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

Abstract

Cell wall inulinase (EC 3.2. I .7) was pirified from K/ywvwt.ws r~~arshr~s var. mtrsicms (formerly K. Jrugilis) and its N-terminal 33-amino acid sequence was established. PCR amplification of cDNA with 2 sets of degenerate primers yielded a gcnomic 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 invertasc-negative S~~clrcrrurr~~~s cerevisicre mutan to grow on inulin. It was shown to contain an inulinase gene (INU I) encoding a 555-amino acid precursor protein with a typical N-terminal signal pcptide. The sequence of inulinase displays a high similarity (67%) IO S. ceruisictr invertasc. suggesting a common evolutionary origin for yeas1 p-fructosidases with different substrate preferences.

The methylotrophic yeast Hansenula polymorpha: a versatile cell factory

“…Other heterologous prepro-sequences, which have successfully been used in H. polymorpha are derived from Schwanniomyces occidentalis glucoamylase [5,45], Carcinus maenas hyperglycemic hormone [45], Aspergillus niger glucoamylase [20], Kluyveromyces lactis killer toxin [20], and Kluyveromyces marxianus inulinase [46] (see Table 1). …”

The MIG1 repressor from Kluyveromyces lactis: cloning, sequencing and functional analysis in Saccharomyces cerevisiae

“…The SUC2 gene encoding invertase in S. cerevisiae, on the other hand, displays two MIG 1-binding sites in its promoter [3] and two putative MIG1 sites are also found [12] upstream of the INU1 gene encoding inulinase (a closely related enzyme) in Kluyveromyces marxianus [13], suggesting that the latter gene also may be repressed by a Migl homologue.…”

“…Moreover, in addition to its galactokinase activity, the K. lactis GALl gene product has a regulatory function required for the induction pathway, upstream of K1LAC9 [11]. Interestingly, a potential MIGl-binding site is found in the GALl promoter of K. lactis suggesting the involvement of a Migl-like protein in glucose repression of galactose-lactose catabolism in K. lactis.The SUC2 gene encoding invertase in S. cerevisiae, on the other hand, displays two MIG 1-binding sites in its promoter [3] and two putative MIG1 sites are also found [12] upstream of the INU1 gene encoding inulinase (a closely related enzyme) in Kluyveromyces marxianus [13], suggesting that the latter gene also may be repressed by a Migl homologue.In this paper, we report the cloning and sequencing of the K. lactis MIG1 gene and we show that the encoded protein is functional in S. cerevisiae. …”

Inulinase production by yeat Kluyveromyces marxianus

“…3.2.1.7), comúnmente conocida como inulinasa, perteneciendo al grupo de clasificación de enzimas hidrolasas glicosidasas (Kushi et al, 2000;Laloux et al, 1991).…”

“…Notoriamente una actividad invertasa de inulinasa es deseable. Laloux et al (1991) sostienen que las inulinasas poseen sitios catalíticos comunes, pero diferentes sitios de enlace para la hidrólisis de inulina y sacarosa.…”