黑曲霉单宁酶的异源表达及其在绿茶同步浸提中的应用
摘要
单宁酶具有多功能性，既可催化水解型单宁中酯键和缩酚酸键的裂解，亦可用于非水相体系合成没食子酸酯类，因此被广泛应用于诸多领域，特别是食品与制药领域。为挖掘新型单宁酶基因，并开发单宁酶的新型应用，本论文从以下三方面进行了研究：（1）黑曲霉单宁酶异源表达
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凯特戴琳斯艳照下载采用PCR扩增技术从黑曲霉中克隆得到两个单宁酶基因（An-Tan1和An-Tan2），基因长度分别为1713 bp和1752 bp，各编码570和583个氨基酸。生物信息学分析表明An-Tan1和An-Tan2都为亲水性蛋白，理论分子量分别为62.96 kDa和63.74 kDa。并成功构建了它们的重组酵母菌株（GS115-An-Tan1和GS115-An-Tan2），摇瓶条件下诱导菌株产酶，培养168 h酶活达最大值分别为0.093 U/mL和0.094 U/mL。进一步于5 L罐上发酵产酶，GS115-An-Tan1和GS115-An-Tan2最大酶活可达4.97 U/mL和7.07 U/mL，相比于摇瓶发酵提高了约53倍和75倍。
（2）重组单宁酶酶学性质研究
对获得的两个重组单宁酶An-Tan1和An-Tan2的酶学性质进行表征。研究发现An-Tan1和An-Tan2的最适温度分别为50 ºC和35 ºC，其中An-Tan1在50 ºC和60 ºC下的半衰期分别为97.1 h和2.8 h，表现出较好
的耐热性；而An-Tan2在4 ºC放置120 min后酶活无损失，并且其在4 ºC和10 ºC下反应的相对酶活分别为49%和60%，表明An-Tan2为适冷型酶。An-Tan1和An-Tan2的最适pH都为5.0，并且它们在pH 2.0-7.0范围内处理24 h 后保留酶活力60%以上，对酸性环境都有良好的耐受性，具有工业应用价值。极性溶剂对An-Tan1和An-Tan2的酶活具有抑制作用，20%甲醛和四氢呋喃使其完全丧失活力。相对于An-Tan1，An-Tan2对高浓度的SDS和EDTA具有较强的耐受性。底物谱结果显示，An-Tan1对儿茶素类物质具有较好降解活性，An-Tan2更偏向于降解单宁酸（TA）和表儿茶素没食子酸酯（ECG）。动力学参数结果表明An-Tan1和An-Tan2对TA的亲和性较强。buy
freaky（3）耐高温单宁酶同步浸提绿茶的新型应用
采用耐高温单宁酶同步浸提（茶叶浸提与酶解同步进行）绿茶茶汤，单因素优化结果表明最优浸提条件为加酶量20 U、温度70 ºC和浸提时间40 min。其次与分步浸提（茶叶浸提与酶解分步进行）所得绿茶茶汤性质对比，结果表明两种方法都可以有效的降解酯型儿茶素，但同步浸提法所得绿茶茶汤的茶多酚含量是分步浸提的1.57倍，并且具有更好的低温储藏稳定性。之后对不同处理后茶汤的抗氧化性进行研究，结果显示同步浸提处理组具有更好的还原能力和对羟基自由基、DPPH自由基和ABTS自由基的清除能力，与分步阿甘正传 下载
浸提处理组相比，其半清除剂量（EC50）分别下降了0.43 mg/mL、0.02 mg/mL和0.03 mg/mL，并且
对猪胰脂肪酶的抑制作用更强，半抑制剂量（IC50）下降了15.8 mg/mL。上述结果表明耐高温单宁酶同步浸提绿茶法在茶饮料加工中具有较好的应用前景。
关键词：单宁酶；黑曲霉；毕赤酵母；绿茶；抗氧化活性
Heterologous Expression of Tanna from Aspergillus niger and Its Application in Simultaneous Extraction of
Green Tea
Abstract
Tanna is a multifunctional enzyme, which not only can catalyze the cleavage of ester and acetal acid bonds in hydrolyzed tannins, but also can be ud to synthesize gallates in organic reaction media. Therefore, tanna has a wide range of applications, especially in the food and pharmaceutical industries. In order to excavate the tanna gene of Aspergillus niger to enrich the gene library of tanna, and to develop novel applications of tanna, this rearch has studied from the following three aspects:
(1) Heterologous expression of Aspergillus niger tanna in Pichia pastoris
Two tanna genes, An-Tan1 and An-Tan2, were cloned from Aspergillus niger FJ0118, which contained 1713 bp and 1752 bp fulllength gene quence length, encoding 570 and 583 amino acids, respectively. Bioinformatics analysis showed that both An-Tan1 and An-Tan2 were hydrophilic proteins with molecular weights of about 62.96 kDa and 63.74 kDa, respectively. Then, the two gene were ligated to pPIC9K expression vector and successfully expresd in Pichia pastoris. After the two recombinant strains were cultured in 500 mL flasks for 168 h , the tanna activity reached the peak (0.093 U/mL and 0.094 U/mL), respectively.Furthermore, through using 5 L fermentation tanks, GS115-An-Tan1 and GS115-An-Tan2 reached the maximum enzyme activity of 4.97 U/mL and 7.07 U/mL, which was incread by 53-fold and 75-fold, respectively.
(2) Enzymatic properties of the recombinant tanna
The two recombinant tanna from Aspergillus niger FJ0118 were purified and characterized. The optimum temperatures of An-Tan1 and An-Tan2 were 50 ºC and 35 ºC, respectively. The calculated half-life values of An-Tan1 at 50 ºC and 60 ºC were 97.1 h and 2.8 h, exhibited the excellent thermal stability. The residual enzyme activity of An-Tan2 was basically unchanged at 4 ºC after 120 min incubation, and the relative enzyme activities at 4 ºC and 10 ºC were 49% and 60%, respectively, indicating that An-Tan2 was a cold-adapted enzyme. Both the optimal pH of An-Tan1 and An-Tan2 w
ere 5.0, and the residual enzyme activities of them were more than 60% after 24 h of treatment at pH 2.0-7.0. The acidophilic tanna had important industrial application value. The polar solvent had significant inhibitory effects on An-Tan1 and An-Tan2, low concentration of formaldehyde and tetrahydrofuran completely inactivated the tanna activity. Compared with An-Tan1, An-Tan2 was more tolerant to high concentrations of骈体
SDS and EDTA. An-Tan1 had better degradation activity on catechins, while An-Tan2 was more inclined to degrade tannin acid (TA) and epicatechin gallate (ECG). The K m values of An-Tan1 and An-Tan2 for the propyl gallate and TA implied that An-Tan1 and An-Tan2 had higher affinity for TA.
(3) Novel application of thermophilic tanna for simultaneous extraction of green tea
The thermophilic tanna was ud for simultaneous extraction of green tea. The optimal extraction conditions were 20 U, 70 ºC and 40 min. Furthermore, comparing with the properties of green tea infusion obtained by different extraction methods, the results showed that both simultaneous extraction and stepwi extraction degraded the catechins effectively. But the green tea infusion obtained by the simultaneous extraction has better low-temperature storage stability, and the content of tea polyphenols were 1.57-fold of that obtain using the stepwi extraction. Moreover, the green te
a infusion obtained by the simultaneous extraction displayed better reducing power and the scavenging abilities towards hydroxyl radicals, DPPH• and ABTS⁺, which EC50 decread by 0.43 mg/mL, 0.02 mg/mL, and 0.03 mg/mL, respectively. The green tea infusion obtained using simultaneous extraction has the better inhibitory effect on pancreatic lipa, and the IC50decread by 15.8 mg/mL compared to that using stepwi extraction. The results indicated that the simultaneous extraction of green tea method had good application potential in tea beverage industry.
Keyword：Tanna；Aspergillus niger；Pichia pastoris；Green tea；Antioxidant activity
目录
第1章引言 (1)
1.1 单宁酶 (1)
1.1.1 单宁酶简介 (1)
lucent1.1.2 单宁酶的性质 (2)
1.1.3 单宁酶的工业应用 (7)hot是什么意思怎么读
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1.1.4 单宁酶的生产 (9)
1.2 茶饮料现状 (12)
1.2.1 绿茶饮料主要营养成分 (13)
1.2.2 目前茶饮料存在的问题 (14)
1.2.3 单宁酶在茶饮料加工中的应用 (15)
1.3 论文的立题依据和研究内容 (17)
1.3.1 研究目的与意义 (17)
1.3.2 研究内容 (17)
第2章黑曲霉单宁酶的异源表达及生物信息学分析 (19)
2.1 实验材料与仪器设备 (19)起英文名的网站
2.1.1 菌株 (19)
2.1.2 引物 (19)
2.1.3 主要试剂 (20)
2.1.4 主要培养基与溶液 (21)
2.1.5 实验仪器 (22)
2.2 实验方法 (23)
2.2.1 菌株活化 (23)
2.2.2 提取基因组DNA (23)
