N-糖基化修饰对泡盛曲霉FCYN206单宁酶分子蛋白折叠结构和热力学稳定性影响机制

Tannase, which can hydrolyse the ester bonds and depside bonds in gallic acid tannins to release gallic acid and glucose, was used to degrade tannin,which is an ati-nutritional factor and producer of bitter taste in food. The taste and quality of the green tea soup would be significantly improved through removing tannin from the summer green tea by tannase. Tannases produced by Aspergillus sp.are natural glyco-proteins.Glycosylation plays an important role to folded structure and function of glyco-proteins, and can improve the thermodynamic stability of glyco-proteins.An Aspergillus awamori FCYN 206 strain with a high yield of tannase has been separated from Yunnan black tea by our work group, and the tannase produced by it has the superiorly high temperature stability. This study aims to separate and purify tannase from Aspergillus awamori FCYN 206, to analyse its enzymatic properties, and to characterize the molecular structure and glycosylation modification. Furthermore, effects of glycosylation on the molecule protein folding and enzymatic activity of tannase will also be studied, and mechanism of glycosylation modification effects on thermodynamic stability of tannase will be discussed at the molecular level. Meanwhile, the enzymatic degradation kinetics model of tannase at high temperature will be established in order to explain high-temperature enzymatic reaction mechanism of Aspergillus awamori tannase. Results will lay the theoretical foundation for industrial production and application of high temperature resistant tannase.

单宁酶能水解单宁的酯键和缩酚羧键，生成没食子酸和葡萄糖，也被用于降解食品中抗营养因子和产生苦涩味的物质- - 单宁。利用单宁酶去除夏季绿茶中的单宁，可显著提高绿茶汤的口感和品质。曲霉属产的单宁酶均是天然糖蛋白，糖基化修饰对糖蛋白的折叠结构和功能起重要的调控作用，可提高酶的热力学稳定性。课题组从云南红茶中筛选到一株高产胞外单宁酶的泡盛曲霉FCYN206，所产胞外单宁酶的高温稳定性能好。本研究拟从该菌株中分离纯化单宁酶，分析其酶学性质，表征其分子结构和糖基化修饰，并研究糖基化修饰对单宁酶的分子蛋白折叠和酶活的影响，从分子水平上阐述糖基化修饰对单宁酶热力学稳定性影响机制；建立单宁酶的高温酶催化降解动力学模型，探讨其高温酶促反应机理，为耐高温泡盛曲霉单宁酶的工业化生产及应用提供理论研究基础。

单宁酶可用于降解速溶茶生产过程中产生苦涩味的单宁，可显著提高绿茶汤的口感和澄清度。绿茶汤提取温度高达70～100℃，而大多数单宁酶热稳定性低、高温易失活。曲霉属产单宁酶是天然糖蛋白，糖基化修饰对其分子折叠结构和功能起重要的调控作用，研究糖基化对单宁酶高温稳定性的影响机制具有重要意义。. 课题组通过硫酸铵分级沉淀、DEAE-Sepharose Fast Flow阴离子交换柱及Sephadex G-150凝胶柱层析分离纯化，所得单宁酶的比酶活为150.63 U/mg，SDS-PAGE凝胶电泳检测该单宁酶由45.0 kDa和36.9 kDa的两个亚基构成。单宁酶最适反应温度为45℃，温度稳定性范围为20-50℃。建立单宁酶高温酶催化单宁降解动力学模型，热失活动力学参数结果表明单宁酶与底物单宁酸的络合反应不能自发进行，其催化水解反应为一吸热过程，单宁酶活性中心的定向有序性随温度的升高逐渐变差。.去N-糖基化后单宁酶活性损失34.42%，其两个亚基中仅有分子量36.9 kDa亚基减小到29. 1kDa，说明N-糖基化在该亚基上。去糖基化结构后单宁酶分子的α-螺旋和无规则卷曲结构比例增加、β结构减少，表明糖基化的共价键破坏后，维系单宁酶的空间折叠作用下降，折叠从有序到无序转化，导致酶活力丧失和高温稳定性下降。去N-糖基化引发单宁酶分子的伸展导致其三级结构改变，其荧光强度增强并伴随蓝移的现象。去N-糖基化后单宁酶分子直径范围从70-260nm下降到60-220nm，平均直径降低20nm，分子高度降低3nm。. 从2个方面提高菌种产单宁酶的能力。通过密码子优化对单宁酶基因进行克隆和毕赤酵母表达，获得一株阳性克隆菌株最高产酶为29.38 U/mL，产酶能力提高54%。另一方面筛选得到一株高产单宁酶的Aspergillus carbonarius FCYN212，比A.awamori FCYN206更高的产酶能力，其代谢产物三没食子酰葡萄糖具有良好的抗氧化活性和抑α-淀粉酶和α-葡萄糖苷酶活性。通过固定化技术将单宁酶固定于磁性纳米粒子上，提高了单宁酶高温稳定性，并将其应用在茶汤澄清中，绿茶茶汤澄清度由46.85%增加至72.17%，而红茶茶汤澄清度由32.66%增加至47.17%。

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