【作者】 华杰；

【导师】 黄可龙；

【作者基本信息】 中南大学 ， 应用化学， 2011， 博士

【摘要】 本文针对5’-磷酸二酯酶水解酵母RNA制备5’-核苷酸工艺中存在的技术问题,开展了对酶制剂制备技术及RNA酶解工艺的改进研究。改进了酶活性的测定方法,研究了从麦芽根中浸取和提纯5’-磷酸二酯酶的技术以及RNA酶解反应动力学,探索了麦芽根5’-磷酸二酯酶的固定化方法,进一步研究了该固定化酶在膜反应器中水解酵母RNA的工艺,并设计了一种新的连续错流萃取色谱分离系统,用于复杂酶解产物的分离。研究工作和主要结果如下：改进了传统的测定5’-磷酸二酯酶酶活的UV-RNA法,提出了HPLC-RNA酶活测定法。该方法的特点是：1)能测定混合酶中5’-磷酸二酯酶的酶活；2)精密度高,性线范围大。当麦芽根的蛋白质浓度在0.02～1.20 mg/mL范围内时,蛋白质浓度与5’-核苷酸产量的线性相关系数R=0.9997,相对标准偏差(R.S.D)为5.7%。采用正交实验法优化了从麦芽根中提取5’-磷酸二酯酶的操作条件。最佳条件为：麦芽根粒径120目,浸取液pH=7,浸取温度10℃,麦芽根：水=1：16(质量比),浸取时间5 h。在该条件下,每克麦芽根提取的总酶活为4470 U,5’-磷酸二酯酶的单位酶活为280 U/mL。采用硫酸铵沉淀、超滤浓缩、透析膜脱盐,以及Sephadex G-25、Sephadex G-75凝胶层析提纯,获得了比酶活为0.02137 mmol/ (mgxmin)的5’-磷酸二酯酶制剂,比未经硫酸铵沉淀的粗酶液的比酶活提高了6倍。探讨了5’-磷酸二酯酶催化水解RNA的反应机理,研究了酶的主要酶学特征值。实验测得米氏常数Km=8.73 mg/mL(底物为RNA),最大反应速度Vmax=14.27×10-3 mg/(mL·min),酶的最适pH值为5.0,最适温度为70℃。考察了酶的稳定性,发现5’-磷酸二酯酶在pH值为5-7的范围内比较稳定,短时间置于较高的温度(50-70℃)下,能保持较高的酶活。在酶液中加入金属离子一股都会导致酶活下降,但镁离子有促进酶活的效应,体现了与桔青霉5’-磷酸二酯酶不同的特性。5’-核苷酸是麦芽根5’-磷酸二酯酶的抑制剂,不断移除RNA酶解反应产物中的5’-核苷酸,有利于酶解反应的进行。采用戊二醛活化的壳聚糖为载体,固定了麦芽根5’-磷酸二酯酶。试验结果表明,当酶固定化温度为30℃、固定化酶pH值为5.0、固定化时间为6h的最适条件下,最高的酶活回收率可达53.6%。所制得的固定化酶的最适温度为75℃,最适pH值为5.5。研究表明,固定化酶比游离酶更纯,酶的固定化过程也起到了提纯酶的作用。研究了固定化5’-磷酸二酯酶的酶学性质,测得固定化酶的米氏常数Km为15.38 mg/mL(底物为RNA)设计装配了一种管式固定床反应器(CPFR)与中空纤维超滤膜相耦合的反应-分离装置。考察了反应温度、pH值、底物浓度等因素对反应进程的影响。酶解反应的最适操作条件为：反应温度为75℃,pH值为5.5,RNA浓度控制在3%以内。在反应-分离耦合装置上进行了较长时间连续酶解RNA的操作,核苷酸的总收率达到了88.3%。反应结束后,固定化5’-磷酸二酯酶能保持原酶74.6%的活性,表明固定化酶具有良好的稳定性。发明了一种连续错流萃取色谱分离系统,推导出了该系统重相出口和轻相出口中的流出色谱图的数学表达式(流出曲线),从理论上证明该系统能够连续、大处理量、高选择性地分离多组分复杂混合物。采用6×6的错流萃取色谱分离系统分离了5’-AMP和苯甲酸的混合物,实验结果与理论计算值相当吻合,证明该系统能实现混合物的高分辨分离。更多还原

【Abstract】 Researches were made in this dissertation to improve the preparation and purification of 5’-phosphodiesterase and innovate the production process for the enzymatic hydrolysis of yeast RNA with this enzyme to produce 5’-nucleotides. A new method for the assay of 5’-phosphodiesterase enzyme activity was set up, a method for the isolation and purification of 5’-phosphodiesterase from barley roots was developed, and the dynamic mechanism of the enzymatic hydrolysis reaction was studied, furthermore, a new method for the immobilization of 5’-phosphodiesterase from barley roots was studied, and this immobilized enzyme was applied in a membrane reactor for the hydrolysis of yeast RNA. A continuous cross-flow chromatograph system was invented to separate complexes mixtures, such as products of enzymatic hydrolysis reactions. The main results of this research work were listed as follows:A novel HPLC-RNA method has been developed for the determination of enzyme activity; this method has some advantages compared with the conventional UV-RNA method:a) It can be used for the determination of enzyme activity of 5’-phosphodiesterase sample containing other undesired enzymes. b) It has fine reproducibility, precision, linearity and large measuring range. The correlation coefficients (r2) and R.S.D for the data was 0.9997 and 5.7%, respectively, when the protein concentrations of 5’-phosphodiesterase were within 0.02～1.20 mg/mL.Optimal conditions for the extraction of 5’-phosphodiesterase from barley rootlets were obtained by using orthogonal assay design. The optimal conditions were as:fineness of barley roots was 120 mesh, pH= 7, temperature was 10℃, ratio of barley roots to water was 1:16 (w/w), and the extraction time was 5 h. Under the condition, the obtained total enzyme activity of the 5’-phosphodiesterase extracted from 1 g barley rootlets was 4470 U, and the enzyme activity of crude 5’-phosphodiesterase was 280 U/mL.Purer 5’-phosphodiesterase with high specific enzyme activity was obtained through ammonium sulphate precipitation, ultrafiltration, dialysis, and Sephadex G-25、Sephadex G-75 chromatography. The specific enzyme activity of the crude enzyme without ammonium sulphate precipitation was 0.00356 mmol/(mgxmin), while the final enzyme had a specific enzyme activity of 0.02137 mmol/(mgxmin),6 times larger than the original one.The reaction mechanisms of enzymatic hydrolysis of RNA catalyzed by 5’-phosphodiesterase were investigated, and the main enzymatic properties of the enzyme were determined. The results showed that the Michaelis constant (Km) was 8.73 mg/mL(with RNA as substrate), maximum reaction velocity (Vmax) was 0.01427 mg/mL·min, optimum pH was 5.0, and optimum temperature was 70℃. This 5’-phosphodiesterase was considerable stable within pH 5～7, and temperature 50～70℃. Generally, most metal ions were inhibitors for this enzyme, while magnesian ion was an accelerant for 5’-phosphodiesterase.5’-nucleotides in the products acted as inhibitors, immediate removal of 5’-nucleotides from the hydrolysate was favorable for the enzymatic reaction.5’-phosphodiesterase was immobilized on chitosan activated by glutaric dialdehyde. Under optimum conditions, a maximum enzyme recovery of 53.6%was reached, when the temperature for the immobilization was 30℃, pH 5.0, and reaction tine was 6 hours. The immobilized enzyme had an optimum pH 5.5, and optimum temperature 75℃. Studies also showed that the immobilized enzyme was purer than free 5’-phosphodiesterase, for the hydrolysate contained more 5’-nucleotides and less other impurities. Michaelis constant (Km) for immobilized enzyme was 15.38 mg/mL(with RNA as substrate)A continuous plug flow reactor(CPFR)coupling with a hollow fiber membrane separator was designed for the hydrolysis of RNA with immobilized 5’-phosphodiesterase. The optimization of the enzymatic reaction showed that the optimum reaction temperature was 75℃, optimum pH was 5.5, and the concentration of RNA should be no more than 3%. A continuous experiment of hydrolysis of RNA was conducted in this coupling device, and 88.3% yield of 5’-nucleotides was achieved. Results showed that the immobilized 5’-phosphodiesterase was rather stable during the reaction process, for it kept 74.6% of the original enzyme activity after the operation.A novel continuous cross-flow extraction chromatograph system was invented for the separation of mixtures, Mathematical expressions for the chromatograph both in heavy phase and light phase outlets were derived, which were used to prove theoretically that this system could separate on large scale complex mixtures with high resolution continuously. A mixture of 5’-AMP and benzoic acid was separated well in a 6×6 cross-flow extraction chromatograph system; the result was in line with the theoretical prediction.更多还原