【作者】 王麟；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 本实验室近年来从不同海洋环境中分离得到了551株酵母菌,经鉴定发现这些酵母菌分别属于22个属、58个种,并以此为基础建立了我国第一个海洋酵母菌种资源库,为每株酵母菌建立了电子档案。通过与中国海洋微生物菌种保藏管理中心合作实现了资源库内所有海洋酵母菌种的实物共享与信息共享,为研究者提供资源支撑平台。通过对本实验室保藏的海洋酵母菌种资源库中的酵母菌进行筛选,我们发现9株海洋酵母菌在含有橄榄油的培养基中能产脂肪酶。经过常规生理生化鉴定和分子生物学方法鉴定,这9株酵母菌分别属于间型假丝酵母(Candida intermedia),季也蒙毕赤酵母(Pichia guilliermondii),近平滑假丝酵母(Candida parapsilosis),长孢洛德酵母(Lodderomyces elongisporus),桔假丝酵母(Candida quercitrusa),普鲁兰短梗霉(Aureobasidium pullulans),解脂耶罗维亚酵母(Yarrowia lipolytica),胶红酵母(Rhodotorula mucilaginosa)和褶皱假丝酵母(Candida rugosa)。这些酵母菌所产的脂肪酶的最适作用pH值介于6.0-8.5之间,最适作用温度分别为35℃和40℃。本研究中的大多数酵母菌产的脂肪酶是与细胞壁结合的,只有普鲁兰短梗霉HN2.3产的脂肪酶只胞外酶。一些酵母菌株产的脂肪酶可以高效地水解不同的油脂,这表明这些菌株在工业上具有潜在的应用价值。通过对本实验室保藏的海洋酵母菌种资源库中的酵母菌进行筛选,我们发现17株海洋酵母菌能够向培养基中分泌嗜杀因子,从而杀死梭子蟹的一种致病菌——二尖梅奇酵母(Metschnikowia bicuspidate)WCY。其中有5株酵母菌(WC91-2,gao1zhong2,YF07b,hcx-1和HN2.3)的嗜杀活力比较高。常规生理生化鉴定和分子生物学鉴定的结果表明,这5株酵母菌分别属于土星拟威尔酵母(Williopsis saturnus),季也蒙毕赤酵母(Pichia guilliermondii),异常毕赤酵母(Pichia anomala),汉逊德巴利酵母(Debaryomyces hansenii)和普鲁兰短梗霉(Aureobasidium pullulans)。我们发现并不是所有的嗜杀因子的最适生产条件和嗜杀因子的最适作用条件与海洋环境及梭子蟹的养殖环境条件相一致。我们还发现由这些酵母菌所产的嗜杀因子能够杀死除了致病酵母WCY之外的其他酵母菌,而且培养基中的NaCl浓度对嗜杀作用有影响。所有的由这些酵母菌所产的嗜杀因子粗酶液均可以水解海带多糖,水解的终产物均为单糖。我们发现从中国东海分离得到的一株海洋酵母菌W14-3能够产核黄素。有趣的是,这株海洋酵母菌在振荡培养的条件下在分别含有木糖、蔗糖、半乳糖和麦芽糖的培养基中能够分泌大量的核黄素。经过常规生理生化鉴定和分子生物学方法鉴定,这株酵母菌是膜醭假丝酵母(Candida membranifaciens)的一个新亚种——Candida membranifaciens subsp. flavinogenie。这株菌中与核黄素生物合成有关的4个基因(分别编码GTP去环化酶II、DRAP脱氨基酶、3,4-二羟基-2-丁酮-4-磷酸合成酶及2,4-二氧四氢蝶啶合成酶)的部分序列被克隆,由这些基因序列推导出的氨基酸序列分别与其它酵母菌中的相关蛋白的氨基酸序列具有高度同源性。培养基中存在Fe3+离子会抑制核黄素的合成并抑制核黄素生物合成途径中相关基因的转录。该实验结果证明了海洋酵母菌W14-3中存在核黄素生物合成途径。这是首个关于从海洋环境中分离得到的C. membranifaciens subsp. flavinogenie W14-3产核黄素的报道。更多还原

【Abstract】 Total 551 marine yeast strains from different marine environments were obtained. The results of routine identification and molecular methods show that they belonged to 22 genera, 58 species. The Marine Yeast Culture Collection, which was the first yeast culture collection in China, was established basing on these strains. An electronic file for each strain was recorded. All the information and strains collected can be shared at the website http://www.mccc.org.cn/ and the collection center.After lipase activity of the yeast cultures was estimated, we found that nine yeast strains obtained in the Marine Yeast Culture Collection grown in the medium with olive oil could produce lipase. The results of routine identification and molecular methods show that they belonged to Candida intermedia YA01a, Pichia guilliermondii N12c, Candida parapsilosis 3eA2, Lodderomyces elongisporus YF12c, Candida quercitrusa JHSb, Candia rugosa wl8, Yarrowia lipolytica N9a, Rhodotorula mucilaginosa L10-2 and Aureobasidium pullulans HN2.3, respectively. The optimal pHs and temperatures of lipases produced by them were between 6.0 and 8.5 and between 35 and 40℃, respectively. Majority of lipases from the yeast strains were cell-bound and only lipase from A. pullulans HN2.3 was extracellular. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry.After killer toxin activity of the yeast cultures was determined, we found that 17 strains obtained in the Marine Yeast Culture Collection could secrete killer toxin into the medium and killed the pathogenic yeast Metschnikowia bicuspidate WCY in crab. However, only five strains (WC91-2,gao1zhong2,YF07b,hcx-1 and HN2.3) among them had higher killer toxin activity against the pathogenic yeast than others. The results of routine identification and molecular methods show that the five yeast strains belonged to Williopsis saturnus WC91-2, Pichia guilliermondii gao1zhong2, Pichia anomala YF07b, Debaryomyces hansenii hcx-1 and Aureobasidium pullulans HN2.3, respectively. We found that not all the optimal conditions for killer toxin production and action of killer toxin by the marine killer yeasts were in agreement with those of marine environments and for crab cultivation. It was found that the killer toxins produced by the killer yeast strains could kill other yeasts in addition to the pathogenic yeast and NaCl concentration in the medium could change killing activity spectra. All the crude killer toxins produced by them could hydrolyze laminarin and the hydrolysis end products were monosaccharides.We found that the marine yeast strain W14-3 isolated from seawater of China Eastern Sea could produce riboflavin. It is interesting to observe that the marine yeast strain produced a large amount of riboflavin in the medium containing xylose, sucrose, galactose and maltose, respectively under the conditions of vigorous shaking. The yeast strain was found to belong to Candida membranifaciens subsp. flavinogenie based on the results of routine and molecular identification. The protein sequences deduced from the partial genes encoding GTP cyclohydrolase II, DRAP deaminase, 3,4-dihydroxy-2-butanone -4-phosphate synthase and lumazine synthase in the yeast exhibited high identity with those of the corresponding enzymes for riboflavin biosynthesis in other yeasts, respectively. Fe3+ available in the medium repressed riboflavin production and expression of the genes responsible for riboflavin biosynthesis in the yeast. The results have evidenced that riboflavin synthesis pathway indeed existed in the yeast. This is the first time to report that C. membranifaciens subsp. flavinogenie W14-3 from the marine environment could produce riboflavin.更多还原