Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis

Abstract

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin ␣-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.

Chitinases Are Essential for Cell Separation in Ustilago maydis

“…Our study revealed a role of chitinases in cell separation of yeast cells in U. maydis. Such a function of chitinases has already been described in ascomycetes (reviewed in references 12 and 49), e.g., in the model yeast S. cerevisiae (11), the opportunistic pathogen Candida albicans (10), and the industrially used yeast Kluyveromyces lactis (50). In these species, single deletions of the corresponding chitinase lead to cell aggregation.…”

Candida albicans CHT3 encodes the functional homolog of the Cts1 chitinase of Saccharomyces cerevisiae

“…1A). The C-terminal sequence of Cts1 encodes a type-2 chitin binding domain that is not present in the Candida chitinases (Colussi et al, 2005;Kuranda and Robbins, 1991). Located in the C-terminal sequence of Cht2 is a potential GPI-anchor site that is not found in the other S. cerevisiae or C. albicans chitinases (analysis with DGPI software at http:// 129.194.185.165/dgpi/index_en.html).…”

Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1,3-Glucanases

“…The Candida albicans genome contains three chitinase genes, CHT1 , CHT2 , and CHT3 , which are homologous to the Saccharomyces cerevisiae CTS1 gene and C. albicans CHT4 , which is homologous to S. cerevisiae CTS2 (Dunkler et al, 2005). Recently a nucleus encoded chitinase (KlCts1p) has been characterized from yeast Kluyveromyces lactis (Colussi et al, 2005 ). This species is an industrially important yeast species due to its ability to grow to a high cell density and abundantly secrete heterologous protein (Rocha et al, 1996 ).…”