【摘要】：有機(jī)相生物催化是工業(yè)生物催化的一個重要方向,提高生物酶在有機(jī)介質(zhì)體系中的催化效率是生物催化技術(shù)目前面臨的重要挑戰(zhàn)之一。沒食子酸丙酯(Propyl gallate,PG)是一種優(yōu)異的的食品抗氧化劑和重要的醫(yī)藥前體。常用的化學(xué)合成法因高能耗、高腐蝕等缺點,已不能滿足PG綠色合成的需要。有機(jī)相生物催化法因其獨特的優(yōu)勢已成為PG綠色合成的重要發(fā)展方向。單寧酶(TAN)作為該催化反應(yīng)中的關(guān)鍵酶,在有機(jī)介質(zhì)體系中活性受到有機(jī)介質(zhì)反應(yīng)體系的約束,導(dǎo)致其表觀催化活性及穩(wěn)定性顯著降低。為此,本文以單寧酸和正丙醇為底物,以一步酯轉(zhuǎn)換合成PG的生物催化反應(yīng)為模式反應(yīng),以有機(jī)介質(zhì)體系為研究對象,具體研究有機(jī)溶劑、酶微環(huán)境對TAN構(gòu)效的影響。在基礎(chǔ)上,進(jìn)一步研究生物印跡誘導(dǎo)TAN催化合成對羥基苯甲酸丙酯(propyl p-hydroxybenzoate,PPHB)的探索性研究,為拓展TAN在化學(xué)品綠色合成中的應(yīng)用提供借鑒。在酯轉(zhuǎn)換化反應(yīng)中,有機(jī)溶劑作為介質(zhì)能有效提升底物溶解度,解除水的抑制作用,抑制逆反應(yīng)。本研究研究多種不同結(jié)構(gòu)類型的有機(jī)溶劑對TAN構(gòu)效的影響,應(yīng)用HPLC、紫外(UV)和紅外(FT-IR)光譜測定TAN微觀結(jié)構(gòu)變化。結(jié)果顯示有機(jī)溶劑的結(jié)構(gòu)、基團(tuán)對TAN催化性能存在較為顯著的影響。如環(huán)狀結(jié)構(gòu)可能對酶的構(gòu)效穩(wěn)定性有負(fù)面影響;烷基團(tuán)通過作用TAN結(jié)構(gòu)中的含氧和含氮基團(tuán),破壞其構(gòu)象穩(wěn)定性。有機(jī)介質(zhì)體系中,酶外周微環(huán)境(緩沖液、金屬離子和表面活性劑等)是影響其表觀活力大小至關(guān)重要的因素。為此,本研究首先研究了磷酸、乙酸和檸檬酸緩沖液及乙酸緩沖液濃度對TAN構(gòu)效的影響,結(jié)果顯示酶活性與其二級結(jié)構(gòu)參數(shù)的變化率呈負(fù)相關(guān)。其中,乙酸和磷酸緩沖液通過將α-螺旋轉(zhuǎn)化為β-折疊的方式提升了 TAN的活性,揭示了一定含量的α-螺旋和β-折疊是TAN維持活性的結(jié)構(gòu)基礎(chǔ)。鋅離子研究發(fā)現(xiàn)離子濃度越大時,酶的增色基團(tuán)越外露。表面活性劑的研究發(fā)現(xiàn)0.24 mM司盤20能顯著激活TAN,PG產(chǎn)量提升了約7倍。生物印跡可以誘導(dǎo)改變酶對底物親和性。為拓展TAN的應(yīng)用,本研究基于生物印跡技術(shù),應(yīng)用對羥基苯甲酸(p-hydroxybenzoic acid,PHBA)誘導(dǎo)TAN,研究印跡TAN催化PHBA到PPHB的酯化反應(yīng)的可行性。經(jīng)HPLC、FT-IR和UV等驗證底物印跡TAN具有催化合成PPHB的活性。進(jìn)一步優(yōu)化有機(jī)介質(zhì),結(jié)果表明正丙醇為該反應(yīng)的最佳反應(yīng)介質(zhì),其最大產(chǎn)量約為860mg/L,底物轉(zhuǎn)化率約為26.5%。本研究有助于進(jìn)一步認(rèn)識酶結(jié)構(gòu)穩(wěn)定性與其活性的關(guān)系,為有效提升酶-有機(jī)介質(zhì)體系的相容性,提升TAN有機(jī)相生物催化性能提供了技術(shù)支撐;所采用的生物印跡技術(shù)對改變生物酶的催化特性具有一定借鑒意義,為有機(jī)相生物合成其他化學(xué)品提供了可借鑒的技術(shù)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 偽 -helix into 尾 -fold, which revealed that 偽 -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.
【學(xué)位授予單位】：安徽工程大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O629.8

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