiae mutant to grow on inulin. It was shown to contain an inulinase gene (INU 1) encoding a 555-amino acid precursor protein with a typical N-terminal signal peptide. The sequence of inulinase displays a high similarity (67%) to S. cerevisiae invertase, suggesting a common evolutionary origin for yeast beta-fructosidases with different substrate preferences.