Isolation and Characterization of the K5-Type Yeast Killer Protein and Its Homology with an Exo-β-1,3-glucanase

Abstract

K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogeneity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinuous gradient SDS-PAGE and had a molecular mass of 49,000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immuno assay revealed that it was a glycosylated protein. Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-beta-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37 degrees C.

Non-Saccharomyces Killer Toxins: Possible Biocontrol Agents Against Brettanomyces in Wine?

“…N-terminal sequencing of the killer toxin of P. anomala NCYC 432 yielded a short sequence with 100% identity to the mature exo-β-1,3-glucanase of P. anomala strain K (İzgü et al, 2006) that is linked to the killer effect of this strain. Similarly, internal amino acid sequencing of the K5-type killer protein of P. anomala NCYC 434 yielded 100% identity with the exo-β-1,3-glucanase of P. anomala strain K (İzgü & Altinbay, 2004). All these studies clearly indicate that exo-glucanases may display some form of killer activity against other yeast species or filamentous fungi.…”

“…14% β-1,3 linked branches; and mannoproteins and chitin (0.6 to 9%) (Kollár et al, 1995;Santos et al, 2000). In recent literature (İzgü & Altinbay, 2004;İzgü et al, 2006;Comitini et al, 2009;Muccilli et al, 2013), growing evidence suggests that the killer activity of some killer toxins occurs through glucanase activity. Fungal β-1,3-glucanases play a role in metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, autolysis during development and differentiation, and in the mobilisation of β-glucans in response to conditions of carbon and energy source exhaustion (Peng et al, 2009).…”

“…Exo-glucanase activity has been detected in killer toxinproducing yeast species of W. anomalus, P. membranifaciens, W. saturnus, P. anomala strain K and Candida oleophila (Jijakli & Lepoivre, 1998;Masih & Paul, 2002;Bar-Shimon et al, 2004;İzgü & Altinbay, 2004;İzgü et al, 2006;Friel et al, 2007;Wang et al, 2007;Xu et al, 2011;Guo et al, 2012). Three killer strains of W. anomalus (BCU24, BS91 and BCA15) exhibited killer activity against a S. cerevisiae wild type strain, while mutants deficient in β-1,6-glucan were resistant to the toxins of the strains.…”

Stratum corneum lipid liposome-encapsulated panomycocin: preparation, characterization, and the determination of antimycotic efficacy against <em>Candida</em> spp. isolated from patients with vulvovaginitis in an in vitro human vaginal epithelium tissue model

“…15,16 In solution, panomycocin is stable and active at the pH range between 3.0 and 5.5 up to 37°C. 12,13 In lyophilized form, it retains its stability and activity up to 38.5°C (unpublished data) and in the vaginal pH range.…”

“…In various studies, panomycocin, which is produced and secreted into the environment by the killer (K+) yeast strain Wickerhamomyces anomalus (formerly known as Pichia anomala) NCYC 434, has been shown as a promising potential antifungal agent in biomedicine. [12][13][14] Panomycocin is a 49 kDa monomeric glycoprotein with an exo-β-1,3-glucanase activity. It hydrolyzes the exo-β-1, 3-glucans which are vital polymers for the integrity of the fungal cell wall, leading to the disruption of the cell wall and death of the target cells.…”