有机溶剂体系对单宁酶构效的影响

发布时间：2018-08-31 12:05

【摘要】：有机相生物催化是工业生物催化的一个重要方向,提高生物酶在有机介质体系中的催化效率是生物催化技术目前面临的重要挑战之一。没食子酸丙酯(Propyl gallate,PG)是一种优异的的食品抗氧化剂和重要的医药前体。常用的化学合成法因高能耗、高腐蚀等缺点,已不能满足PG绿色合成的需要。有机相生物催化法因其独特的优势已成为PG绿色合成的重要发展方向。单宁酶(TAN)作为该催化反应中的关键酶,在有机介质体系中活性受到有机介质反应体系的约束,导致其表观催化活性及稳定性显著降低。为此,本文以单宁酸和正丙醇为底物,以一步酯转换合成PG的生物催化反应为模式反应,以有机介质体系为研究对象,具体研究有机溶剂、酶微环境对TAN构效的影响。在基础上,进一步研究生物印迹诱导TAN催化合成对羟基苯甲酸丙酯(propyl p-hydroxybenzoate,PPHB)的探索性研究,为拓展TAN在化学品绿色合成中的应用提供借鉴。在酯转换化反应中,有机溶剂作为介质能有效提升底物溶解度,解除水的抑制作用,抑制逆反应。本研究研究多种不同结构类型的有机溶剂对TAN构效的影响,应用HPLC、紫外(UV)和红外(FT-IR)光谱测定TAN微观结构变化。结果显示有机溶剂的结构、基团对TAN催化性能存在较为显著的影响。如环状结构可能对酶的构效稳定性有负面影响;烷基团通过作用TAN结构中的含氧和含氮基团,破坏其构象稳定性。有机介质体系中,酶外周微环境(缓冲液、金属离子和表面活性剂等)是影响其表观活力大小至关重要的因素。为此,本研究首先研究了磷酸、乙酸和柠檬酸缓冲液及乙酸缓冲液浓度对TAN构效的影响,结果显示酶活性与其二级结构参数的变化率呈负相关。其中,乙酸和磷酸缓冲液通过将α-螺旋转化为β-折叠的方式提升了 TAN的活性,揭示了一定含量的α-螺旋和β-折叠是TAN维持活性的结构基础。锌离子研究发现离子浓度越大时,酶的增色基团越外露。表面活性剂的研究发现0.24 mM司盘20能显著激活TAN,PG产量提升了约7倍。生物印迹可以诱导改变酶对底物亲和性。为拓展TAN的应用,本研究基于生物印迹技术,应用对羟基苯甲酸(p-hydroxybenzoic acid,PHBA)诱导TAN,研究印迹TAN催化PHBA到PPHB的酯化反应的可行性。经HPLC、FT-IR和UV等验证底物印迹TAN具有催化合成PPHB的活性。进一步优化有机介质,结果表明正丙醇为该反应的最佳反应介质,其最大产量约为860mg/L,底物转化率约为26.5%。本研究有助于进一步认识酶结构稳定性与其活性的关系,为有效提升酶-有机介质体系的相容性,提升TAN有机相生物催化性能提供了技术支撑;所采用的生物印迹技术对改变生物酶的催化特性具有一定借鉴意义,为有机相生物合成其他化学品提供了可借鉴的技术手段。
[Abstract]:Organic phase biocatalysis is an important direction of industrial biocatalysis. Improving the catalytic efficiency of biological enzyme in organic medium system is one of the important challenges of biocatalysis technology. Propyl gallate (Propyl gallate,PG) is an excellent food antioxidant and an important pharmaceutical precursor. Because of the disadvantages of high energy consumption and high corrosion, the common chemical synthesis method can not meet the needs of green synthesis of PG. Organic biocatalysis has become an important direction of green synthesis of PG due to its unique advantages. As the key enzyme in the catalytic reaction, the activity of tannase (TAN) in organic medium system is restricted by the organic medium reaction system, resulting in the apparent catalytic activity and stability decreased significantly. Therefore, using tannic acid and n-propanol as the substrate, the biocatalytic reaction of one-step transesterification to synthesize PG was used as the model reaction, and the organic medium system was used as the research object, the effects of organic solvent and enzyme microenvironment on the structure activity of TAN were studied. On the basis of this study, the further research on the synthesis of propyl p-hydroxybenzoate (propyl) catalyzed by bioblot induced TAN was carried out, which provided a reference for expanding the application of TAN in chemical green synthesis. In transesterification, organic solvents can effectively improve the solubility of the substrate, remove the inhibition of water and inhibit the inverse reaction. In this paper, the effects of different organic solvents on the structure activity of TAN were studied. The microstructure of TAN was determined by HPLC, UV (UV) and FT-IR spectra. The results showed that the structure and group of organic solvents had a significant effect on the catalytic performance of TAN. For example, the ring structure may have a negative effect on the stability of the enzyme, and the alkyl group destroys the conformational stability by acting on the oxygen and nitrogen groups in the TAN structure. In organic media, the microenvironment (buffer, metal ions and surfactants) is the most important factor affecting the apparent activity of enzyme. Therefore, the effects of the concentration of phosphoric acid, acetic acid and citric acid buffer and acetic acid buffer on the structure activity of TAN were studied. The results showed that the activity of the enzyme was negatively correlated with the change rate of its secondary structure parameters. Acetic acid and phosphoric acid buffer enhanced the activity of TAN by converting 伪 -helix into 尾 -fold, which revealed that 伪 -helix and 尾 -folding are the structural basis of maintaining activity of TAN. It was found that the higher the concentration of zinc ion, the more exposed the chromogenic group of enzyme. Surfactant study found that 0.24 mM Span 20 significantly activated TAN,PG production by about 7 times. Bioimprinting can induce changes in enzyme affinity to the substrate. In order to expand the application of TAN, the feasibility of the esterification of PHBA to PPHB catalyzed by imprinted TAN was studied by using p-hydroxybenzoic acid (p-hydroxybenzoic acid,PHBA) -induced TAN, based on bioblot technique. HPLC,FT-IR and UV proved that substrate imprinted TAN had the activity of catalytic synthesis of PPHB. The results show that n-propanol is the best reaction medium, the maximum yield is about 860 mg / L, and the substrate conversion is about 26.5%. This study is helpful to further understand the relationship between enzyme structure stability and its activity and provide technical support for enhancing the compatibility of enzyme-organic medium system and enhancing the biocatalysis performance of TAN organic phase. The biological imprinting technology used in this paper has some reference significance for changing the catalytic properties of bioenzymes and provides a reference for the organic phase biosynthesis of other chemicals.
【学位授予单位】：安徽工程大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.8

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