【作者】 梁丽琨；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2006， 博士

【摘要】 海藻糖在生物圈内无处不在，其独特的物理化学特性使其成为细胞应激代谢物，在不同的逆境胁迫中保护生物大分子。海藻糖在工业中有极好的应用前景，如在干燥和冷冻期间维持细胞膜的完整性；增加多种不稳定物质的稳定性；提高冷冻细胞的生存能力；提高生物物质如酶、疫苗等的稳定性；在食品和制药工业中作为冷冻保护剂和高效保存剂等。 在本实验室前期工作中，从土壤中分离到一株能利用淀粉合成海藻糖的野生型扣囊复膜酵母菌株，该菌株在含淀粉的培养基中经48h发酵可积累占细胞干重18％的海藻糖，但细胞内海藻糖酶活力较高，分解合成的海藻糖，对海藻糖积累不利。本研究以该野生型酵母为出发菌株，经过EMS诱变，得到一株中性和酸性海藻糖酶活力均显著降低的突变株A11，突变株A11在含淀粉培养基中的生长能力并未下降，但经48h发酵，细胞内海藻糖的积累量却比野生型细胞提高了50％。 为了解扣囊复膜酵母A11海藻糖的合成代谢，通过实验确定了该菌株海藻糖的生物合成途径，结果表明其海藻糖合成途径与啤酒酵母和大肠杆菌的一样，即由UDP-葡萄糖与6-磷酸葡萄糖在6-磷酸海藻糖合成酶催化下合成6-磷酸海藻糖，然后由6-磷酸海藻糖磷酸酯酶催化下合成海藻糖。该合成途径的关键酶6-磷酸海藻糖合成酶经过超滤浓缩、Sepharose CL-4B和DEAE-Sepharose Fast Flow阴离子交换层析等系列纯化过程，经SDS-PAGE和Native-PAGE电泳检测，都呈现一条66kDa的条带，证实6-磷酸海藻糖合成酶被纯化，纯化倍数33倍，收率61％。SDS-PAGE和Native-PAGE电泳上显示的结果，说明扣囊复膜酵母6-磷酸海藻糖合成酶与啤酒酵母和其他真核生物的不同。 对纯化的6-磷酸海藻糖合成酶的生化特性进行研究表明，该酶反应的最适温度是37℃，最适pH 6.6，酶的温度和pH稳定性均很差；Ca²⁺、K⁺和Mg²⁺对酶活性有激活作用，最适激活浓度分别是10mmol／L、35mmol／L和35mmol／L，说明该酶是金属酶；而Cu²⁺，Fe³⁺，Hg²⁺，Co²⁺对酶活性有抑制作用，Hg²⁺对酶更多还原

【Abstract】 Trehalose is a non-reducing disaccharide that has been found in many organisms. In addition to being nonreducing, it possesses several unique physical properties, which account for the principal role of trehalose as a stress metabolite. It is now being recognized as a crucial defense mechanism that stabilizes proteins and biological membranes under a variety of stress conditions. Based on its unique properties trehalose has become an important target for biotechnology, where it is produced for food manufacture, vaccine protection, pharmaceuticals and cosmetic products.In the previous studies, we found that Saccharomycopsis fibuligera sdu could accumulate trehalose efficiently (up to 0.18g/g dry weight) at the entrance of stationary phase during growth on starch as the sole carbon source. But the strain can synthesize acid trehalase and neutral trehalase, which can mobilize trehalose accumulated by the cells. To enhance yields of trehalose in this strain, it is important to delete or decrease the activity of the acid and neutral trehalases in the cells. By mutagenesis of EMS (Ethylmethanesulfonate), one mutant (All) that assimilated trehalose slowly, but grew on other carbon sources as fast as its parent strain, was isolated. In shaking 300 mL flask, the content of trehalose in the mutant All was increased by 50% than that of the wild type strain when grown in YPS medium. The activities of acid and neutral trehalases of this mutant were lower than those of the wild type. As a result, trehalose accumulation was distinctly advanced in the mutantAll.Trehalose-6-phosphate synthase (SfTps1) activity was detected in the cell extract of S. fibuligera, suggesting that trehalose synthesis pathway in this strain was as same as that in S. cerevisiae and E. coli. The SfTps1 was purified 33-fold to homogeneity by ultrafiltration and several steps of column chromatography. The purified enzyme has apparent molecular masses of 66 kDa, as determined by Native-PAGE and SDS-更多还原