固定化酶膜反应器藕合错流萃取色谱制备5’-核苷酸
|
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。
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.
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。
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.
引文
[1]孙融融.酶解法制备3’-核苷酸.化学试剂,1983,5(2)：103-106
[2]修俊鹏,丁庆豹,欧伶,邱蔚然.高产耐热3'-磷酸二酯酶菌种的选育及酶性质的研究.华东理工大学学报(自然科学版),2006,32(3)：274-277
[3]Chiwook P. L., Wayne S., Tonald T. R. Contribution of the active site histidine residues of ribonuclease A to nucleic acid biding. Biochemistry,2001,40: 4949-4956
[4]Bradley R K, Tony A K, Mark A B, et al. Hypersensitive substrate for ribonuc leases. Nucleic Acid Research,1999,27 (18):3696-3701
[5]朱兵,李新民,吴亦杰.稀土元素对3’-腺嘌呤及3'-鸟嘌呤核苷酸水解断裂作用.化学学报,1998,56：47-51
[6]Heath H. B., Reineccius G. Flavor Chemistry and Technology. AVI Publishing Company, Imc,1986,319-324
[7]Maga, J.A. Flavor Potentiatora, CRC Crit. Rev.Food Sci. Nutr.1983,18(3):231
[8]崔桂友.谷氨酸一钠和5'-核苷酸的呈味性质.中国烹饪研究,1996(2)：17-20
[9]罗晓燕.抗病毒药阿昔洛韦的合成改进.化学试剂,2001,23(3)：184-185
[10]Morris J. R., Peter W. H., Can J Chem,1982,60 (5):547
[11]詹天荣,娄红祥.核苷类抗HIV药物的研究与开发.药学进展,2005,29(7)：289-295
[12]凌保东.抗病毒药物的研究现状.川北医学院学报,2003,18(2)：159-161
[13]张天民,宗爱珍,王凤山.2007年我国生化药物研究进展.中国药学杂志,2008,43(18)：1364-1368
[14]Tian X L, Zhong J, Li B, et al. Inhibitory effect of 5-aza-2'-deoxycytidine on human gastric cancer xenografts in nude mice. J Shanghai Jiaotong Univ (Med Sci),2007,27 (5):533-536
[15]Jin M L, Jie Z J, Cai Y Y, et al. The effects of CpG-oligode-oxynucleotides on air way inflammation in chronic asthmatic mice. Fudan Univ J (M ed Sci),2007, 34 (3):358-363
[16]吕爱娟,吴皓.中药中核苷类成分的研究进展.中国中医药信息杂志,2006,13(7)：94-97
[17]陈日远,关佩聪,刘诚等.核苷酸及其组合物对冬瓜产量形成及其生理效应的研究.华南农业大学学报,2000,21(3)：9-12
[18]王熹,施一平,金子渔.核酸降解物在水稻生产上的应用及其作用极力的研究：1水稻苗期应用的增产效果和有效组成成分的研究.生物化学与生物物理学报,1975,7(1)：31-39
[19]金子渔,沈守江,孙玉昆.核酸降解物在水稻生产上的应用及其作用极力的研究：2水稻苗期应用的生理作用.生物化学与生物物理学报,1975,7(2)：139-149
[20]邵吉安,孙云详,石兆魁,沈建国.核苷酸复配剂在农业上的应用.土壤,1992,24(4)：210-212
[21]谢宪章,甘蔗糖蜜生产核苷酸的分离方法研究.食品与发酵工业,1993,6：22-26
[22]宁正祥,李玲,胡立侃.核苷酸在柑橘生产上的应用及其作用机理研究.中国柑橘,1986,1：2-6
[23]胡立侃,宁止祥.核苷酸对荔枝生产能力的影响.荔枝科技通讯,1990,2：1-4
[24]应国清,石陆娥,唐振兴.核苷酸类物质的应用研究进展.广州食品工业科技,2004,20(2)：126-128
[25]Ouchi S, Sowa T, Sato K.5'-phosphorylation of unprotected nucleosides. Japan, 7108854,1971
[26]陈耀明,江云,明新.核苷酸的生产方法.中国专利,CN1304939A.2001
[27]陈曾三.发酵法高收率制造5'-肌苷酸的新方法.江苏食品与发酵,1994,(4)：29-34
[28]周秀琴.发酵法制造5'-肌苷酸.发酵科技通讯,1993,22(3)：56-58
[29]杨斌,汤毅珊,高孔荣.呈味核苷酸的生产和应用.中国调味品,1995,3：7-11
[30]王富源,现代食品发酵技术.北京：中国轻工业出版社,2001,427-443
[31]吕东坡,朱仁俊.肌苷酸的研究现状及展望.中国食品添加剂,2007,6：53-56
[32]乔世伟.呈味核苷酸的提取.中国调味品,2001,2：18-19
[33]慕娟,麦芽根在核酸水解中的应用研究,药物生物技术,2002,9(1)：48-52
[34]Benaiges M. D., Lopez-Santin, Sola C. Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA. Enzyme Microb. Technol..1990, (12):86-89
[35]陈洁,王璋,肖刚.啤酒废酵母中酵母抽提物的制备.无锡轻工大学学报,2001,20(4)：356-362
[36]晏志云,赵谋明,彭志英.提高酵母精呈味核苷酸[I+G]含量的研究.中国调味品,1998,(7)：5-8
[37]许剑秋.酶解法生产腺一磷(AMP)的探讨.粮食与饲料工业,1997,(10)：40
[38]宋荣钊,王春林,梁锋.固定化5'一磷酸二酯酶及其在核苷酸生产中的应用.微生物学报,1991,18(4)：211-214
[39]干普行,许惟治,厉雪君.双酶法呈味核苷酸的研制,第3报,双酶法制取呈味核苷酸(I+G).上海调味品,1990,1：19-23
[40]房兴利.呈味核苷酸的生产及其应用.中国调味品,1993, 10：1-4
[41]Kuninaka A., Sakaguchi K. Japanese Patent S.36-5287
[42]石陆娥；酶膜生物反应器制备核苷酸的研究：[博士学位论文].浙江工业大学,2007
[43]徐燚.5'-核苷酸制备工艺的研究：[博士学位论文].南京工业大学：2002
[44]Kinichiro S, Akira K.5'-nucleotides. U.S.3,223,592,1965
[45]Fujimoto M, Fujishima T, Kuninaka A, Activation of nuclease from Penicillium Citrium. Hakko To Taisha.1967,16:20-27
[46]国中明,藤岛铁郎,藤本正雄.Amino Acid and Nucleic Acid,1967,16:28
[47]Fujimoto M, Kuninaka A, Yoshino H. Specificity of a nuclease from Penicillium Citrium. Agri. Biol. Chem.,1969,33(10):1517-1518
[48]国中明.桔青霉固体培养生产核酸.食品工业,1961,11：22-25
[49]Nakiro Y. Agr Biol Chem,1963,27(3):199-204
[50]Ogata K. Agr Biol Chem.,1963,27(2):110-115
[51]普为民,陶元器,丁骅孙.核酸P1酶产生菌选育及其生理生态学研究.云南大学学报(自然科学版),1994,16(2)：174-178
[52]陈信波,罗泽民.核酸酶P1高产菌选育及酶学特性研究.湖南农业大学学报,1995,21(4)：382-385
[53]Zhu Y, Knol W, Smits J P, et al. Biol Medium optimization for nuclease P1 production from Penicillium citrinumin solid-state fermentation using polyurethane foam as inert carrier. J. Enzyme and microbial technology,1996, 18:108
[54]杨加华,李爱芬,李绪青,李磊,毕崇洁.桔青霉发酵生产核酸酶P1的适宜条件.烟台大学学报,1997,10(2)：106-109
[55]娄永江,吴汉民,王海洪.从桔青霉M71生产核酸酶P1及酶活提高途径的研究.宁波大学学报,1997,10(3)：21-28
[56]夏黎明.固定化桔青霉产生核酸酶P1的研究.微生物学报,1998,38(6)：449-453
[57]王克明.双载体固定化桔青霉产生核酸酶P1的研究.中国粮油学报,1999,14(5)：44-46
[58]王克明,许文友,庄树宏.固定化桔青霉气升式反应器生产核酸酶P1的研究.烟台大学学报(自然科学与工程版),2001,14(1)：37-41
[59]李科德,韩木兰,柏建玲,贺鹰抟.5'-磷酸二酯酶高产菌株的选育和发酵培养条件的优化.微生物学杂志,2001,21 (3)：28-30
[60]吕浩,应汉杰.核酸酶P1的纯化和酶学性质研究.南京工业大学学报,2002,24(4)：66-69
[61]徐正军,肖林平,吕浩,谢宁昌,应汉杰.实验设计法优化核酸酶P1的发酵培养基.过程工程学报,2003,3(5)：433-437
[62]莫晓燕,宋威,张芹,廖红东.桔青霉生产核酸酶P1发酵条件优化研究.西安交通大学学报,2003,37(10)：1083-1085
[63]Desai N A, Shanker V. Single-strand-specific nucleases, FEMS Microbiology Review J.2003,26:457
[64]廖红东,莫晓燕,宋威.核酸酶P1的分离纯化及部分酶学性质研究.中国医药工业杂志,2005,36(9)：536-544
[65]Fujimoto M, Kuninaka A, Yoshino H. Some Physical and Chemical Properties of Nuclease P 1. Agri. Biol. Chem.,1975,39(10):1991-1997
[66]Rokugawa K, Fujimoto M. Kuninaka A. Yoshino H. The Role of Zinc Atoms in Nuclease P1. Agri. Biol. Chem.,1980,44(8):1987-1988
[67]Fujimoto M, Kuninaka A. Yoshino H. Identity of phosphodiesterase and phosphomonoesterase Activities with Nuclease P1. Agri. Biol. Chem.1974, 38(4):785-790;
[68]陈珊珊,曹剑虹.Zn2+对5'-磷酸二酯酶活性的影响.福建医药杂志.1994,16(3)：49
[69]Volbeda A, Lahm A, Sakiyama F, Suck D. Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution. The EMBO Journal,1991,10(7): 1607-1618
[70]Maekawa K, Tsunasawa S, Dibo G. Primary structure of Nuclease P1 from Penicillium citrinum. Eur. J. Biochem.,1991,200:651-661
[71]Fujimoto M, Kuninaka A, Yoshino H. Secondary Structure of Nuclease P1. Agri. Biol. Chem.,1975,39(11):2145-2148
[72]Lahm A, Volbeda A, Suck D. Crystallization Crystallographic Analysis of P1 Nuclease from Penicillium Citrinum. J. Mol. Biol,1990,215:207-210
[73]Romier C, Lahm A, Suck D. Recognition of single-stranded DNA by nuclease PI:High resolution crystal structure of complexes with substrate analogs. Proteins,1998,32:414-424
[74]Fujimoto M, Kuninaka A,Yoshino H, et al. Identity of Phosphodiesterase and phosphomonoesterase activities with nuclease P1. J. Agric Biol Chem,1974, 38:785
[75]郭春香,邵昌平,郭和夫.金属离子对核酸酶P1催化水解RNA反应的影响及其机理探讨.催化学报,1992,13(4)：316-319
[76]Briggs D E, Hongn I S, Young T W, Stevens R. Malting and Brewing Science, Chapman & Hall,1981,141～142
[77]Rodriguez-Lopez M, Baroja-Fernandez E, Zandueta-Criado A, Moreno-Bruna B, Munoz FJ, Akazawa T, Pozueta-Romero J. Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein. FEBS letters,2001, 490:44-48
[78]Wang AY, Juang RH, Chang CT, Sung HY. Purification and characterization of 5'-phosphodiesterase from barley rootlets. Biochemistry and Molecular Biology International,1993,29:1095-1102
[80]Bowles LK.5'-phosphodiesterase enzyme preparation and method for its production, United States Patent,5,034,325,1991
[81]N. Prentice. Comparison of malts for nuclease and nucleo-base potentials. Journal of the American Society of Brewing Chemists,1983,41 (4):133～144
[82]N. Prentice, Characterization of a Nuclease from Malted Barley Roots. Journal of Cereal Science,1987 (5):175-187
[83]Laufer L, Gutcho S. Hydrolysis of RNA to 5'-nucleotide by seed sprouts, particularly malt sprouts. Biotechnology and Bioengineering,1968,10:257-275
[84]Tanekawa T; Takashima H; Hachiya T. US Patent 4303,680,1981
[85]吴应文.大麦芽中酸性磷酸酯酶的分离及性质.兰州大学学报,1992,28(4)：117-119
[86]李德莹,邱蔚然,丁庆豹.麦芽根中5'-磷酸二酯酶的制备及性质.华东理工大学学报,2002,28(4)：376-379
[87]陈洁,王璋.麦芽根中核酸酶的提取及制备.食品工业科技,2000,21(1)：19-21
[88]李祥.5'-磷酸二酯酶的制备及其在酵母抽提物生产中的应用.中国调味品,2000,4：12-14
[89]陈洁,王璋.麦芽根中核酸酶的分离纯化及性质研究.无锡轻工大学学报,2001,20(3)：270-274
[90]段作营,刘军昌,于瑞嵩,毛忠贵.麦芽根中5'-磷酸二酯酶的提取及制备.中国调味品,2003,2：36-39
[91]Beluhan S, Karmelic I, Novak S, Maric V. Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts. BiotechnologyLetters,2003,25:1099-1103
[92]Pietrzak M, Cudny H, Maluszynski Miroslaw. Purification and properties of two ribonucleases and a nuclease from barley seeds. Biochimica et Biophysica Acta,1980,614:102-112
[93]Deoda A J, Singhal R S.5'-Phosphodiesterase (5'-PDE) from germinated barley for hydrolysis of RNA to produce flavour nucleotides. Bioresource Technology, 2003,88:245-250
[94]Benaiges M D, Lopez-Santin J, Sola C. Partial purification of 5'-phosphodiesterase activity from barley rootlets. Enzyme and Microbial Technology,1989,11(7):444-451
[95]Dhule S S, Shetty P R, Iyer J L, Singhal R S.Purification and characterization of 5'-phosphodiesterase from germinated barley. Process Biochemistry,2006, 41:1899-1902
[96]Strater N. Ecto-5'-nucleotidase:Structure function relationships. Purinergic Signalling,2006.2:343-350
[97]Valerio A A, Corradini A C, Panunto P C, Mello S M, Hyslop S. Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom, Journal of Protein Chemistry,2002,21:495-503
[98]Mori N, Nikai T, Sugihara H. Phosphodiesterase from the venom of crotalus ruber rubber. IntJ. Biochem.,1987,19(2):115-119
[99]罗艳萍,陈远志,黎肇炎.短尾蝮蛇毒磷脂酶A2的分离纯化及抗血小板聚集作用.蛇志,2009,21(2)：90-93
[100]Ouyang C, Huang T F. Inhibition of platelet aggregation by 5-nucleotidase purified from Trimeresurus gramineus snake venom. Toxicon,1983,21 (4): 491-501.
[101]余晓东,黄立农,熊郁良.竹叶青(Trimeresurus Stejnegeri)蛇毒5-核苷酸酶对血小板聚集功能的抑制机制研究.重庆师范学院学报(自然科学版),1997,14(2)：61-68
[102]Dhananjaya BL, Nataraju A, Rajesh R, Gowda CDR, Sharath BK,Vishwanath BS, D'Souza CJM. Anticoagulant effect of Naja naja venom 5-nucleotidase: demonstration through the use of novel specific inhibitor, vanillic acid. Toxicon, 2006.48 (4):411-421
[103]陈夏,余晓东,邓敏,李卉,林亦心,和七一,柳建平.白唇竹叶青蛇毒5'-核苷酸酶的分离纯化及性质.动物学研究,2008,29(4)：399-404
[104]Fujimoto H, Fujiyama K, Midorikawa Y, Fujishima T, Kuninaka A, Yoshino H. Production of 5'-PDE from Aspergillus niger. Agricultural and Biological Chemistry,1977,41:737-74
[105]Taketo Y, Taketo A. Phosphoesterases of hemolytic Streptococci. The Japanese Journal of Experimental Medicine,1974,44:291-299.
[106]Fitt PS, Baddoo P. (1978) Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum. The Biochemical Journal,181,347-353.
[107]Ito K, Yamamoto T, Minamiura N. Phosphodiesterase I in Human Urine: Purification and Characterization of the Enzyme.J. Biochem.,1987, 102:359-367
[108]Wilson, C.M. Plant Nucleases. I. Separation and Purification of Two Ribonucleases and One Nuclease from Corn. Plant Physiol.,1968,43: 1332-1338
[109]Wilson C.M. Purification of a corn ribonuclease. J. Biol. Chem.1967,242: 2260-2263
[110]Wilson C.M. Plant Nucleases. II. Properties of Com Ribonucleases I and II and Com Nuclease I., Plant Physiol.,1968,43:1339-1346
[111]Torti G, Mapelli S, Soave C. Acid ribonuclease from wheat germ:Purification, properties and specificity. Biochim Biophys Acta,1973,324:254-266
[112]Wyen NV, Erdei S, Farkas GL. Isolation from Avena leaf tissues of a nuclease with the same type of specificity towards RNA and DNA. Accumulation of the enzyme during leaf senescence. Biochim Biophys Acta.,1971,232(3):472-483
[113]Barker GR, Bray CM, Walter TJ. The Development of Ribonuclease and Acid Phosphatase During Germination of Pisum arvense. Biochem. J.,1974,142: 211-219
[114]Jervis L. Partial purification and characterization of two Nicotiana tabacum leaf ribonucleases, Phytochemistry,1974,13:709-714
[115]Noguchi S, Shimura G, Kimura K, Samejima H. Production of 5'-mononu cleotides using immobilized 5'-phosphodiesterase and 5'-AMP deaminase. Journal of Solid-phase Biochemistry,1976,1 (2):105-118
[116]张德安,年燕兰,张今.固定化5'-磷酸二酯酶的制备.吉林大学自然科学学报,1979,1：74-76
[117]袁中一,刘树煌,汪静英.固定化核酸酶P1应用于5'-核苷酸生产.科学通报,1980,25(14)：654-657
[118]Rokugawa K, Fujishima T, Kuninaka A. Studies on immobilized enzymes(I):Immobilization of nuclease P1 on cellulose. Journal of Fermentation Technology,1979,57:570-573
[119]Rokugawa K, Fujishima T, Kuninaka A. Immobilized enzymes (Ⅱ): Immobilization of nuclease P 1 on ion exchange resins by titanium complex method. Journal of Fermentation Technology,1980,58:423-429
[120]Rokugawa K, Fujishima T, Kuninaka A. Studies on immobilized enzymes (Ⅲ): Immobilization of nuclease P 1 on inorganic supports by titanium complex method. Journal of Fermentation Technology,1980,58:509-515
[121]Chen WP. Use of immobilized enzymes for the Production of 5'-nucleotides from yeast RNA(Ⅰ):Immobilization of 5'-phosphodiesterase.Kexue Fazhan Yuekan,1983, 11(3):228-235
[122]Lo MC, Chang HS, Chen WP. Studies on immobilization of 5'-Phosphod iesterase on chitin, Shipin Kexue (Taipei, Taiwan),1985,12(3-4):151-162
[123]Suh W.C, Lim B.S, Chun M. Immobilization of nuclease P1 from Penicillium citrinum and Production of 5'-nucleotides by bioreactor. Han'guk Saenghwa Hakhoechi,1987,20(1):17-23
[124]Keller R., Schlingmann M, Woernle R. Production of 5'-ribonucleotides using immobilized 5'-phosphodiesterase. Methods in Enzymology,1987, 136:517-522
[125]刘红,潘红春,丁洪,王晓兰.核酸酶P1固定化研究.食品与发酵工业,1994,4：44-48
[126]Olmedo F, Iturbe F, Gomez-Hemandez J. Continuous production of 5'-ribo nucleotides from yeast RNA by hydrolysis with immobilized 5'-phosphod iesterase and 5'-adenylate deaminase. Enzyme Microb technol.,1994, 10(1):36-40
[127]Serrat JM, Benaiges MD, Lopez-santin J, Immobilization of 5'-Phosphodiesterase from vegetal origin by covalent binding on activated Celite.Biocatalysis,1992,6(1):51-59
[128]杨大令,高涵,张守海,吴迪,蹇锡高.固定化酶膜中凝胶层膜的制备与性能研究.化工时刊,2008,22(5)：1-4
[129]Manoharan M, Schwille RM, Schwille PO. Adenosine nucleotides in rat bone measured by ion-pair reversed-phase high-performance liquid chromatography: effect of hemorrhagic shock,with and without retransfusion of blood. Journal of Chromatography B,2001,761:159-166
[130]Pierro D Di, Tavazzi B, Perno CF, Bartolini M, Balestra E, Calio R, Giardina B, Lazzarino G. An Ion-Pairing High-Performance Liquid Chromatographic Method for the Direct Simultaneous Determinationof Nucleotides, Deoxynucleotides, Nicotinic Coenzymes, Oxypurines, Nucleosides, and Bases in Perchloric Acid Cell Extracts, Analytical Biochemistry,1995,231:407-412
[131]Zheng YF, Xu GW, Yang J, Zhao XJ, Pang Tao, Kong HW. Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography, Journal of Chromatography B,2005,819:85-90
[132]Gill BD, Indyk HE. Development and application of a liquid chromatographic method for analysis of nucleotides and nucleosides in milk and infant formulas. International Dairy Journal,2007,17:596-605
[133]Lopez SLB, Moal J, Serrano FSJ. Development of a method for the analysis of nucleotides from the mantle tissue of the mussel Mytilus galloprovincialis, Journal of Chromatography A,2000,891:99-107
[134]Guo FQ, Li A, Huang LF, Liang YZ, Chen BM. Identification and determination of nucleosides in Cordyceps sinensis and its substitutes by high performance liquid chromatography with mass spectrometric detection, Journal of Pharmaceutical and Biomedical Analysis,2006,40:623-630
[135]Gilar M, Fountain KJ, Budman Y, Neue U D, Yardley KR, Rainville PD, Russell RJ, Gebler JC. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:Retention prediction. Journal of Chromatography A,2002,958:167-182
[136]Gao JL, Leung KSY, Wang YT, Lai CM, Li SP, Hu LF, Lu GH, Jiang ZH, Yu ZL. Qualitative and quantitative analyses of nucleosides and nucleobases in Ganoderma spp. by HPLC-DAD-MS. Journal of Pharmaceutical and Biomedical Analysis,2007,44:807-811
[137]Ferreira IMPLVO, Mendes E, Gomes AMP, Faria MA, Ferreira MA. The determination and distribution of nucleotides in dairy products using HPLC and diode array detection. Food Chemistry,2001,74:239-244
[138]霍小敏,屠春燕,宋慧敏,欧阳平凯.HPLC法测定酵母RNA降解的4种核苷酸.南京工业大学学报,2002,24(4)：61-64
[139]黄晓兰,李科德,陈云华.15种核酸水解产物的高效液相色谱分离及其在酵母抽提物分析中的应用.分析化学,2000,28(12)：1504-1507
[140]Huang H X, Zhang YK, Lu P. Z. Chromatography,1992,10:25
[141]赵红玲,贾乃堃,刘朵花,黎高沃.一种利用麦芽根复合磷酸酯酶制备5’-核苷酸的方法,中国专利,CN200710063910.4
[142]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry,1976,72:248-254.
[143]李建武,萧能庚,余瑞元,袁明秀,陈丽蓉,陈雅蕙,陈来同.生物化学实验原理和方法.北京大学出版社,1994,
[144]Yarnell A.The power of promiscuity: enzymes'ability to catalyze alternative reactions may provide a springboard for evolution. Chem Eng News 2003; 81:33-35
[145]O'Brien PJ, Herschlag D. Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase. Biochemistry,2001,40:5691-5699
[146]Krajewska B. Application of chitin-and chitosan-based materials for enzyme immobilizations:a review. Enzyme and Microbial Technology,2004,35: 126-139
[147]宋建彬,任孝修.以壳聚糖为载体固定化青霉素酰化酶的研究.化工进展,2004,23(2)：181-184
[148]Wei X, Zhang M, Gorski W. Coupling the lactate oxidase to electrodes by ionotropic gelation of biopolymer. Anal Chem,2003,75:2060-2064
[149]梁足培,桑明心,李建,曹长青,王广建.戊二醛交联壳聚糖球固定化脲酶的制备.青岛科技大学学报,2006,27(2)：123-126
[150]王永健,孙彦.适于蛋白质吸附的交联壳聚糖树脂的制备.天津大学学报,2001,34(6)：819-822
[151]唐振兴,石陆娥,钱俊青.壳聚糖凝胶吸附蛋白质机理研究.精细化工2004,21(11)：833-836
[152]刘满英,王春霞,刘杏恋,孙领霞.壳聚糖吸附提取分离蛋白质.离子交换与吸附,1997,15(5)：525-531
[153]Yu YH, He BL. A new type of AL SS-The preparation of crosslinked chitosan resin and its adsorption properties for bilirubin. Reactive & Functional Polymers,1996,31 (3):195～200.
[154]余艺华,孙彦,何炳林.交联壳聚糖树脂的制备工艺及性能表征.天津大学学报,2000,33(1)：113-117
[155]姜忠义,陈洪钫.酶膜反应器研究进展.高分子材料科学与工程,2004,20(1)：14-17
[156]杨利敏,王亚明,张松,唐红玉,钟莉.酶膜反应器的应用研究.化工时刊,2005,19(5)：62-65
[157]石陆娥,应国清,唐振兴,易喻,熊文悦.酶膜生物反应器中酶的固定化方法研究及其应用进展.药物生物技术,2006,13(4)：310-314
[158]陈静,张和平.膜分离式酶解反应器的研究及其在食品工业上的应用.内蒙古农业大学学报,2004,25(2)：124-126
[159]孪雪辉,王乐夫.新型反应器一酶膜反应器.天然气化工,2001,26：48-52
[160]Sethi S, Wiesner MR, Modeling of transient permeate flux in cross-flow membrane filtration incorporating multiple particle transport mechanisms, Journal of Membrane Science,1997,136(1-2):191-205
[161]Lahoussine-Turcaud V, Fouling in tangential-flow ultrafitration:The effect of colloid size and coagulation pretreatment, Membrane Science,1990,52 (2): 173-190
[162]Field RW, Wu D, Howell JA, Critical flux concept for micro filtration fouling, Journal of Membrane Science,1995,100(3):259～272
[163]John A, Sub-critical flux operation of microfiltration, Journal of Membrane Science,1995,107(1-2):165～171
[164]Thomassen JK, Faraday DBF, Underwood BO, The effect of varying transmembrane pressure and crossflow velocity and the microfiltration fouling of a model beer, Separation and Purification Technology,2005,41(1):91-100
[165]Defiance L, Jafrin MY,Comparison between filtration at fixed transmembrane pressure and fixed permeate flux:Application to a membrane bioreactor used for wastewater treatment, Journal of Membrane Science,1999,152(2):203-210
[166]Ueda T, Hata K, Kiknoka Y, Effect of aeration on suction pressure in a submerged membrane bioreactor, Water Research,1997,31(3):489-494
[167]Choo KH,Lee CH,Membrane fouling mechanisms in the membrane-coupled anaerobic bioreactor, Water Research,1996,30(8):1771-1780
[168]Choo KH,Lee CH,Effect of anaerobic digestion broth composition in membrane permeability, Water Science and Technology,1996,34(9):173-179
[169]Cihlar T, Rosenberg I. Efficient separation of natural ribonucleotides by low-pressure anion-exchange chromatography. J.Chromatog,1993,644(2): 299-305.
[170]肖林平,徐正军,何明芳.NH-1分离5'-核苷酸的研究.离子交换与吸附,2003,19(5)：430-436
[171]李黎,莫晓燕,陈雪玉.混合5'-核苷酸的分离与检测.中国医药指南,2010,8(21)：45-48
[2]修俊鹏,丁庆豹,欧伶,邱蔚然.高产耐热3'-磷酸二酯酶菌种的选育及酶性质的研究.华东理工大学学报(自然科学版),2006,32(3)：274-277
[3]Chiwook P. L., Wayne S., Tonald T. R. Contribution of the active site histidine residues of ribonuclease A to nucleic acid biding. Biochemistry,2001,40: 4949-4956
[4]Bradley R K, Tony A K, Mark A B, et al. Hypersensitive substrate for ribonuc leases. Nucleic Acid Research,1999,27 (18):3696-3701
[5]朱兵,李新民,吴亦杰.稀土元素对3’-腺嘌呤及3'-鸟嘌呤核苷酸水解断裂作用.化学学报,1998,56：47-51
[6]Heath H. B., Reineccius G. Flavor Chemistry and Technology. AVI Publishing Company, Imc,1986,319-324
[7]Maga, J.A. Flavor Potentiatora, CRC Crit. Rev.Food Sci. Nutr.1983,18(3):231
[8]崔桂友.谷氨酸一钠和5'-核苷酸的呈味性质.中国烹饪研究,1996(2)：17-20
[9]罗晓燕.抗病毒药阿昔洛韦的合成改进.化学试剂,2001,23(3)：184-185
[10]Morris J. R., Peter W. H., Can J Chem,1982,60 (5):547
[11]詹天荣,娄红祥.核苷类抗HIV药物的研究与开发.药学进展,2005,29(7)：289-295
[12]凌保东.抗病毒药物的研究现状.川北医学院学报,2003,18(2)：159-161
[13]张天民,宗爱珍,王凤山.2007年我国生化药物研究进展.中国药学杂志,2008,43(18)：1364-1368
[14]Tian X L, Zhong J, Li B, et al. Inhibitory effect of 5-aza-2'-deoxycytidine on human gastric cancer xenografts in nude mice. J Shanghai Jiaotong Univ (Med Sci),2007,27 (5):533-536
[15]Jin M L, Jie Z J, Cai Y Y, et al. The effects of CpG-oligode-oxynucleotides on air way inflammation in chronic asthmatic mice. Fudan Univ J (M ed Sci),2007, 34 (3):358-363
[16]吕爱娟,吴皓.中药中核苷类成分的研究进展.中国中医药信息杂志,2006,13(7)：94-97
[17]陈日远,关佩聪,刘诚等.核苷酸及其组合物对冬瓜产量形成及其生理效应的研究.华南农业大学学报,2000,21(3)：9-12
[18]王熹,施一平,金子渔.核酸降解物在水稻生产上的应用及其作用极力的研究：1水稻苗期应用的增产效果和有效组成成分的研究.生物化学与生物物理学报,1975,7(1)：31-39
[19]金子渔,沈守江,孙玉昆.核酸降解物在水稻生产上的应用及其作用极力的研究：2水稻苗期应用的生理作用.生物化学与生物物理学报,1975,7(2)：139-149
[20]邵吉安,孙云详,石兆魁,沈建国.核苷酸复配剂在农业上的应用.土壤,1992,24(4)：210-212
[21]谢宪章,甘蔗糖蜜生产核苷酸的分离方法研究.食品与发酵工业,1993,6：22-26
[22]宁正祥,李玲,胡立侃.核苷酸在柑橘生产上的应用及其作用机理研究.中国柑橘,1986,1：2-6
[23]胡立侃,宁止祥.核苷酸对荔枝生产能力的影响.荔枝科技通讯,1990,2：1-4
[24]应国清,石陆娥,唐振兴.核苷酸类物质的应用研究进展.广州食品工业科技,2004,20(2)：126-128
[25]Ouchi S, Sowa T, Sato K.5'-phosphorylation of unprotected nucleosides. Japan, 7108854,1971
[26]陈耀明,江云,明新.核苷酸的生产方法.中国专利,CN1304939A.2001
[27]陈曾三.发酵法高收率制造5'-肌苷酸的新方法.江苏食品与发酵,1994,(4)：29-34
[28]周秀琴.发酵法制造5'-肌苷酸.发酵科技通讯,1993,22(3)：56-58
[29]杨斌,汤毅珊,高孔荣.呈味核苷酸的生产和应用.中国调味品,1995,3：7-11
[30]王富源,现代食品发酵技术.北京：中国轻工业出版社,2001,427-443
[31]吕东坡,朱仁俊.肌苷酸的研究现状及展望.中国食品添加剂,2007,6：53-56
[32]乔世伟.呈味核苷酸的提取.中国调味品,2001,2：18-19
[33]慕娟,麦芽根在核酸水解中的应用研究,药物生物技术,2002,9(1)：48-52
[34]Benaiges M. D., Lopez-Santin, Sola C. Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA. Enzyme Microb. Technol..1990, (12):86-89
[35]陈洁,王璋,肖刚.啤酒废酵母中酵母抽提物的制备.无锡轻工大学学报,2001,20(4)：356-362
[36]晏志云,赵谋明,彭志英.提高酵母精呈味核苷酸[I+G]含量的研究.中国调味品,1998,(7)：5-8
[37]许剑秋.酶解法生产腺一磷(AMP)的探讨.粮食与饲料工业,1997,(10)：40
[38]宋荣钊,王春林,梁锋.固定化5'一磷酸二酯酶及其在核苷酸生产中的应用.微生物学报,1991,18(4)：211-214
[39]干普行,许惟治,厉雪君.双酶法呈味核苷酸的研制,第3报,双酶法制取呈味核苷酸(I+G).上海调味品,1990,1：19-23
[40]房兴利.呈味核苷酸的生产及其应用.中国调味品,1993, 10：1-4
[41]Kuninaka A., Sakaguchi K. Japanese Patent S.36-5287
[42]石陆娥；酶膜生物反应器制备核苷酸的研究：[博士学位论文].浙江工业大学,2007
[43]徐燚.5'-核苷酸制备工艺的研究：[博士学位论文].南京工业大学：2002
[44]Kinichiro S, Akira K.5'-nucleotides. U.S.3,223,592,1965
[45]Fujimoto M, Fujishima T, Kuninaka A, Activation of nuclease from Penicillium Citrium. Hakko To Taisha.1967,16:20-27
[46]国中明,藤岛铁郎,藤本正雄.Amino Acid and Nucleic Acid,1967,16:28
[47]Fujimoto M, Kuninaka A, Yoshino H. Specificity of a nuclease from Penicillium Citrium. Agri. Biol. Chem.,1969,33(10):1517-1518
[48]国中明.桔青霉固体培养生产核酸.食品工业,1961,11：22-25
[49]Nakiro Y. Agr Biol Chem,1963,27(3):199-204
[50]Ogata K. Agr Biol Chem.,1963,27(2):110-115
[51]普为民,陶元器,丁骅孙.核酸P1酶产生菌选育及其生理生态学研究.云南大学学报(自然科学版),1994,16(2)：174-178
[52]陈信波,罗泽民.核酸酶P1高产菌选育及酶学特性研究.湖南农业大学学报,1995,21(4)：382-385
[53]Zhu Y, Knol W, Smits J P, et al. Biol Medium optimization for nuclease P1 production from Penicillium citrinumin solid-state fermentation using polyurethane foam as inert carrier. J. Enzyme and microbial technology,1996, 18:108
[54]杨加华,李爱芬,李绪青,李磊,毕崇洁.桔青霉发酵生产核酸酶P1的适宜条件.烟台大学学报,1997,10(2)：106-109
[55]娄永江,吴汉民,王海洪.从桔青霉M71生产核酸酶P1及酶活提高途径的研究.宁波大学学报,1997,10(3)：21-28
[56]夏黎明.固定化桔青霉产生核酸酶P1的研究.微生物学报,1998,38(6)：449-453
[57]王克明.双载体固定化桔青霉产生核酸酶P1的研究.中国粮油学报,1999,14(5)：44-46
[58]王克明,许文友,庄树宏.固定化桔青霉气升式反应器生产核酸酶P1的研究.烟台大学学报(自然科学与工程版),2001,14(1)：37-41
[59]李科德,韩木兰,柏建玲,贺鹰抟.5'-磷酸二酯酶高产菌株的选育和发酵培养条件的优化.微生物学杂志,2001,21 (3)：28-30
[60]吕浩,应汉杰.核酸酶P1的纯化和酶学性质研究.南京工业大学学报,2002,24(4)：66-69
[61]徐正军,肖林平,吕浩,谢宁昌,应汉杰.实验设计法优化核酸酶P1的发酵培养基.过程工程学报,2003,3(5)：433-437
[62]莫晓燕,宋威,张芹,廖红东.桔青霉生产核酸酶P1发酵条件优化研究.西安交通大学学报,2003,37(10)：1083-1085
[63]Desai N A, Shanker V. Single-strand-specific nucleases, FEMS Microbiology Review J.2003,26:457
[64]廖红东,莫晓燕,宋威.核酸酶P1的分离纯化及部分酶学性质研究.中国医药工业杂志,2005,36(9)：536-544
[65]Fujimoto M, Kuninaka A, Yoshino H. Some Physical and Chemical Properties of Nuclease P 1. Agri. Biol. Chem.,1975,39(10):1991-1997
[66]Rokugawa K, Fujimoto M. Kuninaka A. Yoshino H. The Role of Zinc Atoms in Nuclease P1. Agri. Biol. Chem.,1980,44(8):1987-1988
[67]Fujimoto M, Kuninaka A. Yoshino H. Identity of phosphodiesterase and phosphomonoesterase Activities with Nuclease P1. Agri. Biol. Chem.1974, 38(4):785-790;
[68]陈珊珊,曹剑虹.Zn2+对5'-磷酸二酯酶活性的影响.福建医药杂志.1994,16(3)：49
[69]Volbeda A, Lahm A, Sakiyama F, Suck D. Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution. The EMBO Journal,1991,10(7): 1607-1618
[70]Maekawa K, Tsunasawa S, Dibo G. Primary structure of Nuclease P1 from Penicillium citrinum. Eur. J. Biochem.,1991,200:651-661
[71]Fujimoto M, Kuninaka A, Yoshino H. Secondary Structure of Nuclease P1. Agri. Biol. Chem.,1975,39(11):2145-2148
[72]Lahm A, Volbeda A, Suck D. Crystallization Crystallographic Analysis of P1 Nuclease from Penicillium Citrinum. J. Mol. Biol,1990,215:207-210
[73]Romier C, Lahm A, Suck D. Recognition of single-stranded DNA by nuclease PI:High resolution crystal structure of complexes with substrate analogs. Proteins,1998,32:414-424
[74]Fujimoto M, Kuninaka A,Yoshino H, et al. Identity of Phosphodiesterase and phosphomonoesterase activities with nuclease P1. J. Agric Biol Chem,1974, 38:785
[75]郭春香,邵昌平,郭和夫.金属离子对核酸酶P1催化水解RNA反应的影响及其机理探讨.催化学报,1992,13(4)：316-319
[76]Briggs D E, Hongn I S, Young T W, Stevens R. Malting and Brewing Science, Chapman & Hall,1981,141～142
[77]Rodriguez-Lopez M, Baroja-Fernandez E, Zandueta-Criado A, Moreno-Bruna B, Munoz FJ, Akazawa T, Pozueta-Romero J. Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein. FEBS letters,2001, 490:44-48
[78]Wang AY, Juang RH, Chang CT, Sung HY. Purification and characterization of 5'-phosphodiesterase from barley rootlets. Biochemistry and Molecular Biology International,1993,29:1095-1102
[80]Bowles LK.5'-phosphodiesterase enzyme preparation and method for its production, United States Patent,5,034,325,1991
[81]N. Prentice. Comparison of malts for nuclease and nucleo-base potentials. Journal of the American Society of Brewing Chemists,1983,41 (4):133～144
[82]N. Prentice, Characterization of a Nuclease from Malted Barley Roots. Journal of Cereal Science,1987 (5):175-187
[83]Laufer L, Gutcho S. Hydrolysis of RNA to 5'-nucleotide by seed sprouts, particularly malt sprouts. Biotechnology and Bioengineering,1968,10:257-275
[84]Tanekawa T; Takashima H; Hachiya T. US Patent 4303,680,1981
[85]吴应文.大麦芽中酸性磷酸酯酶的分离及性质.兰州大学学报,1992,28(4)：117-119
[86]李德莹,邱蔚然,丁庆豹.麦芽根中5'-磷酸二酯酶的制备及性质.华东理工大学学报,2002,28(4)：376-379
[87]陈洁,王璋.麦芽根中核酸酶的提取及制备.食品工业科技,2000,21(1)：19-21
[88]李祥.5'-磷酸二酯酶的制备及其在酵母抽提物生产中的应用.中国调味品,2000,4：12-14
[89]陈洁,王璋.麦芽根中核酸酶的分离纯化及性质研究.无锡轻工大学学报,2001,20(3)：270-274
[90]段作营,刘军昌,于瑞嵩,毛忠贵.麦芽根中5'-磷酸二酯酶的提取及制备.中国调味品,2003,2：36-39
[91]Beluhan S, Karmelic I, Novak S, Maric V. Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts. BiotechnologyLetters,2003,25:1099-1103
[92]Pietrzak M, Cudny H, Maluszynski Miroslaw. Purification and properties of two ribonucleases and a nuclease from barley seeds. Biochimica et Biophysica Acta,1980,614:102-112
[93]Deoda A J, Singhal R S.5'-Phosphodiesterase (5'-PDE) from germinated barley for hydrolysis of RNA to produce flavour nucleotides. Bioresource Technology, 2003,88:245-250
[94]Benaiges M D, Lopez-Santin J, Sola C. Partial purification of 5'-phosphodiesterase activity from barley rootlets. Enzyme and Microbial Technology,1989,11(7):444-451
[95]Dhule S S, Shetty P R, Iyer J L, Singhal R S.Purification and characterization of 5'-phosphodiesterase from germinated barley. Process Biochemistry,2006, 41:1899-1902
[96]Strater N. Ecto-5'-nucleotidase:Structure function relationships. Purinergic Signalling,2006.2:343-350
[97]Valerio A A, Corradini A C, Panunto P C, Mello S M, Hyslop S. Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom, Journal of Protein Chemistry,2002,21:495-503
[98]Mori N, Nikai T, Sugihara H. Phosphodiesterase from the venom of crotalus ruber rubber. IntJ. Biochem.,1987,19(2):115-119
[99]罗艳萍,陈远志,黎肇炎.短尾蝮蛇毒磷脂酶A2的分离纯化及抗血小板聚集作用.蛇志,2009,21(2)：90-93
[100]Ouyang C, Huang T F. Inhibition of platelet aggregation by 5-nucleotidase purified from Trimeresurus gramineus snake venom. Toxicon,1983,21 (4): 491-501.
[101]余晓东,黄立农,熊郁良.竹叶青(Trimeresurus Stejnegeri)蛇毒5-核苷酸酶对血小板聚集功能的抑制机制研究.重庆师范学院学报(自然科学版),1997,14(2)：61-68
[102]Dhananjaya BL, Nataraju A, Rajesh R, Gowda CDR, Sharath BK,Vishwanath BS, D'Souza CJM. Anticoagulant effect of Naja naja venom 5-nucleotidase: demonstration through the use of novel specific inhibitor, vanillic acid. Toxicon, 2006.48 (4):411-421
[103]陈夏,余晓东,邓敏,李卉,林亦心,和七一,柳建平.白唇竹叶青蛇毒5'-核苷酸酶的分离纯化及性质.动物学研究,2008,29(4)：399-404
[104]Fujimoto H, Fujiyama K, Midorikawa Y, Fujishima T, Kuninaka A, Yoshino H. Production of 5'-PDE from Aspergillus niger. Agricultural and Biological Chemistry,1977,41:737-74
[105]Taketo Y, Taketo A. Phosphoesterases of hemolytic Streptococci. The Japanese Journal of Experimental Medicine,1974,44:291-299.
[106]Fitt PS, Baddoo P. (1978) Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum. The Biochemical Journal,181,347-353.
[107]Ito K, Yamamoto T, Minamiura N. Phosphodiesterase I in Human Urine: Purification and Characterization of the Enzyme.J. Biochem.,1987, 102:359-367
[108]Wilson, C.M. Plant Nucleases. I. Separation and Purification of Two Ribonucleases and One Nuclease from Corn. Plant Physiol.,1968,43: 1332-1338
[109]Wilson C.M. Purification of a corn ribonuclease. J. Biol. Chem.1967,242: 2260-2263
[110]Wilson C.M. Plant Nucleases. II. Properties of Com Ribonucleases I and II and Com Nuclease I., Plant Physiol.,1968,43:1339-1346
[111]Torti G, Mapelli S, Soave C. Acid ribonuclease from wheat germ:Purification, properties and specificity. Biochim Biophys Acta,1973,324:254-266
[112]Wyen NV, Erdei S, Farkas GL. Isolation from Avena leaf tissues of a nuclease with the same type of specificity towards RNA and DNA. Accumulation of the enzyme during leaf senescence. Biochim Biophys Acta.,1971,232(3):472-483
[113]Barker GR, Bray CM, Walter TJ. The Development of Ribonuclease and Acid Phosphatase During Germination of Pisum arvense. Biochem. J.,1974,142: 211-219
[114]Jervis L. Partial purification and characterization of two Nicotiana tabacum leaf ribonucleases, Phytochemistry,1974,13:709-714
[115]Noguchi S, Shimura G, Kimura K, Samejima H. Production of 5'-mononu cleotides using immobilized 5'-phosphodiesterase and 5'-AMP deaminase. Journal of Solid-phase Biochemistry,1976,1 (2):105-118
[116]张德安,年燕兰,张今.固定化5'-磷酸二酯酶的制备.吉林大学自然科学学报,1979,1：74-76
[117]袁中一,刘树煌,汪静英.固定化核酸酶P1应用于5'-核苷酸生产.科学通报,1980,25(14)：654-657
[118]Rokugawa K, Fujishima T, Kuninaka A. Studies on immobilized enzymes(I):Immobilization of nuclease P1 on cellulose. Journal of Fermentation Technology,1979,57:570-573
[119]Rokugawa K, Fujishima T, Kuninaka A. Immobilized enzymes (Ⅱ): Immobilization of nuclease P 1 on ion exchange resins by titanium complex method. Journal of Fermentation Technology,1980,58:423-429
[120]Rokugawa K, Fujishima T, Kuninaka A. Studies on immobilized enzymes (Ⅲ): Immobilization of nuclease P 1 on inorganic supports by titanium complex method. Journal of Fermentation Technology,1980,58:509-515
[121]Chen WP. Use of immobilized enzymes for the Production of 5'-nucleotides from yeast RNA(Ⅰ):Immobilization of 5'-phosphodiesterase.Kexue Fazhan Yuekan,1983, 11(3):228-235
[122]Lo MC, Chang HS, Chen WP. Studies on immobilization of 5'-Phosphod iesterase on chitin, Shipin Kexue (Taipei, Taiwan),1985,12(3-4):151-162
[123]Suh W.C, Lim B.S, Chun M. Immobilization of nuclease P1 from Penicillium citrinum and Production of 5'-nucleotides by bioreactor. Han'guk Saenghwa Hakhoechi,1987,20(1):17-23
[124]Keller R., Schlingmann M, Woernle R. Production of 5'-ribonucleotides using immobilized 5'-phosphodiesterase. Methods in Enzymology,1987, 136:517-522
[125]刘红,潘红春,丁洪,王晓兰.核酸酶P1固定化研究.食品与发酵工业,1994,4：44-48
[126]Olmedo F, Iturbe F, Gomez-Hemandez J. Continuous production of 5'-ribo nucleotides from yeast RNA by hydrolysis with immobilized 5'-phosphod iesterase and 5'-adenylate deaminase. Enzyme Microb technol.,1994, 10(1):36-40
[127]Serrat JM, Benaiges MD, Lopez-santin J, Immobilization of 5'-Phosphodiesterase from vegetal origin by covalent binding on activated Celite.Biocatalysis,1992,6(1):51-59
[128]杨大令,高涵,张守海,吴迪,蹇锡高.固定化酶膜中凝胶层膜的制备与性能研究.化工时刊,2008,22(5)：1-4
[129]Manoharan M, Schwille RM, Schwille PO. Adenosine nucleotides in rat bone measured by ion-pair reversed-phase high-performance liquid chromatography: effect of hemorrhagic shock,with and without retransfusion of blood. Journal of Chromatography B,2001,761:159-166
[130]Pierro D Di, Tavazzi B, Perno CF, Bartolini M, Balestra E, Calio R, Giardina B, Lazzarino G. An Ion-Pairing High-Performance Liquid Chromatographic Method for the Direct Simultaneous Determinationof Nucleotides, Deoxynucleotides, Nicotinic Coenzymes, Oxypurines, Nucleosides, and Bases in Perchloric Acid Cell Extracts, Analytical Biochemistry,1995,231:407-412
[131]Zheng YF, Xu GW, Yang J, Zhao XJ, Pang Tao, Kong HW. Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography, Journal of Chromatography B,2005,819:85-90
[132]Gill BD, Indyk HE. Development and application of a liquid chromatographic method for analysis of nucleotides and nucleosides in milk and infant formulas. International Dairy Journal,2007,17:596-605
[133]Lopez SLB, Moal J, Serrano FSJ. Development of a method for the analysis of nucleotides from the mantle tissue of the mussel Mytilus galloprovincialis, Journal of Chromatography A,2000,891:99-107
[134]Guo FQ, Li A, Huang LF, Liang YZ, Chen BM. Identification and determination of nucleosides in Cordyceps sinensis and its substitutes by high performance liquid chromatography with mass spectrometric detection, Journal of Pharmaceutical and Biomedical Analysis,2006,40:623-630
[135]Gilar M, Fountain KJ, Budman Y, Neue U D, Yardley KR, Rainville PD, Russell RJ, Gebler JC. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:Retention prediction. Journal of Chromatography A,2002,958:167-182
[136]Gao JL, Leung KSY, Wang YT, Lai CM, Li SP, Hu LF, Lu GH, Jiang ZH, Yu ZL. Qualitative and quantitative analyses of nucleosides and nucleobases in Ganoderma spp. by HPLC-DAD-MS. Journal of Pharmaceutical and Biomedical Analysis,2007,44:807-811
[137]Ferreira IMPLVO, Mendes E, Gomes AMP, Faria MA, Ferreira MA. The determination and distribution of nucleotides in dairy products using HPLC and diode array detection. Food Chemistry,2001,74:239-244
[138]霍小敏,屠春燕,宋慧敏,欧阳平凯.HPLC法测定酵母RNA降解的4种核苷酸.南京工业大学学报,2002,24(4)：61-64
[139]黄晓兰,李科德,陈云华.15种核酸水解产物的高效液相色谱分离及其在酵母抽提物分析中的应用.分析化学,2000,28(12)：1504-1507
[140]Huang H X, Zhang YK, Lu P. Z. Chromatography,1992,10:25
[141]赵红玲,贾乃堃,刘朵花,黎高沃.一种利用麦芽根复合磷酸酯酶制备5’-核苷酸的方法,中国专利,CN200710063910.4
[142]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry,1976,72:248-254.
[143]李建武,萧能庚,余瑞元,袁明秀,陈丽蓉,陈雅蕙,陈来同.生物化学实验原理和方法.北京大学出版社,1994,
[144]Yarnell A.The power of promiscuity: enzymes'ability to catalyze alternative reactions may provide a springboard for evolution. Chem Eng News 2003; 81:33-35
[145]O'Brien PJ, Herschlag D. Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase. Biochemistry,2001,40:5691-5699
[146]Krajewska B. Application of chitin-and chitosan-based materials for enzyme immobilizations:a review. Enzyme and Microbial Technology,2004,35: 126-139
[147]宋建彬,任孝修.以壳聚糖为载体固定化青霉素酰化酶的研究.化工进展,2004,23(2)：181-184
[148]Wei X, Zhang M, Gorski W. Coupling the lactate oxidase to electrodes by ionotropic gelation of biopolymer. Anal Chem,2003,75:2060-2064
[149]梁足培,桑明心,李建,曹长青,王广建.戊二醛交联壳聚糖球固定化脲酶的制备.青岛科技大学学报,2006,27(2)：123-126
[150]王永健,孙彦.适于蛋白质吸附的交联壳聚糖树脂的制备.天津大学学报,2001,34(6)：819-822
[151]唐振兴,石陆娥,钱俊青.壳聚糖凝胶吸附蛋白质机理研究.精细化工2004,21(11)：833-836
[152]刘满英,王春霞,刘杏恋,孙领霞.壳聚糖吸附提取分离蛋白质.离子交换与吸附,1997,15(5)：525-531
[153]Yu YH, He BL. A new type of AL SS-The preparation of crosslinked chitosan resin and its adsorption properties for bilirubin. Reactive & Functional Polymers,1996,31 (3):195～200.
[154]余艺华,孙彦,何炳林.交联壳聚糖树脂的制备工艺及性能表征.天津大学学报,2000,33(1)：113-117
[155]姜忠义,陈洪钫.酶膜反应器研究进展.高分子材料科学与工程,2004,20(1)：14-17
[156]杨利敏,王亚明,张松,唐红玉,钟莉.酶膜反应器的应用研究.化工时刊,2005,19(5)：62-65
[157]石陆娥,应国清,唐振兴,易喻,熊文悦.酶膜生物反应器中酶的固定化方法研究及其应用进展.药物生物技术,2006,13(4)：310-314
[158]陈静,张和平.膜分离式酶解反应器的研究及其在食品工业上的应用.内蒙古农业大学学报,2004,25(2)：124-126
[159]孪雪辉,王乐夫.新型反应器一酶膜反应器.天然气化工,2001,26：48-52
[160]Sethi S, Wiesner MR, Modeling of transient permeate flux in cross-flow membrane filtration incorporating multiple particle transport mechanisms, Journal of Membrane Science,1997,136(1-2):191-205
[161]Lahoussine-Turcaud V, Fouling in tangential-flow ultrafitration:The effect of colloid size and coagulation pretreatment, Membrane Science,1990,52 (2): 173-190
[162]Field RW, Wu D, Howell JA, Critical flux concept for micro filtration fouling, Journal of Membrane Science,1995,100(3):259～272
[163]John A, Sub-critical flux operation of microfiltration, Journal of Membrane Science,1995,107(1-2):165～171
[164]Thomassen JK, Faraday DBF, Underwood BO, The effect of varying transmembrane pressure and crossflow velocity and the microfiltration fouling of a model beer, Separation and Purification Technology,2005,41(1):91-100
[165]Defiance L, Jafrin MY,Comparison between filtration at fixed transmembrane pressure and fixed permeate flux:Application to a membrane bioreactor used for wastewater treatment, Journal of Membrane Science,1999,152(2):203-210
[166]Ueda T, Hata K, Kiknoka Y, Effect of aeration on suction pressure in a submerged membrane bioreactor, Water Research,1997,31(3):489-494
[167]Choo KH,Lee CH,Membrane fouling mechanisms in the membrane-coupled anaerobic bioreactor, Water Research,1996,30(8):1771-1780
[168]Choo KH,Lee CH,Effect of anaerobic digestion broth composition in membrane permeability, Water Science and Technology,1996,34(9):173-179
[169]Cihlar T, Rosenberg I. Efficient separation of natural ribonucleotides by low-pressure anion-exchange chromatography. J.Chromatog,1993,644(2): 299-305.
[170]肖林平,徐正军,何明芳.NH-1分离5'-核苷酸的研究.离子交换与吸附,2003,19(5)：430-436
[171]李黎,莫晓燕,陈雪玉.混合5'-核苷酸的分离与检测.中国医药指南,2010,8(21)：45-48