茶葉多酚氧化酶同工酶分離鑒定及其酶促合成茶黃素研究
發(fā)布時(shí)間：2018-10-12 21:36
【摘要】：多酚氧化酶(Polyphenol Oxidase, PPO)是自然界普遍存在一種氧化還原酶,由Cu2+輔基和主酶組成,茶葉PPO對(duì)茶樹(shù)生理代謝和茶葉加工均有重要意義。國(guó)內(nèi)外許多研究將茶葉PPO作為一個(gè)整體,圍繞其細(xì)胞定位、生理遺傳、酶學(xué)特性、酶源篩選與利用、提取與分離、酶促氧化合成茶黃素以及酶固定化等作了大量而系統(tǒng)的研究,取得了顯著研究成果。但在PPO同工酶的分離鑒定、定向酶促合成茶黃素及其組分等方面未能取得突破性進(jìn)展。本研究集成系列現(xiàn)代分離純化技術(shù),擬通過(guò)分離鑒定政和大白茶鮮葉中PPO同工酶單體,并在探明其酶學(xué)性質(zhì)的基礎(chǔ)上,深入研究其定向酶促合成茶黃素的效果,以期為茶葉PPO同工酶單體的分離制備及其定向酶促合成茶黃素提供科學(xué)依據(jù)。主要研究結(jié)果如下：1、以政和大白一芽二葉為材料,先后通過(guò)丙酮粉干燥、緩沖液勻漿提取、硫酸銨分級(jí)沉淀、透析、Q-Sepharose Fast Flow強(qiáng)陰離子交換色譜、Sephadex G-75凝膠柱層析、超濾膜濃縮脫鹽等步驟,獲得了電泳純PPO同工酶單體PPO1和PP02,其比活力分別為10850.00 U/mg和24102.31 U/mg,純化倍數(shù)分別達(dá)到22.03倍和48.94倍。2、經(jīng)MALDI-TOF/TOF質(zhì)譜鑒定,發(fā)現(xiàn)PPO1酶蛋白分子量為85089 Da,等電點(diǎn)pI為6.1,與Methionine synthase isozyme酶各肽片段匹配氨基酸序列有YLFAGVVDGR、AGINVIQIDEAALR. YGAGIGPGVYDIHSPP、LQEELDIDVLVHGEP ER; PPO2酶蛋白分子量為42531.7 Da,等電點(diǎn)pI為5.49,與Phosphoglycerate kinase酶各肽片段匹配氨基酸序列有YSLKPLVPR、LASLAD LYVNDAFGTAHR、DLNVPL DDNLNITDDTR。3、對(duì)PPO1和PP02的酶學(xué)性質(zhì)研究發(fā)現(xiàn),PPO1與底物結(jié)合能力順序?yàn)榻剐詻](méi)食子酸EGC鄰苯二酚EGCGL-酪氨酸,PP02則為焦性沒(méi)食子酸EGCG鄰苯二酚EGCL-酪氨酸；最適溫度分別為30℃、38℃,超過(guò)45℃后兩種同工酶均失活加速；最適pH值分別為5.5和6.0,在pH值4.5-6.5范圍內(nèi),均能保持40%以上的相對(duì)酶活性；PPO1的熱穩(wěn)定性稍強(qiáng)于PP02；金屬離子Ba2+、Fe3+對(duì)兩種同工酶無(wú)明顯影響,而Cu2+、Al3+對(duì)其均有抑制作用,Mg2+對(duì)PPO1有抑制作用,而對(duì)PP02表現(xiàn)促進(jìn)作用,Ca2+在10mmol/L濃度時(shí),對(duì)兩種PPO同工酶均具有促進(jìn)作用；抑制劑亞硫酸鈉、抗壞血酸、半胱氨酸對(duì)兩種同工酶均有強(qiáng)抑制性,EDTA、草酸和檸檬酸對(duì)PPO1表現(xiàn)抑制性,而對(duì)PP02具有促進(jìn)性。4、酶促合成茶黃素中,PPO1酶促合成產(chǎn)物為單一的茶黃素組TF,而PP02則酶促合成TF、TF-3-G.TF-3'-G和TFDG四種茶黃素組分,同時(shí)在pH值5-6、溫度35℃左右時(shí),兩種同工酶酶促合成茶黃素的效率較高,通過(guò)改變底物中簡(jiǎn)單兒茶素和酯型兒茶素的含量及比例,可對(duì)合成產(chǎn)物茶黃素組分和含量進(jìn)行調(diào)控,其中PPO1酶促合成茶黃素濃度范圍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，
本文編號(hào)：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，
本文編號(hào)：2267679
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