【作者】 张颖；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2010， 硕士

【摘要】 海藻糖(Trehalose)广泛存在于微生物、无脊椎动物、脊椎动物和植物中。研究显示海藻糖不仅可以作为细胞碳源储存物,还可用作高效保护剂增加细胞组分对不良环境条件(如高温、饥饿、冷冻、脱水、辐射、有毒试剂、高渗透压和高酒精浓度)的抗性。海藻糖不仅可以在生物体内起作用,而且外加的海藻糖同样可以对生物大分子具有保护作用,这使其在很多方面具有广泛的应用,如在医学、分子生物学和微生物学中作为细胞防冻剂、化妆品的有效成分、诊断试剂和生物产品的稳定剂,以及用作新鲜食品的保鲜剂。因此,用廉价高效的的方法生产海藻糖已成为当前研究热点。淀粉价格低廉,来源广泛,以淀粉为原料,酶法制备海藻糖,起始物价格便宜,转化率高,很有应用前景,被认为是用来生产海藻糖的最佳原料。我们在过去的研究中也发现Saccharomycopsis fibuligera sdu是利用淀粉来转化生产海藻糖的很好菌株。但该酵母菌体内能合成酸性和中性海藻糖酶,这些酶在细胞中可以分解合成的海藻糖,这将不利于海藻糖的累积。因此,为了提高海藻糖的产量,从该菌株中去除或降低酸性和中性海藻糖酶活力是必要的。后来,利用化学诱变技术获得酸性和中性海藻糖酶活力大大降低的A11突变株,该突变株在可溶性淀粉培养基中培养菌体内海藻糖的累积量达到19.1%(w／w),在玉米淀粉培养基中培养菌体内海藻糖的累积量达到24.0%(w／w)。本研究发现用木薯淀粉代替玉米淀粉来生产海藻糖能使海藻糖的生产成本更低,A11突变株在2.0%(w／v)木薯淀粉培养基2-L发酵罐中发酵48 h菌体内海藻糖的累积量达到25.8%(w／w)。使用可溶性淀粉和玉米淀粉作为碳源来转化海藻糖是不经济的。木薯作为一种非粮作物,被用来当做食品和发酵工业的淀粉材料,在中国南方很多省市大范围种植。研究中,我们发现在摇瓶试验中,A11菌株利用木薯淀粉转化海藻糖量高于可溶性淀粉、玉米淀粉等。且在2.0%木薯淀粉、4.0%豆饼粉水解液、pH 5.5·和30℃条件下培养48 h,海藻糖累积量达24.8%(w／w)。本论文还利用2-L全自动发酵罐研究该菌株发酵木薯淀粉生产海藻糖,A11菌株48 h发酵罐中海藻糖积累量为25.8%,残留还原糖浓度和残留总糖浓度分别为1.2 mg/mL和2.1 mg/mL。这是目前所报道的海藻糖积累量最高的菌株。在海藻糖提取精制工艺方面,本论文主要用了三氯乙酸和乙醇沉淀法提取菌体中的海藻糖,提取液经过进一步分离纯化,得到海藻糖晶体。针对目前海藻糖的提取方法都存在不足,本研究用化学诱变方法得到突变株A83,该菌株在37℃条件下细胞壁通透性发生变化能够自动释放海藻糖,且海藻糖释放率达到44.7%。此研究是在目前环境污染、资源消耗形势严峻的社会背景下,为响应节能环保的号召以及为低成本无毒性的海藻糖的工业生产做出了积极的探索和尝试。更多还原

【Abstract】 Trehalose (α-D-glucopyranosyl-1-1-α-glucopyranose) is widely present in bacteria, yeasts, and fungi as well as some insects, invertebrates,vertebrates and plants. Recent results have shown that trehalose does not only primarily function as a reserve carbohydrate but also as a highly efficient protectant, enhancing the resistance of cellular components against adverse conditions such as high temperature, starvation, radiation, freezing, dehydration, high osmotic pressure,toxic reagents and high concentration of ethanol. These unique protective properties of trehalose make it an interesting compound for several applications, for example as cryoprotectant for cells in medicine, molecular biologyand microbiology, as an effective component in cosmetics, as a stabilizer for clinical reagents and bioproducts, and even as a preservative for fresh foodstuff. Therefore, much attention has been directed to inexpensive and effective means for producing trehalose.It is thought that starch is the best substrate for production of trehalose due to its low price, high conversion rate and easily obtained raw material.It was shown that S. fibuligera is a good producer of trehalose from starch. But the yeast strain can synthesize acid trehalase and neutral trehalase which can mobilize trehalose accumulated by the cells. Therefore, it is very necessary to delete or decrease the activity of acid and neutral trehalase in the yeast in order to increased trehalose production. Later, using chemical mutagenesis techniques to obtain a mutant strain which acidic and neutral trehalase activity significantly reduce. Trehalose yield was 19.1 g per 100g of cell dry weight used use soluble starch as the substrate, and trehalose yield was 24.0 g per 100g of cell dry weight used use corn starch as the substrate. It is not economical to use soluble starch or corn starch as the substrate for production of trehalose in a large scale. As one of the non-food crops, cassava is widely cultivated in some of the southern provinces of China as a source of starchy material for food and fermentation industry. In this study, we found that strain A11 accumulated more trehalose in the media containing corn starch than in those containg souble starch and corn strach. The results indicated that trehalose accumulation by S. fibuliger a A11 was optimal in the medium comprised of 2.0% cassava starch,4.0% hydrolysate of soybean cake at pH 5.5 and 30℃for 48 h. At the flask level, trehalose yield was 24.8 g per 100g of cell dry weight.At the end of 2-Lfermation, trehalose yield was 25.8 g per 100 g of cell dry weight. This is the highest trehalose yield accumulated in the yeast cells reported so far. At the same time,0.12 mg/mL of reducing sugar and 0.21 mg/mL of total sugar were left in the fermented medium. In this paper, TCA and alcohol used to extract trehalose from the cells of strain All. After isolation and purification, the crystal trehalose was obtained from the culture.Based on the shortcomings of current extraction methods of trehalose from the yeast cells by using the toxic chemicals, a mutant A83 which was thermosensitive autolytic was obatined by chemical mutagenesis, the trehalose could be automatically releasedfrom the mutant cells under the conditions of the cell wall permeability changes at 37℃, and trehalose release rate was 44.7%.更多还原