Screening For The Marine Killer Yeast Against Pathogenic Yeast In A Crab (Portunus Trituberculatus), Purification Of The Killer Toxin And Cloning Of The Killer Toxin Gene From The Marine Killer Yeast YF07b

A pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus was identified to be Metschnikowia bicuspidate according to the results of routine yeast identification and 18S rDNA and ITS sequences. After screening of more than 300 yeast strains from different sources in marine environments, it was found that strain YF07b had the highest ability to produce killer toxin against the pathogenic yeast. Strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The optimal conditions for killer toxin production by strain YF07b were the production medium with 2.0% NaCl,pH 4.5,cultivation temperature of 20 oC and the optimal conditions for action of the crude killer toxin against the pathogenic yeast were the assay medium with 6.0% NaCl,pH 4.5 and temperature 15 oC.The gene encoding the killer toxin from the marine killer yeast YF07b was cloned and the accession number of the gene in the GenBank was EF029071.The gene was 1589bp long which included a ORF from 209bp to 1492bp. The deduced protein from the gene had 427 amino acids which contained signal peptide of 17 amino acids.The killer toxin in the supernatant of cell culture of the marine killer yeast YF07b was purified to homogeneity with a 180-fold increase in specificβ-1, 3-D-glucanase activity as compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (SephadexTM G-75) and anion exchange chromatography (DEAE Sepharose Fast Flow Anion Exchange). The molecular weight of the purified killer toxin was estimated to be 47.0 kDa acording to the SDS-PAGE. The optimal pH and temperature of the purified killer toxin were 4.5 and 40°C, respectively. The toxin was activated by Ca2+, K+, Na+, Mg2+ and Co2+. However, Fe2+, Fe3+, Hg2+, Cu2+, Mn2+, Zn2+ and Ag+ acted as inhibitors in decreasing activity of the purified killer toxin. The killer toxin was strongly inhibited by Phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid EDTA, and 1,10-phenanthroline. The Km and Vmax values of the purified killer toxin for larminarin were 1.17 mg/ml and 72.5μmol/min.mg, respectively. A large amount of monosaccharides and disaccharides were detected after the hydrolysis of larminarin, indicating the purified killer toxin had a high exo-β-1, 3-D-glucanase activity. The purified killer toxin also actively killed the whole cells of the pathogenic yeast in crab.