【作者】 刘洋；

【导师】 谢秋宏； 相宏宇；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 糖化酶，全名葡萄糖淀粉酶（Glucoamylase），是一种具有外切酶活性的酸性单链糖苷水解酶。它可以从淀粉或低聚糖的非还原末端水解α-1,4-糖苷键生成葡萄糖。糖化酶被广泛地应用于淀粉糖、食品、医药、酿造等工业生产中，是我国产量最大、应用范围最广的酶制剂。目前，工业用糖化酶存在的缺点之一是热稳定性差。淀粉在高温液化后必须经过一个降温过程才能添加糖化酶进行糖化；此外，长时间的糖化过程也会造成糖化酶活力的损失，大大增加了生产成本。与此同时，关于糖化酶热稳定机制的研究还不完善，热稳定糖化酶的开发尚处于研究阶段。为了推动这一问题的解决，我们以一株经诱变获得的糖化酶高产菌株Aspergillus niger B-30为材料，对其产生的糖化酶进行了分离纯化及诸酶学性质分析，并对温度诱导糖化酶去折叠机制进行了详细研究。本文中也阐述了添加稳定剂提高糖化酶的热稳定性及其稳定机制，尝试了对纯化后糖化酶的固定化，并对固定化条件及固定化酶学性质进行探讨和分析。首先，对A. niger B-30产生的糖化酶进行纯化和酶学性质分析。通过硫酸铵沉淀、DEAE FF离子交换层析和Superdex G-75凝胶过滤层析对A. niger B-30液体发酵产生的糖化酶进行分离纯化，获得两个不同的单一的糖化酶组分，分别命名为GAM-1和GAM-2。经SDS-PAGE检测其分子量分别为97.2kDa和78.3kDa。同时，通过MALDI-TOF-MS检测其分子量分别为80.5kDa和70.4kDa。GAM-1和GAM-2均为糖蛋白，其碳水化合物含量分别为10.4%和13.4%。GAM-1和GAM-2的最适反应pH和最适反应温度相同，分别为4.0-4.6和70℃。两糖化酶均有较好的热稳定性和pH稳定性，且GAM-2优于GAM-1。GAM-1和GAM-2酶学性质研究显示二者均适于淀粉质原料的糖化过程。其次，本论文对温度诱导糖化酶去折叠机制进行了探讨。通过圆二色谱技术、荧光光谱技术、紫外吸收光谱、动态光散射等技术以及Native-PAGE等分析方法系统地研究了温度对GAM-1和GAM-2构象的影响。结果表明二者去折叠机制相似，即温度诱导GAM-1和GAM-2去折叠时，α-螺旋含量逐渐减少，β-折叠、β-转角和无规则卷曲含量逐渐增加；疏水基团外露且伴随着蛋白质的聚集；在加热过程中，GAM-2的结构比GAM-1更稳定是其表现出较高热稳定性的重要原因。本论文还进行了稳定剂对糖化酶热稳定性影响及热稳定机制的研究。为了稳定糖化酶构象，使其在高温下维持活力，研究中向糖化酶反应体系内分别加入1M山梨醇和海藻糖，发现稳定剂在低温时不会明显提高酶活力，只有在高温反应时，才具有稳定作用。为解析山梨醇和海藻糖使糖化酶热稳定性提高的机制，我们向GAM-1和GAM-2中分别加入1M山梨醇和海藻糖后，在75℃和80℃下测定了两糖化酶的热动力学参数，结果显示热失活常数明显减小，半衰期增加，热失活自由能△G增大。同时，我们又通过光谱学方法解析了其热稳定机制。结果表明25℃时，加入稳定剂对其构象无显著影响，而在高温时可以很大程度上阻止糖化酶二级结构、三级结构的变化以及热聚集，维持其天然构象的稳定，使其在高温下保持较高的酶活力，且海藻糖对糖化酶蛋白的稳定能力高于山梨醇。最后，对糖化酶进行了固定化研究。我们以两种性质优良的树脂SepabeadsEC-OD和Sepabeads EC-HA为载体探索了GAM-1的最适固定化条件。结果发现两种树脂固定化条件一致：最适固定化酶浓度为1mg/mL；最适固定化温度为25℃；最适固定化pH为4.6；最适固定化离子强度为25mM； SepabeadsEC-HA交联糖化酶需要的最适戊二醛浓度为2%。我们通过激光粒度分析发现两固定化酶EC-OD-GA和EC-HA-GA分散度较好，平均粒径分别为259.9±41.10μm和229.2±44.04μm；通过扫描电镜观察表面形态发现，GAM-1成功固定到两树脂上。固定化酶性质分析的结果表明，两固定化糖化酶的最适温度与游离酶相同，均为70℃；但与游离酶相比固定化酶在高温下活力更高，且EC-HA-GA高于EC-OD-GA；与游离酶相比，EC-OD-GA的最适pH稍偏酸性，而EC-HA-GA没发生变化；两固定化酶的热稳定性和pH稳定性也均有提高，且EC-HA-GA的热稳定性高于EC-OD-GA；GAM-1通过两种树脂固定化后，均有较好的循环使用性，在重复使用8次后，EC-HA-GA和EC-OD-GA分别可保持接近60%和28%的残留活性；与游离酶相比，两固定化酶的储藏稳定性均有较大提升。更多还原

【Abstract】 Glucoamylase is an acidic glucoside hydrolase with exo-acting enzymeactivity. Glucoamylase could catalyze the hydrolysis of α-1,4glycosidic linkagesfrom the non-reducing ends of starch and oligosaccharide to produce glucose.Glucoamylase is an industrially important biocatalyst and has the most extensiveuses in the manufacture of starch sugar, food, medicine and brewing, and it hasthe largest production in China.At present, the glucoamylase has the disadvantage of low thermal stability.Hence, the starch after liquefying at high temperature has to be cooled tocontinue the following saccharification process by glucoamylase. In addition, thelong-time saccharification also causes greater loss of glucoamylase, which hasgreatly increased the cost of production. However, the mechanism for the thermalstability of glucoamylase is not clear, and meanwhile the exploitation ofglucoamylase with high thermostability is still on the experimental level. Tosolve this problem, the glucoamylases from a mutant strain Aspergillus nigerB-30were purified, and the properties and unfold mechanism induced bytemperature of these two enzymes were also characterized. Meanwhile, thethermal stability of glucoamylases was improved by adding stabilizer, and themechanism was also eluciadted. Finally, the glucoamylase were immobilized,and the properties of the immobilized glucoamylase were analyzed.First, the glucoamylases from A. niger B-30were purified and the propertiesof these enzymes were also characterized. The two different glucoamylasesGAM-1and GAM-2were purified by ammonium sulfate precipitation, DEAE Fast Flow and Superdex G-75gel filtration columns from the fermentation brothof A. niger B-30. The molecular weight values of GAM-1and GAM-2weredetermined as97.2kDa and78.3kDa by SDS-PAGE, while the correspondingvalues of GAM-1and GAM-2were determined to be80.5kDa and70.4kDa byMALDI-TOF MS, respectively. Both the enzymes were glycosylated, with10.4%and13.4%carbohydrate content. The optimal pH and temperature of these twoenzymes were4.0-4.6and70℃. Both the two glucoamylases displayed highthermal stability and pH stability, and the GAM-2was more stable than GAM-1.Thus, GAM-1and GAM-2are suitable for starch saccharification due to theirhigh catalytic acitivty and stability.Secondly, the folding and unfolding mechanisms of glucoamylases inducedby temperature were studied. The effect of temperature on the conformation ofGAM-1and GAM-2was analyzed by the method of circular dichroism spectrum,fluorescence spectrum, ultraviolet absorption spectrum, dynamic light scatteringand Native-PAGE. The results suggested that both GAM-1and GAM-2displayedsimilar unfolding mechanism. During the process of GAM-1and GAM-2, theα-helix content decreased, and β-fold, β-turn and random coil contents increased,and hydrophobic groups exposed with the aggregation of the protein. Thestructure of GAM-2was more stable during heating, which was the probablereason for that GAM-2showed higher thermal stability.Then the effect of stabilizer on the thermal stability and the mechanism werestudied.1M sorbitol and mycose were respectively added into glucoamylasereaction mixture to stabilize the conformation to maintain the activity at hightemperature. The results suggested that the activity was not obviously increasedat low temperature. On the contrary, the glucoamylase with stabilizer remainedhigher activity at high temperature compared to the negative control. To analyze the thermal stability mechanism of the stabilizer, the thermodynamics data with1M sorbitol and mycose was calculated at75℃and80℃, respectively. Theresults indicated that the kinetics data of heat inactivation obviously reduced afterthe adding of stabilizers, and the half-life and△G increased. The glucoamylaseconformation was not influenced by the stabilizer at25℃, however, at hightemperature the stabilizer could restrain the changing of the secondary andtertiary structure and aggregating of glucoamylase to maintain the naturalconformation, which would lead to higher activity, and the mycose was moreefficient than sorbitol.Finally, the glucoamylase was immobilized on two resins Sepabeads EC-ODand Sepabeads EC-HA, and the properties of the immobilized glucoamylase wereanalyzed. The results suggested that the optimal conditions of immobilizing theGAM-1onto these two supports were uniform. The optimal concentration ofenzyme, temperature, pH and ionic strength were determined as1mg/mL,25℃,4.6and25mM, respectively. The optimal concentration of glutaraldehyde usedto cross-link Sepabeads EC-HA was2%. The two immobilized enzymeEC-OD-GA and EC-HA-GA exhibited a uniform disperse. Particle sizes of thetwo immobilized enzymes were259.9±41.10and229.2±44.04μm by laserscattering analysis, respectively. The surface morphologies of the carriers and theimmobilized enzyme were studied using SEM analysis. The results provided adirect evidence of the successful adsorption of the enzyme on the resins. Theproperties analysis of the immobilized glucoamylase suggested that the optimaltemperature of immobilized and free enzyme were both70℃, but the activity ofimmobilized enzyme was higher than free enzyme at high tempreture.Furthermore, the activity of the EC-OD-GA was higher than EC-HA-GA.Compared with free GAM-1, the optimal pH of the EC-OD-GA is more acidic, and the optimal pH of EC-HA-GA did not change. Both the thermostability andpH stability of immobilized enzyme were improved, and the EC-HA-GA wasmore stable than EC-OD-GA. After immobilization using two supports, theGAM-1showed a good ability to be recycled. After eight times repeated use, theEC-HA-GA and EC-OD-GA could maintained60%and28%relative activity,respectively. Compared with the free enzyme, the storage stability of theimmobilized enzyme was largely improved.更多还原