茶叶多酚氧化酶同工酶分离鉴定及其酶促合成茶黄素研究
发布时间：2018-10-12 21:36
【摘要】：多酚氧化酶(Polyphenol Oxidase, PPO)是自然界普遍存在一种氧化还原酶,由Cu2+辅基和主酶组成,茶叶PPO对茶树生理代谢和茶叶加工均有重要意义。国内外许多研究将茶叶PPO作为一个整体,围绕其细胞定位、生理遗传、酶学特性、酶源筛选与利用、提取与分离、酶促氧化合成茶黄素以及酶固定化等作了大量而系统的研究,取得了显著研究成果。但在PPO同工酶的分离鉴定、定向酶促合成茶黄素及其组分等方面未能取得突破性进展。本研究集成系列现代分离纯化技术,拟通过分离鉴定政和大白茶鲜叶中PPO同工酶单体,并在探明其酶学性质的基础上,深入研究其定向酶促合成茶黄素的效果,以期为茶叶PPO同工酶单体的分离制备及其定向酶促合成茶黄素提供科学依据。主要研究结果如下：1、以政和大白一芽二叶为材料,先后通过丙酮粉干燥、缓冲液匀浆提取、硫酸铵分级沉淀、透析、Q-Sepharose Fast Flow强阴离子交换色谱、Sephadex G-75凝胶柱层析、超滤膜浓缩脱盐等步骤,获得了电泳纯PPO同工酶单体PPO1和PP02,其比活力分别为10850.00 U/mg和24102.31 U/mg,纯化倍数分别达到22.03倍和48.94倍。2、经MALDI-TOF/TOF质谱鉴定,发现PPO1酶蛋白分子量为85089 Da,等电点pI为6.1,与Methionine synthase isozyme酶各肽片段匹配氨基酸序列有YLFAGVVDGR、AGINVIQIDEAALR. YGAGIGPGVYDIHSPP、LQEELDIDVLVHGEP ER; PPO2酶蛋白分子量为42531.7 Da,等电点pI为5.49,与Phosphoglycerate kinase酶各肽片段匹配氨基酸序列有YSLKPLVPR、LASLAD LYVNDAFGTAHR、DLNVPL DDNLNITDDTR。3、对PPO1和PP02的酶学性质研究发现,PPO1与底物结合能力顺序为焦性没食子酸EGC邻苯二酚EGCGL-酪氨酸,PP02则为焦性没食子酸EGCG邻苯二酚EGCL-酪氨酸；最适温度分别为30℃、38℃,超过45℃后两种同工酶均失活加速；最适pH值分别为5.5和6.0,在pH值4.5-6.5范围内,均能保持40%以上的相对酶活性；PPO1的热稳定性稍强于PP02；金属离子Ba2+、Fe3+对两种同工酶无明显影响,而Cu2+、Al3+对其均有抑制作用,Mg2+对PPO1有抑制作用,而对PP02表现促进作用,Ca2+在10mmol/L浓度时,对两种PPO同工酶均具有促进作用；抑制剂亚硫酸钠、抗坏血酸、半胱氨酸对两种同工酶均有强抑制性,EDTA、草酸和柠檬酸对PPO1表现抑制性,而对PP02具有促进性。4、酶促合成茶黄素中,PPO1酶促合成产物为单一的茶黄素组TF,而PP02则酶促合成TF、TF-3-G.TF-3'-G和TFDG四种茶黄素组分,同时在pH值5-6、温度35℃左右时,两种同工酶酶促合成茶黄素的效率较高,通过改变底物中简单儿茶素和酯型儿茶素的含量及比例,可对合成产物茶黄素组分和含量进行调控,其中PPO1酶促合成茶黄素浓度范围0.973～11.807μg/mL。
[Abstract]:Polyphenol oxidase (Polyphenol Oxidase, PPO) is a common redox enzyme in nature, which is composed of Cu2 cogroups and main enzymes. Tea PPO is of great significance to the physiological metabolism and tea processing of tea plants. Many studies on tea PPO as a whole have focused on its cellular location, physiological inheritance, enzymatic properties, screening and utilization of enzyme sources, extraction and separation, enzymatic oxidation synthesis of theaflavins and enzyme immobilization. Remarkable research results have been obtained. However, no breakthrough has been made in the separation and identification of PPO isozymes and the synthesis of theaflavins and their components by directed enzymes. This study integrates a series of modern separation and purification techniques to identify the PPO isozyme monomer in fresh leaves of Zhenhe and Daqing tea, and to study the effect of its directed enzymatic synthesis of theaflavins on the basis of the study of its enzymatic properties. To provide a scientific basis for the separation and preparation of tea PPO isozyme monomer and its directed enzymatic synthesis of theaflavins. The main results were as follows: 1. Acetone powder drying, buffer homogenate extraction, ammonium sulfate precipitation, dialysis, Q-Sepharose Fast Flow strong anion exchange chromatography, Sephadex G-75 gel column chromatography were used as materials. The specific activity of pure PPO isozyme monomer PPO1 and PP02, were 10850.00 U/mg and 24102.31 U / mg, and the purification times were 22.03 and 48.94 times, respectively. The specific activity of PPO1 and PP02, were identified by MALDI-TOF/TOF mass spectrometry. It was found that the molecular weight of PPO1 protein was 85089 Da, isoelectric point pI was 6.1. The amino acid sequence matched with the peptide fragment of Methionine synthase isozyme enzyme was YLFAGVVDGR,AGINVIQIDEAALR.. The molecular weight of YGAGIGPGVYDIHSPP,LQEELDIDVLVHGEP ER; PPO2 protein was 42531.7 Da, isoelectric point pI was 5.49. The amino acid sequence matched with each peptide fragment of Phosphoglycerate kinase enzyme was YSLKPLVPR,LASLAD LYVNDAFGTAHR,DLNVPL DDNLNITDDTR.3, and the enzymatic properties of PPO1 and PP02 were studied. It was found that the binding ability of PPO1 to the substrate was in the order of EGC o-phenylpyrogallic acid. Diphenol EGCGL- tyrosine and PP02 EGCG catechol EGCL- tyrosine; The optimum temperature is 30 鈩，
本文编号：2267679
[Abstract]:Polyphenol oxidase (Polyphenol Oxidase, PPO) is a common redox enzyme in nature, which is composed of Cu2 cogroups and main enzymes. Tea PPO is of great significance to the physiological metabolism and tea processing of tea plants. Many studies on tea PPO as a whole have focused on its cellular location, physiological inheritance, enzymatic properties, screening and utilization of enzyme sources, extraction and separation, enzymatic oxidation synthesis of theaflavins and enzyme immobilization. Remarkable research results have been obtained. However, no breakthrough has been made in the separation and identification of PPO isozymes and the synthesis of theaflavins and their components by directed enzymes. This study integrates a series of modern separation and purification techniques to identify the PPO isozyme monomer in fresh leaves of Zhenhe and Daqing tea, and to study the effect of its directed enzymatic synthesis of theaflavins on the basis of the study of its enzymatic properties. To provide a scientific basis for the separation and preparation of tea PPO isozyme monomer and its directed enzymatic synthesis of theaflavins. The main results were as follows: 1. Acetone powder drying, buffer homogenate extraction, ammonium sulfate precipitation, dialysis, Q-Sepharose Fast Flow strong anion exchange chromatography, Sephadex G-75 gel column chromatography were used as materials. The specific activity of pure PPO isozyme monomer PPO1 and PP02, were 10850.00 U/mg and 24102.31 U / mg, and the purification times were 22.03 and 48.94 times, respectively. The specific activity of PPO1 and PP02, were identified by MALDI-TOF/TOF mass spectrometry. It was found that the molecular weight of PPO1 protein was 85089 Da, isoelectric point pI was 6.1. The amino acid sequence matched with the peptide fragment of Methionine synthase isozyme enzyme was YLFAGVVDGR,AGINVIQIDEAALR.. The molecular weight of YGAGIGPGVYDIHSPP,LQEELDIDVLVHGEP ER; PPO2 protein was 42531.7 Da, isoelectric point pI was 5.49. The amino acid sequence matched with each peptide fragment of Phosphoglycerate kinase enzyme was YSLKPLVPR,LASLAD LYVNDAFGTAHR,DLNVPL DDNLNITDDTR.3, and the enzymatic properties of PPO1 and PP02 were studied. It was found that the binding ability of PPO1 to the substrate was in the order of EGC o-phenylpyrogallic acid. Diphenol EGCGL- tyrosine and PP02 EGCG catechol EGCL- tyrosine; The optimum temperature is 30 鈩，
本文编号：2267679
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