【作者】 王亚军；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2004， 博士

【摘要】 具有免疫活性的（1→3）-β-D-葡聚糖是葡萄糖通过（1→3）-β-D-糖苷键连接而成的一类高分子化合物，广泛分布在细菌、酵母、蘑菇、真菌、谷物、海藻中。Saccharomyces cerviseae是重要的一类单细胞真菌，外被一坚硬的刚性细胞壁包围，（1→3）-β-D-葡聚糖是构成细胞壁的主要组分之一。（1→3）-β-D-葡聚糖通过与巨噬细胞、嗜中性粒细胞和NK细胞上的受体相互作用，诱导生物活性，提高宿主免疫功能，具有抗肿瘤和辐射保护作用，是哺乳动物、无脊椎动物的免疫促进物，还可用于治疗细菌、病毒、真菌和寄生虫引发的疾病。 S.cerevisiae FL1因具有较厚的细胞壁而被选择为（1→3）-β-D-葡聚糖的生产菌株。在液体YEPD培养基的生长，S.cerevisiae FL1最大比生长速率μ_max0.51h^-1，细胞倍增时间约1.34h。在利用单因素摇瓶实验考察营养和环境条件对S.cerevisiae FL1生物量影响的基础上，得到了较优的培养条件：8％（w／v）葡萄糖，4％（w／v）蛋白胨，1％（w／v）酵母浸提物，培养温度30℃，种龄5～8h，接种量2～4％（v／v），摇床转速220r／min，装液量30％（v／v）。随后，考虑到单因素试验设计的局限性，采用了响应面法对发酵培养基进行了优化。响应面法系数学与统计学的结合，它有助于快速建模、缩短优化时间和提高工程应用可信度。通过响应面法建立了S.cerevisiae FL1生物量与培养基组分蔗糖、硫酸铵及酵母浸出物浓度之间的函数关系 y=9.8634+1.0066x₁-0.2782x₂+2.3718x₃+0.3187x₁x₂+1.1812x₁x₃-0.1688x₂x₃,-0.2414x₁²-0.0735x₂²-1.0191x₃² 其中，x₁、x₂、x₃分别表示蔗糖、硫酸铵和酵母浸出物的编码值浓度。该模型与实际符合程度较高（R²=0.997）。变量分析揭示，在考察范围内，酵母浸出物对生物量收率影响程度最高，蔗糖次之，硫酸铵最弱。优化得到的发酵培养基为：115.5g／L蔗糖，10.0g/L硫酸铵，25.0g/L酵母浸出物，0.1g/L NaCl，0.1/LCaCl₂·2H₂O，0.5g/L MgSO₄·7H₂O，1.0/L KH₂SO₄。上罐发酵，S.cerevisiae FL1的最大生物量42.96g/L。 传统的（1→3）-β-D-葡聚糖制备方法是基于酸碱反复萃取，这种方法的明显不足在于酸、碱试剂用量大、产物杂质含量高、劳动强度大和环境污染大。本工作提出了使用次氯酸钠氧化S.cerevisiae FL1自溶细胞制备（1→3）-β-D-葡聚糖的思路。在考察了各种理化因素对细胞自溶率的影响后发现，52℃下，S.cerevisiae FL1暴露在1.5％（v／v）乙酸乙酯（pH5.5）溶液中，S.cerevisiae FL1自溶率达到最值。自溶36h后，自溶进行得比较彻底。而且与平衡期的S.cerevisiaeFL1细胞壁相比较，自溶细胞的细胞壁基本保持不变。利用自溶过程降解、去浙江大学博士学位论文除细胞内的大分子物质，继而使用稀次氯酸钠溶液氧化5. cerevisia。FLI自溶细胞制备真菌（l一3）一p一D一葡聚糖。借助于元素分析、红外吸收光谱技术和‘HNMR，得到的多糖系（l峥3）一p一D一葡聚糖。（1分3）一p一D一葡聚糖’H NMR谱图分辨率与温度相关，温度越高，谱图分辨率提高;（1分3）一p一D·葡聚糖的水溶性较差。 细胞在其自然栖息环境和工业培养过程如食品、制药工业、绿色化学合成、深海微生物学和有机相生物转化以及细胞固定化工程中，常常要忍受高盐、高糖渗透应激作用。为了揭示渗透作用机制，考察了氯化钠、山梨醇、甘油和聚乙二醇（PEG600）等4种化合物对模式生物5. cerevisiae细胞生长、形态结构和代谢的影响。24h细胞培养结果表明，渗透抑制作用随着渗透强度的增加一致加剧，抑制细胞比生长速率。当甘油浓度上升到4.52 mol/L时，细胞增殖完全被抑制。增加环境中甘油浓度能增加细胞平均体积，并随着甘油浓度的提高，L:B值趋近于1.18左右，细胞形状趋于球形，这减少了细胞的比表面积，有利于减少胞内水分的损失流量，从而保护了细胞。在生理可接受的渗透强度内，与对照组相比较，0.5 mo比氯化钠和0.33m。比PEG600不影响最终残糖水平和生物量浓度。5 cerevisiae FLI由于具有刚性的细胞壁能耐受低渗应激处理，尝试通过了低渗应激诱导5. cerevisiaeFLI合成（l峥3）一p一D一葡聚糖。与对照组，等渗环境的5. cerevista。FLI相比，低渗环境抑制了细胞壁的增长，但低渗、等渗交替作用对细胞壁的效应最明显。低渗应激处理40h后，细胞p一葡聚糖含量7.2%，而低渗、等渗交替作用40h后，细胞壁p一葡聚含量提高了18.1%。低渗、等渗交替作用能够刺激5. cerevisiaoFLI合成（1分3）一p一D一葡聚糖。 通过简化5. cerevisiaeFLI菌落形态和引入未饱和系数、渗透作用，得到了一个描述不同渗透环境下5. cerevisiae FLI在YEPD固体培养基上的增殖模型m（t）0 .00004lx0.006728加.000041“+（0.006728“一0.000041”）。一，聆其中，刀表示环境因子的效应。卢越大，环境越利于细胞的增殖，环境越友好;Z为适小，环境越不利于细胞的增殖，环境越恶劣。使用该模型，我们建立了一个定量描述不同渗透剂在不同浓度时的渗透抑制作用。对照组YEPD平板、o.smol几氯化钠、0.巧mol几甘油、o.smol/L氯化钠和。，15mol几甘油更多还原

【Abstract】 Immunologically active glucans are (l→3)-β-D-linked glucose polymers that occur as a primary component in the cell walls of bacteria, fungi, cereals, mushrooms, seaweed or are secreted extracellularly by various fungi. Fungi are almost entirely multicellular, with yeast, Saccharomyces cerviseae, being a prominent unicellular fungus. S. cerviseae are surrounded by a tough, rigid cell wall. Cell walls are dynamic structures during normal cell expansion. β-Glucans are the conserved structure of cell wall, which are (l→3)-linked β-D-glucopyranan backbone with (1 →6)-β-branches.The beneficial effects of glucans have been attributed to modulation of immune function, increased bacterial clearance, increased bactericidal activity, and other nonspecific effects. One strain of S. cerevisiae FL1 screened from the nature, rather than commercial spent yeast, was selected as the starting fungus for β-D-glucan production as their cell walls were thick under transmission electron microscopy. Influences of environmental conditions and media components on S. cerevisiae FL1 biomass were evaluated in shake flasks. Results indicated that cells multiplied and sugar dropped quickly with μmax of approximate 0.51h-1; influences of temperature, nutrient usage and agitation on biomass were significant; a satisfactory biomass productivity was obtained under the, condition: 30℃, 8% (w/v) glucose, 4% (w/v) peptone, and agitation speed of 220 r/min, medium volumetric ratio of 30% (v/v). In addition, sucrose as well as ammonium sulfate could be utilized by S. cerevisiae FL1 to meet their requirement for nutrients while inoculation ratio, in the observed range, had negligible effect on biomass. Due to inherent drawbacks of the traditional single factor experimental design, response surface methodology was further employed to optimize the growth media for S. cerevisiae FL1. The relationship between biomass and nutrients was founded, which correlated experiment well with R2 of 0.997. It wasy = 9.8634 + 1.0066x1, -0.2782x2 +2.3718x3 + 0.3187x1x2 +1.1812x1x2 -0.1688x2x3 -0.2414x12 -0.0735x22 -1.0191x32, where x1, x2, x3, denoted the concentration of sucrose, ammonium sulfate and yeast extract, respectively. Analysis of variables indicated that sucrose, ammonium sulfateand yeast extract significantly influenced biomass density. Further mathematical analysis was conducted. From the contour plot of the fitted mode, yeast extract and sucrose had significant positive effects on S. cerevisiae FL1 biomass while ammonium sulfate had negative effect on the biomass.(l→3)-β-D-Glucan has received much attention from 1940s. Traditional method for preparing (l→3)-β-D-glucan from S. cerevisiae is based on the repeated extractions with acids and alkali. However, there are some drawbacks in this method, such as large amount of chemicals required, time consuming, many impurities and much wastewater produced. Herein, a novel method to extract (1→3)-β-D-glucan from S. cerevisiae cell wall was proposed, which was based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal condition was 52℃, pH5.5 and 1.5% (v/v) ethyl acetate. The kinetics behavior of yeast autolytic process under the optimized condition was further studied. After 36h of autolysis, 42.0% (w/w) cellular substances were released while cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL1 was treated with the novel method. Infrared spectrum and H NMR spectra conferred the prepared polysaccharide was (l→3)-β-D-glucan. However, the aqueous solubility of (l→3)-β-D-glucan was still poor.In biochemical process with whole cell used, S. cerviseae may encounter several environmental stresses that adversely affect c更多还原