| The extracellular?-1,3-glucanases in the supernatant of cell culture of the marine yeast W. saturnus WC91-2 was purified. According to the data from SDS-PAGE, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. One peptide fragment of the protein was measured by MALDITOF/TOF MS and the amino acid sequence was YIEAQLDAFEKR,its proved that the purified protein was exo-?-1,3-glucanase.The optimal pH and temperature of the purified?-1,3-glucanase were 4.0 and 40?, respectively. The enzyme was activated by Ba2+, Ni2+, Li+ and inhibited by Zn2+, Mg2+,Ca2+, Na+,Fe3+, Fe2+,K+, Co2+,Hg2+, Cu2+, Mn2+ and Ag+.The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 1,10-phenanthroline. The Km and Vmax values of the purified?-1,3-glucanase for laminarin were 3.07 mg/ml and 4.02 mg/(min*ml), respectively.The extracellular killer toxin in the supernatant of cell culture of the marine yeast W. saturnus WC91-2 was purified. The molecular mass of the purified killer toxin was estimated to be 11.0 kDa according to the data from SDS-PAGE. The purified killer toxin had killer activity, but could not hydrolyze laminarin, had no?-1,3-glucanase activity. The optimal conditions for action of the purified killer toxin against the pathogenic yeast WCY were the assay medium with 10.0% NaCl, pH 3-3.5 and temperature 16?.The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378 bp.The deduced protein from the gene had 125 amino acids with calculated molecular weight of 11.6 kDa. It contained a signal peptide of 19 amino acids.The purified?-1,3-glucanase from W. saturnus WC91-2 could inhibit the activity of the WC91-2 toxin produced by the same yeast. We found that the mechanisms of the inhibition may be that the?-1,3-glucanase competed for binding to?-1,3-glucan on the sensitive yeast cell wall with the WC91-2 toxin, causing decrease in the amount of the WC91-2 toxin bound to?-1,3-glucan on the sensitive yeast cell wall and the activity of the WC91-2 toxin against the sensitive yeast cells.The purified WC91-2 toxin from the yeast strain WC91-2 could not kill the protoplasts of the sensitive yeast cells.This may imply that the binding receptor of the WC91-2 toxin indeed exists in the cell wall of the sensitive yeast, and it probably is cell wall?-1,3-glucan. The killing mechanisms of the killer toxin may be that the killer toxin produced by W.saturnus WC91-2 acted on the cell wall of the sensitive yeast cells, causing defective integrity of the cell wall and cell death.The exo-?-1,3-glucanase structural gene(WsEXG1 gene, accession number: FJ875997.2) was isolated from both the genomic DNA and cDNA of W. saturnus WC91-2 by degenerate PCR, inverse PCR and RT-PCR. An open reading frame of 1,254 bp encoding a 417 amino-acid protein (isoelectric point:4.5)with calculated molecular weight of 46.2 kDa was characterized. The promoter of the gene (intronless) was located from-28 to-77 and had one TATA box while its terminator contained the sequence AAGAACAATAAACAA from+1,386 to+1,401.The protein had the Family 5 glycoside hydrolase signature IGLELLNEPL and a fragment with the sequence of NLCGEWSAA, where the Glu-310 (E) was considered to be the catalytic nucleophile.The WsEXG1 gene was cloned into pINA1317 expression vector and overexpressed in Y. lipolytica Po1h, the transformant named 1317-G80 with high?-1,3-glucanase activity was obtained. The recombinant WsEXG1 secreted into the medium was purified by Ni2+ affinity chromatography. The purified rWsEXG1 was analyzed by SDS-PAGE and Western blotting. A specific band with molecular mass of approximatly 46 kDa was found. The optimal pH and temperature of the purified rWsEXG1 were 5.0 and 40?, respectively. Only Ba2+ had an activating effect on the recombinant?-1,3-glucanase activity, while Mg2+,Ca2+, Na+, K+ and Li+ had no significant effect on the recombinant?-1,3-glucanase activity.However, Co2+, Zn2+, Mn2+, Fe2+, Fe3+, Hg2+, Cu2+ Ag+ and Ni2+ tested had negative influence on the recombinant (3-1,3-glucanase activity.The recombinant enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1,10-phenanthroline. The Km and Vmax values of the purified recombinant?-1,3-glucanase for laminarin were 3.95 mg/ml and 2.3 mg/(min*ml), respectively. The purified rWsEXGl had high exo-?-1,3-glucanase activity, it could hydrolyze laminarin into monosaccharides. |
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