【作者】 周海燕；

【导师】 吴永尧；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 目的： 葡甘低聚糖是一种具有多种特殊生理功能的寡聚糖，可以预防肥胖症、糖尿病、龋齿等多种疾病的发生，减轻人体因摄入过多热量而产生的负担，是新型的功能低聚糖。 本论文运用现代发酵技术及酶工程学原理，利用微生物产酶水解魔芋精粉来制备葡甘低聚糖。在前期工作的基础上，对自行筛选的一株产高活性葡甘聚糖酶的细菌进行鉴定，摸索细菌生长和产酶的规律，优化产酶条件，并以摇瓶培养结果为依据，用5 L发酵放大培养细菌，进一步优化放大发酵条件，此外，对发酵液中的葡甘聚糖酶进行提取并纯化，探索出一套纯化葡甘聚糖酶的理想方案，建立完整的葡甘低聚糖制备工艺，试图为工业生产葡甘聚糖酶提供理论依据，为工业化生产葡甘低聚糖提供技术指导。 方法： 首先运用常规方法观察细菌形态，并对20多种生理生化特征进行鉴定，根据《伯杰细菌鉴定手册》和《常见细菌系统鉴定手册》对细菌特征进行检索，为细菌分类到种并命名。为了了解细菌生长和产酶规律，在摇瓶培养过程中摸索细菌生长的最适培养基及最佳培养时间，通过正交试验确定摇瓶培养条件的最佳配伍，并在此基础上进行细菌的放大生产，再次利用正交试验对放大发酵条件进行优化。发酵得到的葡甘聚糖酶经过盐析、分子筛层析和离子交换层析三个步骤进行分离纯化，纯化后的酶蛋白用聚丙烯酰胺常规电泳进行纯度鉴定，变性电泳测定其平均分子量，同时对酶作用的最适pH和最适温度等特性进行研究，调整酶反应环境，提高酶效。综合试验数据，在其指导下进行葡甘低聚糖的生产小试，对发酵液的凝聚剂种类和凝聚条件进行研究，同时对粗提酶用于葡甘低聚糖生产的实际可行性进行模拟，了解粗酶作用比率、时间、温度、pH等条件，测定水解产物的总糖含量和还原糖含量，薄板层析法分析产品的寡糖种类，计算原料转化率和增值率。 结果： 1．通过鉴定，该菌属于芽孢杆菌属，具有耐盐、耐热、液化明胶、水解淀粉、可运动、好氧兼性厌氧等特点，与芽孢杆菌属中其他种相比较应该是一个新种，目前暂时将其命名为耐热芽孢杆菌Bacillus thermodurans。 2．Bacillus thermodurans产酶的最适培养基为蛋白胨1％、牛肉膏粉0.3％、魔芋精粉2％、NaCl0.5％、K₂HPO₄0.2％，培养条件的最适温度为40℃，pH7.2，振荡速度120rpm，培养时间为24h。该条件下培养的获得的粗酶液平均比活力可达3859U／mg。n摘要 3.正交试验分析结果显示，SL发酵罐培养的最适条件为温度50℃，PH 6.2，发酵初期搅拌速度为200 rpm，通气量为40眺，6h后变为搅拌速度100 rpm，通气量20眺，得到的最高酶比活力可达4403U/mg。刀狡‘illus thermodurans生长周期较短，6h后即进入对数期，26h后进入稳定期，期间出现二次生长现象。与生物量积累情况相比，所产酶的活性上升速度更快，直至24h后才开始有明显下降，整个过程中pH值变化幅度极小，而溶氧值由于发酵液粘度变化及需氧量变化起落较大。酶蛋白的产量与菌体积累量密切相关，发酵动力学属于偶联型。 4.盐析浓度在朋⁸0%范围内葡甘聚糖酶的活性较高，将该浓度下沉淀的蛋白质经Sepharose 6B分子筛层析和DEAE一52离子交换层析分离出达到电泳纯的酶蛋白。纯化倍数达到6.7，酶活回收率为67.14%。该酶降解魔芋精粉时的Km=lomg/血L，Vmax=6.67mg/血Lmln。SDS一PAGE测定结果表明纯化后的葡甘聚糖酶由三种亚基组成，推断其中可能含有其他分子量接近的蛋白质或为葡甘聚糖同工酶，结果有待进一步验证。 葡甘聚糖酶在温度为50℃，PH为5.6一5.8时活力最高。低温对酶活影响不大，40℃以下酶可在Zoh内保持较高活性，而pH对酶活影响相对显著，在pHS^PH7范围内酶的活力可保持大约10h左右，PHS时则大大降低了酶的活性。金属离子和有机物往往对酶反应有比较大的影响，对该葡甘聚糖酶来说，znZ十和FeZ+对酶的反应有明显的抑制作用，其他试验的离子和有机物则未见显著的抑制或激活性。 5.在SL发酵罐上对葡甘低聚糖进行实验性生产的主要工艺为用0.05%（岁1 00 mL）氯化钙和0.1%（留1 00 mL）磷酸氢二钠处理培养24h的发酵液，去除菌体和固体颗粒后在低温下进行40⁸0%浓度的盐析，得到葡甘聚糖粗酶，脱盐后直接与魔芋精粉以1:300（w八V）的比例混合，50℃下保温10h，充分水解后4 000 rpm下离心，去除未降解的魔芋粉，得到葡甘低聚糖浆，最后由喷雾干燥得到低聚糖粉。经测定葡甘聚糖的转化率为66.6%，糖粉中总糖含量为90.11%，13.09%，薄板层析结果显示糖粉中单糖浓度很低，聚糖酶可能是一种内切酶。小试生产的葡甘低聚糖产品有较奸的复溶性，还原糖含量为所产生的葡甘液体透明无异味，不需对产品进行脱色和除单糖等特别处理，生产工艺比较简单，所使用的仪器和试剂成本也较低，生产流程不对环境造成污染，是一个经济效益和社会效益都相当可观的课题项目。更多还原

【Abstract】 Objects:To identify a strain producing high activity konjac glucomannanase, investigate theregularity of bacterial growth and fermentation, verify the conditions of producingglucomannanase, probe into the purification methods and the characteristics of glucomannanase, found a process producing konjac oligosaccharide.Methods:The strain was identified with the characteristics of morph and physiological biochemistry. According to Bergey’s Manual of Determinative Bacteriology and Determinative Manual of Common Bacteria, the strain was grouped into genus or even species. Based on shake flasks the optimum culture and fermentation time were studied and then the fermentation conditions were verified on 5 L fermentator with the orthogonal experiment. Konjac glucomannanase was separated and purified through ammonium sulphate precipitation, gel filtration chromatography(Sepharose 6B) and ion exchange chromatography(DEAE-52), then the protein purify was determinated by PAGE and the molecular weight by SDS-PAGE. Furthermore, the enzyme characteristics were investigated about the optimum pH and temperature and so on to improve enzyme activity through adjusting the reaction environments. Based on the above results, the small scale of KOS production is studied including the agglutinator and the crude enzyme’s reaction conditions. Meanwhile the parameters of KOS specimen, such as the sugar contents and components, transformation efficiency and so on, are determined.Results:1. According to morph characteristics and physiological biochemistry analysis, this strain is identified as Bacillus Cohn. 6ue to its deviation with other species of Bacillus Colin, this strain maybe a new species, for the time being it was named Bacillus thermodurans.2. On the shake flasks, the optimum culture is peptone 1%, beef extract 0.3%, konjac glucomannan 2%, NaCl 0.5%, K2HPO4 0.2%, the optimum temperature 40℃, pH 7.2, rotating velocity 120 rpm. The crude enzyme specific activity is average to 3859 U/mg.3. Through the orthogonal experiment, the optimum magnification conditions on 5 L fermentator are temperature 50℃, pH 6, at prophase stirring velocity 200 rpm, aeration rate 40 L/h, then after 6 h stirring velocity 100 rpm, aeration rate 20 L/h. The average specific activityis 4403 U/mg. when monitored on line, the growth of bacteria entered logarithmic phase after 6 h and stationary phase after 26 h. The enzyme specific activity decreased after 24 h. In this course, DO changed largely while pH was stable. In view of the relation between the bacteria growth and the production, the fermentation was coupling kinetics.4. Purification of Konjac glucomannanase includes three steps: ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography.Glucomannanase precipitation concentration is at the range of 40~80%0 Sepharose 6B and DEAE-52 have better resolving power. After the three-step treatment, purification fold is 6.7 with 67.14% enzyme activity recovery and single band shows by PAGE determination. When substrate is konjac glucomannan, enzyme has Fmax of 6mg/mL-min and Km of lOmg/mL.According to the SDS-PAGE result, the purified enzyme has three subunits and it will be studied deeply whether the enzyme has single protein or it is another isoenzyme. This glucomannanase shows the maximal activity at temperature 50癈 and pH 5.6~5.8. When temperature is below 40癈, It remained 90% activity for 20 hours. At the range of pH 5’~7 enzyme activity can remain for 10 hours or so. Zn2+ and Fe2+ have great inhibition on enzyme activity while other trial ion and organic compound have little effect on activity.5. After fermented mash was pretreated by the mixture of 0.05% CaCl2 and 0.1% Na2HPO4, The bacteria and solid particles were separated and then salted out at 40%~80% under 0℃. the crude enzyme was dialyzed and mixed konjac glucomannan at the scale of 1:300 in water, then acted under 50℃ for 10 hours. At last, the hydrolyzate was spray-dried to the konjac oligosaccharide powder. In this cour更多还原