氨基酰化酶法拆分制备手性氨基酸
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摘要
氨基酸是构成蛋白质的基础，同时也是一种十分重要的营养物质，它在生命活动中起着举足轻重的作用。
在传统的20种天然氨基酸中，除没有手性中心的甘氨酸外，其他19种氨基酸均为L构型。随着研究的不断深入，越来越多非天然构型—D型氨基酸的作用被发现，它们的需求也随之产生。对于L型和D型都有利用价值的氨基酸，化学合成结合手性拆分的方法，具有低成本、适于大规模生产等优点。而高效、绿色、安全的氨基酰化酶法拆分，是拆分氨基酸的最佳方法之一。
蛋氨酸和苯丙氨酸，都是人体必需的氨基酸。蛋氨酸是一种重要的饲料添加剂。L-苯丙氨酸是合成抗病毒和抗癌药物及人工甜味剂的原料，D-苯丙氨酸能增强人体的免疫功能，具有出色的镇痛作用。
首先，选取了氨基酰化酶含量较为丰富且方便易得的米曲霉3042作为酶源。通过硫酸铵分级沉淀、Sephadex G50凝胶层析和DEAE-Sepharose阴离子交换层析从米曲霉3042中提取到了米曲氨基酰化酶，该酶的纯化倍数为54.29，比活为647.66U／mg，总收率为49.53％。以N-乙酰-DL-蛋氨酸为底物的酶促反应最适pH为7.5-8.0，最适反应温度随催化反应时间的延长而降低，缓冲体系中的离子对酶活有抑制作用，而低浓度的CO~(2+)对酶活有激活作用。
将游离酶用于拆分Met和Phe。最佳拆分条件和拆分结果分别为：
Met：反应温度为37℃；pH7.5；Co~(2+)浓度为5×10~(-4)mol／L；初始底物浓度为0.3mol／L。产品：L-Met，％O.P=96.2％，收率70.5％；D-Met，％O.P=95.3％，收率50.5％。
Phe：反应温度为37℃；pH7.0；Co~(2+)浓度为5×10~(-4)mol／L；初始底物浓度为0.2mol／L。产品：L-Phe，％O.P=98.6％，收率68.2％；D-Phe，％O.P=97.1％，收率60.1％。
之后，采用了离子交换凝胶吸附法和海藻酸钙微胶囊包埋法对酶进行固定化。
吸附法所选用的固定化介质是国产DEAE-Sepharose凝胶。研究表明，固定化DSA的热稳定性明显优于游离酶。利用DSA柱进行Met的连续拆分，稳定工作30天后，酶活仍保留60％以上。经初步估算，氨基酰化酶固定化后，酶活利用率提高10倍以上，具有良好的工业应用前景。
包埋有氨基酰化酶的海藻酸钙凝胶珠分别与聚阳离子PMCG和PDMDACC形成微胶囊。对于本体系，两种聚阳离子具有相似的效果。两种微胶囊酶的固定化率都较高，同时酶的泄漏问题也得到了较好的解决。但是随着反应次数的增加，
浙江大学硕士学位论文
摘要
微胶囊的操作稳定性随之下降。在应用之前，还需解决微胶囊的易损坏问题。
最后，用明胶和戊二醛固定化液态发酵得到的含有胞内酶的米曲霉菌。比较
了游离菌体和固定化菌体的性质，结果显示固定化后菌体的稳定性明显提高。
Amino acids are the basic building blocks for proteins and nutritionally important key compounds. They play significant roles in the life. Among 20 basic amino acids, glycine is the only one not exhibiting chirality, and other 19 are all L-amino. acids. With the development of amino acids industry, the functions of D-amino acids are being discovered. It is economical to produce amino acids in large scale by chemical synthesis and chiral resolution as both L- and D- amino acids are valuable. And resolution by aminoacylase is one of the best methods in the resolution of amino acids. It is a high efficient, green and safe method.
Methionine and Phenylalanine are essential amino acids for human development. Methionine is one of the most important feed supplements. L-phenylalanine can be used to synthesis new medicament and artificial sweetener (aspartame). D-phenylalanine has analgesic activity.
Firstly, Aspergillus oryzae 3042 which produces higher aminoacylase was chosen for the production of aminoacylase in this research. The aminoacylase from Aspergillus oryzae 3042 was partially purified by ammonium sulfate fractionation, cloumn chromatography on Sephadex G50 and DEAE-Sepharose. The specific activity of aminoacylase was 647.66U/mg. The purification ratio and recovery was 54.29 and 49.53% respectively. The optimal pH of aminoacylase was 7.5-8.0, and with the increase of catalysis time, the optimal reaction temperature decreased correspondingly. The ions in the buffer lowered the activity of aminoacylase, but the Co2+ in low concentration activated aminoacylase.
Optimal conditions and results of optical resolution of Met and Phe by free aminoacylase are as follows respectively:
Met:Temperature 37℃; pH7.5; Concentration of Co2+ 5 X 10'4mol/L; Concentration of initial substrate 0.3mol/L. Products: L-Met, %O.P=96.2%, yields 70.5 %; D-Met, %O.P=95.3%, yields 50.5 %.
Phe:Temperature 37℃; pH7.0; Concentration of Co2+ 5 X 10'4mol/L; Concentration of initial substrate is 0.2mol/L. Products: L-Phe, %O.P=98.6%, yields 68.2%; D-Met, %O.P=97.1%, yields 60.1 %.
Secondly, aminoacylase was immobilized by two methods, ionic binding to DEAE-Sepharose(DSA) and entrapping into Alginate-PMCG/PDMDAAC microcapsules.
Domestic DEAE-Sepharose was used to decrease the cost of the production. The stability of DSA was much better than that of the free aminoacylase. Then continuous resolution of Met was performed by DSA column. Over 60% of the aminoacylase activity remained after being operated for 30days. Generally, total amounts of the products obtained by DSA were 10 folds more than that by free aminoacylase. It is suitable to be potentially applied in the industrial production of chiral amino acids.
The leakage of aminoacylase., operation stability and properties of Alginate-PMCG microcapsules and Alginate-PDMDAAC microcapsules are quite similar. The operation stability of microcapsules was decreased as the operation times increased. But the breakage of microcapsules should be solved before application.
Finally, Aspergillus oryzae 3042 was immobilized in gelatin, then crosslinked by glutaraldehyde. The enzymic characteristics of the immobilized cells were also studied. Optimal pH and temperature of the free and immobilized pellets were determined. The immobilized cells were more stable over broader temperature and pH ranges. In addition, the immobilized cells showed stable activity under operation and storage conditions.
在传统的20种天然氨基酸中，除没有手性中心的甘氨酸外，其他19种氨基酸均为L构型。随着研究的不断深入，越来越多非天然构型—D型氨基酸的作用被发现，它们的需求也随之产生。对于L型和D型都有利用价值的氨基酸，化学合成结合手性拆分的方法，具有低成本、适于大规模生产等优点。而高效、绿色、安全的氨基酰化酶法拆分，是拆分氨基酸的最佳方法之一。
蛋氨酸和苯丙氨酸，都是人体必需的氨基酸。蛋氨酸是一种重要的饲料添加剂。L-苯丙氨酸是合成抗病毒和抗癌药物及人工甜味剂的原料，D-苯丙氨酸能增强人体的免疫功能，具有出色的镇痛作用。
首先，选取了氨基酰化酶含量较为丰富且方便易得的米曲霉3042作为酶源。通过硫酸铵分级沉淀、Sephadex G50凝胶层析和DEAE-Sepharose阴离子交换层析从米曲霉3042中提取到了米曲氨基酰化酶，该酶的纯化倍数为54.29，比活为647.66U／mg，总收率为49.53％。以N-乙酰-DL-蛋氨酸为底物的酶促反应最适pH为7.5-8.0，最适反应温度随催化反应时间的延长而降低，缓冲体系中的离子对酶活有抑制作用，而低浓度的CO~(2+)对酶活有激活作用。
将游离酶用于拆分Met和Phe。最佳拆分条件和拆分结果分别为：
Met：反应温度为37℃；pH7.5；Co~(2+)浓度为5×10~(-4)mol／L；初始底物浓度为0.3mol／L。产品：L-Met，％O.P=96.2％，收率70.5％；D-Met，％O.P=95.3％，收率50.5％。
Phe：反应温度为37℃；pH7.0；Co~(2+)浓度为5×10~(-4)mol／L；初始底物浓度为0.2mol／L。产品：L-Phe，％O.P=98.6％，收率68.2％；D-Phe，％O.P=97.1％，收率60.1％。
之后，采用了离子交换凝胶吸附法和海藻酸钙微胶囊包埋法对酶进行固定化。
吸附法所选用的固定化介质是国产DEAE-Sepharose凝胶。研究表明，固定化DSA的热稳定性明显优于游离酶。利用DSA柱进行Met的连续拆分，稳定工作30天后，酶活仍保留60％以上。经初步估算，氨基酰化酶固定化后，酶活利用率提高10倍以上，具有良好的工业应用前景。
包埋有氨基酰化酶的海藻酸钙凝胶珠分别与聚阳离子PMCG和PDMDACC形成微胶囊。对于本体系，两种聚阳离子具有相似的效果。两种微胶囊酶的固定化率都较高，同时酶的泄漏问题也得到了较好的解决。但是随着反应次数的增加，
浙江大学硕士学位论文
摘要
微胶囊的操作稳定性随之下降。在应用之前，还需解决微胶囊的易损坏问题。
最后，用明胶和戊二醛固定化液态发酵得到的含有胞内酶的米曲霉菌。比较
了游离菌体和固定化菌体的性质，结果显示固定化后菌体的稳定性明显提高。
Amino acids are the basic building blocks for proteins and nutritionally important key compounds. They play significant roles in the life. Among 20 basic amino acids, glycine is the only one not exhibiting chirality, and other 19 are all L-amino. acids. With the development of amino acids industry, the functions of D-amino acids are being discovered. It is economical to produce amino acids in large scale by chemical synthesis and chiral resolution as both L- and D- amino acids are valuable. And resolution by aminoacylase is one of the best methods in the resolution of amino acids. It is a high efficient, green and safe method.
Methionine and Phenylalanine are essential amino acids for human development. Methionine is one of the most important feed supplements. L-phenylalanine can be used to synthesis new medicament and artificial sweetener (aspartame). D-phenylalanine has analgesic activity.
Firstly, Aspergillus oryzae 3042 which produces higher aminoacylase was chosen for the production of aminoacylase in this research. The aminoacylase from Aspergillus oryzae 3042 was partially purified by ammonium sulfate fractionation, cloumn chromatography on Sephadex G50 and DEAE-Sepharose. The specific activity of aminoacylase was 647.66U/mg. The purification ratio and recovery was 54.29 and 49.53% respectively. The optimal pH of aminoacylase was 7.5-8.0, and with the increase of catalysis time, the optimal reaction temperature decreased correspondingly. The ions in the buffer lowered the activity of aminoacylase, but the Co2+ in low concentration activated aminoacylase.
Optimal conditions and results of optical resolution of Met and Phe by free aminoacylase are as follows respectively:
Met:Temperature 37℃; pH7.5; Concentration of Co2+ 5 X 10'4mol/L; Concentration of initial substrate 0.3mol/L. Products: L-Met, %O.P=96.2%, yields 70.5 %; D-Met, %O.P=95.3%, yields 50.5 %.
Phe:Temperature 37℃; pH7.0; Concentration of Co2+ 5 X 10'4mol/L; Concentration of initial substrate is 0.2mol/L. Products: L-Phe, %O.P=98.6%, yields 68.2%; D-Met, %O.P=97.1%, yields 60.1 %.
Secondly, aminoacylase was immobilized by two methods, ionic binding to DEAE-Sepharose(DSA) and entrapping into Alginate-PMCG/PDMDAAC microcapsules.
Domestic DEAE-Sepharose was used to decrease the cost of the production. The stability of DSA was much better than that of the free aminoacylase. Then continuous resolution of Met was performed by DSA column. Over 60% of the aminoacylase activity remained after being operated for 30days. Generally, total amounts of the products obtained by DSA were 10 folds more than that by free aminoacylase. It is suitable to be potentially applied in the industrial production of chiral amino acids.
The leakage of aminoacylase., operation stability and properties of Alginate-PMCG microcapsules and Alginate-PDMDAAC microcapsules are quite similar. The operation stability of microcapsules was decreased as the operation times increased. But the breakage of microcapsules should be solved before application.
Finally, Aspergillus oryzae 3042 was immobilized in gelatin, then crosslinked by glutaraldehyde. The enzymic characteristics of the immobilized cells were also studied. Optimal pH and temperature of the free and immobilized pellets were determined. The immobilized cells were more stable over broader temperature and pH ranges. In addition, the immobilized cells showed stable activity under operation and storage conditions.
引文
1 沈同，王镜岩主编．生物化学(第二版)上册 北京，高等教育出版社．1990，pp79．
2 Chambers I, Frampton J, Goldfarb P, Affara N, McBain W, Harrison PR. The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA. EMBO J, 1986, 5(6): 1221～1227.
3 Zinoni F, Birkmann A, Stadtman TC, Bock A. Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc Natl Acad Sci U S A, 1986, 83(13): 4650～4654.
4 Srinivasan G, James C M, Krzycki J A. Pyrrolysine encoded by UAG in archaea: Charging of a UAG-decoding specialized tRNA. Science, 2002, 296:1459～1462.
5 Hao B, Gong W, Ferguson T K. A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science, 2002, 296: 1462～1466.
6 梅乐和，姚善泾，关怡新，林东强．D-氨基酸的生物转化与拆分技术研究的新进展．中国医药工业杂志，1999，5：235～238．
7 王大慧，韦萍，欧阳平凯．D—色氨酸研究进展．化工进展，2002，21(2)：103～105，115．
8 柴多里．D-色氨酸．精细与专用化学品，2003，11(5)：24．
9 王颖，李云政．D—丙氨酸的用途及制备方法．辽宁化工，2003，32(2)：58～60．
10 齐欣，赵温涛，聂建明，汤红梅．DL—苯丙氨酸合成新方法．化学工业与工程，2002，19(5)：394～397．
11 Ikeda M. Amino acid production processes. Adv Biochem Eng Biotechnol,2003,79:1～35.
12 陈燕珠．氨基酸的应用与发展趋势．化工时刊，2001,15(7)：4～7．
13 戴伟国．中国氨基酸工业生产现状差距及今后方向．中国制药信息，2002，18(8)：10～12．
14 徐铮奎．20世纪世界氨基酸工业回顾与展望．中国制药信息，2001，17(3)：1～4．
15 徐铮奎．我国氨基酸工业现状与前景展望．中国制药信息，2000，16(1)：3～5．
16 毛忠贵．国际氨基酸产业发展动态．中国食品添加剂，2001(1)：55～60．
17 http://www.ajinomoto.com
18 董占华．蛋氨酸的生产及应用．科技情报(吉林)，1998(2)：28～31．
19 温占玺．蛋氨酸的制备和应用．化工之友，1998(3)：29．
20 王敏．L—苯丙氨酸开发前景诱人．化工生产与技术，2003，10(1)．23．
21 Kitade T, Odahara Y, Stfinohara S, Ikeuchi T, Sakai T, Morikawa K, Minamikawa M, Toyota S, Kawachi A, Hyodo M. Studies on the enhanced effect of acupuncture analgesia and acupuncture anesthesia by D-phenylalanine (2nd report)--schedule of administration and clinical effects in low back pain and tooth extraction. Acupunct Electrother Res, 1990,15(2): 121～135.
22 杨友泌，罗明富，李兰芳，裴玉珍，王萍萍．艾灸和D-苯丙氨酸对小鼠移植性肿瘤-180的抑制作用观察．中国针炙，1991，2：37～38．
23 Brown JL, Roberts WK. Evidence that approximately eighty per cent of the soluble proteins from Ehrlich ascites cells are Nalpha-acetylated. J Biol Chem, 1976,251(4):1009～1014.
24 Cook RM, Burke B J, Buchhagen DL, Minna JD, Miller YE. Human arninoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer. J Biol Chem, 1993,268:17010～17017.
25 Uttamsingh V, Keller DA, Anders MW. Acylase I-catalyzed deacetylation of N-Acetyl-L-cysteine and S-Alkyl-N-acetyl-L-cysteines. Chem Res Toxicol, 1998; 11(7); 800～809.
26 BRENDA: The Comprehensive Enzyme Information System. (http://www.brenda.uni-koeln.de)
27 Lindner H, Hpfner S, Tfler-Naumann M, Miko M, Konrad L.The distribution of aminoacylase Ⅰ among mammalian species and localization of the enzyme in porcine kidney. Biochimie, 2000, 82(2): 129～137.
28 Endo Y. N-Acyl-L-aromatic amino acid deacylase in animal tissues. Biochim Biophys Acta, 1978,523:207～214.
29 D'Adamo AF, Smith JC, Woiler C. The occurrence of N-acetylaspartate amidohydrolase (aminoacylase Ⅱ) in the developing rat. J Neurochem, 1973,20:1275～1278.
30 Szewczuk A. The occurrence of Co~(2+)-activated acylase in animal tissues. Arch Immunol Exp Ther, 1971,19: 389～402.
31 Fujimoto D, Koyama T, Tamyia N. N-Acetyl-β-alanine deacetylase in hog lddney. Biochim Biophys Acta, 1968,167:407～413.
32 Goldstein FB. Aminohydrolases of brain. Enzymatic hydrolysis of N-acetyl-L-aspartate and other N-acetyl-L-amino acids. J Neurochem, 1976,26:45～49.
33 Lugay JC, Aronson JN. Palo Verde (Parldnsonia aculeata L.) seed aminoacylase.
Biochim Biophys Acta, 1969,191(2):397～414.
34 施华芳 黄伟达．水稻氨基酰化酶的分离纯化与性质分析．生物化学杂志，1997，13(1)：54～58．
35 Chibata I, Ishikawa T, Yarnada S. L-amino acid production by aminacylase from Aspergillus. Bull Agr Chem Soc Japan, 1957, 21:300～303.
36 Tosa T, Mori T, Fuse N, Chibata I. Studies on continuous enzyme reactions I.Sereening of carriers for preparation of water-insoluble aminoaeylase. Enzymologia, 1966,31:214～224.
37 Yang YB, Hu, HL, Chang MC, Li H, Tsai YC. Purification and characterization of L-aminoacylase from Alcaligenes denitrificans DA181. Biosci Biotechnol Biochem, 1994,58:204～205.
38 Cho HY, Tanizawa K, Tanaka H, Soda K. Thermostable aminoacylase from Bacillus thermoglucosidius: Purification and characterization. Agric Biol Chem, 1987,51:2793～2800.
39 Sakanyan V, Desmarez L, Legrain C, Charlier D; Mett I, Kochikyan A, Savchenko A, Boyen A, Falmagne P, Pierard A, Glansdorff N. Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus. Appl Environ Microbiol, 1993,59:3878～3888.
40 Park R.W, Fox S.W. An acylase system related to the utilization of benzoylamino acids by Lactobacillus arabinosus. J Biol Chem, 1960,235:3193～3197.
41 Matsumoto J, Nagai S. Amidohydrolases for N-short and long chain fatty acyl-L-amino acids from Mycobacteria. J Biochem, 1972,72:269～279.
42 Wakayama M, Shiiba E, Sakai, K, Moriguchi M.Purification and characterization of L-aminoacylase from Pseudomonas maltophila B1. J Ferment Bioeng, 1998,85:278～282.
43 Krdel W, Schneider F. Chemical investigations on pig kidney aminoacylase. Biochim Biophys Acta, 1976,445(2):446～457.
44 Krdel W, Selmeider F. Renal aminoacylase, a zinc enzyme. Z Naturforsch [C], 1977,32(5-6): 342～344.
45 Szajani B, Kiss A. Boross L.Investigation of the active center and catalytic mechanism of porcine kidney aminoacylase: a model of the active center. Acta Biochim Biophys Acad Sci Hung, 1980,15(1): 29～37.
46 Wang Z X. Kinetic of the course of inactivation of aminoacylase by 1,10-phenanthroline: Biochem J, 1992,281:285～290.
47张彤，周海梦．锌离子对氨基酰化酶构象及其稳定性的影响．生物物理学报．1994，10(2)：198～202．
48 Krdel W, Schneider E Chemical modification of two tryptophan residues abolishes the catalytic activity of aminoacylase. Hoppe Seylers Z Physiol Chem, 1976, 357(8): 1109～1115.
49 Krdel W, Schneider E Identification of essential histidine residues of aminoacylase by photooxidation and by reaction with diethylpyroearbonate. Z Naturforsch [C], 1977,32(5-6): 337～341.
50 Loffler HG, Schneider F.Inhibition of aminoacylase from hog lddney by 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline. Biol Chem Hoppe Seyler, 1987,368(5): 481～485.
51 周海梦，王希成．氨基酰化酶Ⅰ的必需半胱氨酸巯基．生物化学杂志，1991，7(6)：702～706．
52 Gilles I, Loffier HG, Schneider F. New isolation procedure for swine Kidney acylase. Kinetics of Co~(2+), Mn~(2+), Ni~(2+) and Cd~(2+)-enzymes. Z Naturforsch [C], 1984,39(9-10):1017～1020.
53 张英侠，王希成．锰或镍离子取代的氨基酰化酶性质的研究．清华大学学报(自然科学版)，1993,33(6)：85～90．
54 Gentzen I., Lffler HG, Schneider F. Aminoacylase from Aspergillus oryzae. Comparison with the pig lddney enzyme. Z Naturforsch [C], 1980, 35: 544～550.
55 Wu HB, Tsou CL. A comparison of Zn(Ⅱ) and Co(Ⅱ) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme. Biochem J, 1993, 296: 435～441.
56 Chibata I, Tosa T, Sato T. Preparation and industrial applications of immobilized aminoacylases. Proc.IV IFS: Ferment. Technol. Today, 1972:383～389.
57 李民勤，王道宾．高分子载体对米曲霉氨基酰化酶的固定化研究．离子交换与吸附，1993，9(3)：199～203．
58 李民勤，何炳林．功能基化聚丙烯酸甲酯固定化氨基酰化酶的研究．生物工程学报，1993，9(3)：282～286．
59 王道宾，何炳林．用固定化氨基酰化酶拆分DL-对氯苯丙氨酸．纪学通报，1994(1)：50～52．
60 王晓平，刘一鸣．米曲氨基酰化酶的纯化及固定化酶法拆分D,L-丙氨酸．吉林大学自然科学学报，1998(4)：81～84．
61 张一竹，彭晓．DL-苯丙氨酸酶法拆分．氨基酸和生物资源，1999，21(2)：19～20．
62姜忠义，陈洪钫．苯丙氨酸光学异构体的酶法拆分．应用化学,2001，18(3)：23 1～232．
63姚文兵，吴梧桐．猪肾和牛肾氨基酰化酶拆分DL-Ala及制备D-Ala的实验研究．中国药科大学学报，1997，28(6)：358～361．
64姚文兵，侯振清．固定化牛肾氨基酰化酶拆分法制备D-丙氨酸．中国药科大学学报，2000，31(4)：297～300．
65Meiller F, Mirabel B. Support-aminoacylase complexes. USP 4,132,596. 1979
66Chibata I, Tosa T, Mori T, Fujimura M. Immobilized aminoacylase. USP 4,390,626. 1983
67Szajani B, Kiss J, Ivony J, Huber I, Boros L, Daroczi I. Immobilized aminoacylase enzyme. USP 4,608,340. 1986
68Bódalo Santoyo A, Bastida Rodríguez J, Gómez Carrasco JL, Gómez Gómez E,Alcaraz Rojo I, Asanza Teruel ML. Immobilization of Pseudomonas sp. BA2 by entrapment in calcium alginate and its application for the production of -alanine, Enzyme and Microbial Technology, 1996,19(3): 176～180.
69Bódalo A, Bastida J, Gómez JL, Gómez E, Alcaraz I, Asanza ML. Stabilization studies of aminoacylase-producing Pseudomonas sp. BA2 immobilized in calcium alginate gel. Enzyme and Microbial Technology, 1997,21 (1):64～69.
70张龙翔等编著．生化实验方法和技术(第二版)．北京：高等教育出版社．1997．7，pp163～165．
71李建武等编著．生物化学实验原理和方法．北京：北京大学出版社．1994．9，pp174～176．
72徐克勋主编．精细化工原料及中间体手册．化学工业出版社．1997．
73国家药典委员会编．中华人民共和国药典(2000年版二部)．北京：化学工业出版社．2000．1，pp161．
74国家药典委员会编．中华人民共和国药典(2000年版二部)．北京：化学工业出版社．2000．1，pp364-365．
75Dictionary of Organic Compounds. 6th Ed. Chapman&Hall. 1996. pp5190
76禹邦超，刘德立编著．应用酶学导论．武汉：华中师范大学出版社．1994．12，pp233～234．
77Chibata I, Tetsuya Tosa, Tadashi Sato. Production of L-aminoAcids by aminoacylase absorbed on DEAE-Sephadex. Methods in Enzymology, Academic Press, 1976,44(51):746～759.
78徐东，曹竹安．国产大孔树脂固定氨基酰化酶．清华大学学报(自然科学版)，
1990，30(6)：33～38．
79韩际宏，姜坪．氨基酰化酶的的固定化及其对D，L-蛋氨酸的光学拆分．离子交换与吸附，1993，9(2)：107～113．
80李建武等编著．生物化学实验原理和方法．北京：北京大学出版社．1994．9，pp171～173．
81王淑豪等．米曲霉3042的液态培养特性及其产氨基酰化酶的特性研究．食品科学，2000，20(7)：9～13．
82Scardi V. Immobilization of enzymes and microbial cells in gelatin. Methods of Enzymology, New York, Academic Press. 1987, 135, pp293～299.
2 Chambers I, Frampton J, Goldfarb P, Affara N, McBain W, Harrison PR. The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA. EMBO J, 1986, 5(6): 1221～1227.
3 Zinoni F, Birkmann A, Stadtman TC, Bock A. Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc Natl Acad Sci U S A, 1986, 83(13): 4650～4654.
4 Srinivasan G, James C M, Krzycki J A. Pyrrolysine encoded by UAG in archaea: Charging of a UAG-decoding specialized tRNA. Science, 2002, 296:1459～1462.
5 Hao B, Gong W, Ferguson T K. A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science, 2002, 296: 1462～1466.
6 梅乐和，姚善泾，关怡新，林东强．D-氨基酸的生物转化与拆分技术研究的新进展．中国医药工业杂志，1999，5：235～238．
7 王大慧，韦萍，欧阳平凯．D—色氨酸研究进展．化工进展，2002，21(2)：103～105，115．
8 柴多里．D-色氨酸．精细与专用化学品，2003，11(5)：24．
9 王颖，李云政．D—丙氨酸的用途及制备方法．辽宁化工，2003，32(2)：58～60．
10 齐欣，赵温涛，聂建明，汤红梅．DL—苯丙氨酸合成新方法．化学工业与工程，2002，19(5)：394～397．
11 Ikeda M. Amino acid production processes. Adv Biochem Eng Biotechnol,2003,79:1～35.
12 陈燕珠．氨基酸的应用与发展趋势．化工时刊，2001,15(7)：4～7．
13 戴伟国．中国氨基酸工业生产现状差距及今后方向．中国制药信息，2002，18(8)：10～12．
14 徐铮奎．20世纪世界氨基酸工业回顾与展望．中国制药信息，2001，17(3)：1～4．
15 徐铮奎．我国氨基酸工业现状与前景展望．中国制药信息，2000，16(1)：3～5．
16 毛忠贵．国际氨基酸产业发展动态．中国食品添加剂，2001(1)：55～60．
17 http://www.ajinomoto.com
18 董占华．蛋氨酸的生产及应用．科技情报(吉林)，1998(2)：28～31．
19 温占玺．蛋氨酸的制备和应用．化工之友，1998(3)：29．
20 王敏．L—苯丙氨酸开发前景诱人．化工生产与技术，2003，10(1)．23．
21 Kitade T, Odahara Y, Stfinohara S, Ikeuchi T, Sakai T, Morikawa K, Minamikawa M, Toyota S, Kawachi A, Hyodo M. Studies on the enhanced effect of acupuncture analgesia and acupuncture anesthesia by D-phenylalanine (2nd report)--schedule of administration and clinical effects in low back pain and tooth extraction. Acupunct Electrother Res, 1990,15(2): 121～135.
22 杨友泌，罗明富，李兰芳，裴玉珍，王萍萍．艾灸和D-苯丙氨酸对小鼠移植性肿瘤-180的抑制作用观察．中国针炙，1991，2：37～38．
23 Brown JL, Roberts WK. Evidence that approximately eighty per cent of the soluble proteins from Ehrlich ascites cells are Nalpha-acetylated. J Biol Chem, 1976,251(4):1009～1014.
24 Cook RM, Burke B J, Buchhagen DL, Minna JD, Miller YE. Human arninoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer. J Biol Chem, 1993,268:17010～17017.
25 Uttamsingh V, Keller DA, Anders MW. Acylase I-catalyzed deacetylation of N-Acetyl-L-cysteine and S-Alkyl-N-acetyl-L-cysteines. Chem Res Toxicol, 1998; 11(7); 800～809.
26 BRENDA: The Comprehensive Enzyme Information System. (http://www.brenda.uni-koeln.de)
27 Lindner H, Hpfner S, Tfler-Naumann M, Miko M, Konrad L.The distribution of aminoacylase Ⅰ among mammalian species and localization of the enzyme in porcine kidney. Biochimie, 2000, 82(2): 129～137.
28 Endo Y. N-Acyl-L-aromatic amino acid deacylase in animal tissues. Biochim Biophys Acta, 1978,523:207～214.
29 D'Adamo AF, Smith JC, Woiler C. The occurrence of N-acetylaspartate amidohydrolase (aminoacylase Ⅱ) in the developing rat. J Neurochem, 1973,20:1275～1278.
30 Szewczuk A. The occurrence of Co~(2+)-activated acylase in animal tissues. Arch Immunol Exp Ther, 1971,19: 389～402.
31 Fujimoto D, Koyama T, Tamyia N. N-Acetyl-β-alanine deacetylase in hog lddney. Biochim Biophys Acta, 1968,167:407～413.
32 Goldstein FB. Aminohydrolases of brain. Enzymatic hydrolysis of N-acetyl-L-aspartate and other N-acetyl-L-amino acids. J Neurochem, 1976,26:45～49.
33 Lugay JC, Aronson JN. Palo Verde (Parldnsonia aculeata L.) seed aminoacylase.
Biochim Biophys Acta, 1969,191(2):397～414.
34 施华芳 黄伟达．水稻氨基酰化酶的分离纯化与性质分析．生物化学杂志，1997，13(1)：54～58．
35 Chibata I, Ishikawa T, Yarnada S. L-amino acid production by aminacylase from Aspergillus. Bull Agr Chem Soc Japan, 1957, 21:300～303.
36 Tosa T, Mori T, Fuse N, Chibata I. Studies on continuous enzyme reactions I.Sereening of carriers for preparation of water-insoluble aminoaeylase. Enzymologia, 1966,31:214～224.
37 Yang YB, Hu, HL, Chang MC, Li H, Tsai YC. Purification and characterization of L-aminoacylase from Alcaligenes denitrificans DA181. Biosci Biotechnol Biochem, 1994,58:204～205.
38 Cho HY, Tanizawa K, Tanaka H, Soda K. Thermostable aminoacylase from Bacillus thermoglucosidius: Purification and characterization. Agric Biol Chem, 1987,51:2793～2800.
39 Sakanyan V, Desmarez L, Legrain C, Charlier D; Mett I, Kochikyan A, Savchenko A, Boyen A, Falmagne P, Pierard A, Glansdorff N. Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus. Appl Environ Microbiol, 1993,59:3878～3888.
40 Park R.W, Fox S.W. An acylase system related to the utilization of benzoylamino acids by Lactobacillus arabinosus. J Biol Chem, 1960,235:3193～3197.
41 Matsumoto J, Nagai S. Amidohydrolases for N-short and long chain fatty acyl-L-amino acids from Mycobacteria. J Biochem, 1972,72:269～279.
42 Wakayama M, Shiiba E, Sakai, K, Moriguchi M.Purification and characterization of L-aminoacylase from Pseudomonas maltophila B1. J Ferment Bioeng, 1998,85:278～282.
43 Krdel W, Schneider F. Chemical investigations on pig kidney aminoacylase. Biochim Biophys Acta, 1976,445(2):446～457.
44 Krdel W, Selmeider F. Renal aminoacylase, a zinc enzyme. Z Naturforsch [C], 1977,32(5-6): 342～344.
45 Szajani B, Kiss A. Boross L.Investigation of the active center and catalytic mechanism of porcine kidney aminoacylase: a model of the active center. Acta Biochim Biophys Acad Sci Hung, 1980,15(1): 29～37.
46 Wang Z X. Kinetic of the course of inactivation of aminoacylase by 1,10-phenanthroline: Biochem J, 1992,281:285～290.
47张彤，周海梦．锌离子对氨基酰化酶构象及其稳定性的影响．生物物理学报．1994，10(2)：198～202．
48 Krdel W, Schneider E Chemical modification of two tryptophan residues abolishes the catalytic activity of aminoacylase. Hoppe Seylers Z Physiol Chem, 1976, 357(8): 1109～1115.
49 Krdel W, Schneider E Identification of essential histidine residues of aminoacylase by photooxidation and by reaction with diethylpyroearbonate. Z Naturforsch [C], 1977,32(5-6): 337～341.
50 Loffler HG, Schneider F.Inhibition of aminoacylase from hog lddney by 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline. Biol Chem Hoppe Seyler, 1987,368(5): 481～485.
51 周海梦，王希成．氨基酰化酶Ⅰ的必需半胱氨酸巯基．生物化学杂志，1991，7(6)：702～706．
52 Gilles I, Loffier HG, Schneider F. New isolation procedure for swine Kidney acylase. Kinetics of Co~(2+), Mn~(2+), Ni~(2+) and Cd~(2+)-enzymes. Z Naturforsch [C], 1984,39(9-10):1017～1020.
53 张英侠，王希成．锰或镍离子取代的氨基酰化酶性质的研究．清华大学学报(自然科学版)，1993,33(6)：85～90．
54 Gentzen I., Lffler HG, Schneider F. Aminoacylase from Aspergillus oryzae. Comparison with the pig lddney enzyme. Z Naturforsch [C], 1980, 35: 544～550.
55 Wu HB, Tsou CL. A comparison of Zn(Ⅱ) and Co(Ⅱ) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme. Biochem J, 1993, 296: 435～441.
56 Chibata I, Tosa T, Sato T. Preparation and industrial applications of immobilized aminoacylases. Proc.IV IFS: Ferment. Technol. Today, 1972:383～389.
57 李民勤，王道宾．高分子载体对米曲霉氨基酰化酶的固定化研究．离子交换与吸附，1993，9(3)：199～203．
58 李民勤，何炳林．功能基化聚丙烯酸甲酯固定化氨基酰化酶的研究．生物工程学报，1993，9(3)：282～286．
59 王道宾，何炳林．用固定化氨基酰化酶拆分DL-对氯苯丙氨酸．纪学通报，1994(1)：50～52．
60 王晓平，刘一鸣．米曲氨基酰化酶的纯化及固定化酶法拆分D,L-丙氨酸．吉林大学自然科学学报，1998(4)：81～84．
61 张一竹，彭晓．DL-苯丙氨酸酶法拆分．氨基酸和生物资源，1999，21(2)：19～20．
62姜忠义，陈洪钫．苯丙氨酸光学异构体的酶法拆分．应用化学,2001，18(3)：23 1～232．
63姚文兵，吴梧桐．猪肾和牛肾氨基酰化酶拆分DL-Ala及制备D-Ala的实验研究．中国药科大学学报，1997，28(6)：358～361．
64姚文兵，侯振清．固定化牛肾氨基酰化酶拆分法制备D-丙氨酸．中国药科大学学报，2000，31(4)：297～300．
65Meiller F, Mirabel B. Support-aminoacylase complexes. USP 4,132,596. 1979
66Chibata I, Tosa T, Mori T, Fujimura M. Immobilized aminoacylase. USP 4,390,626. 1983
67Szajani B, Kiss J, Ivony J, Huber I, Boros L, Daroczi I. Immobilized aminoacylase enzyme. USP 4,608,340. 1986
68Bódalo Santoyo A, Bastida Rodríguez J, Gómez Carrasco JL, Gómez Gómez E,Alcaraz Rojo I, Asanza Teruel ML. Immobilization of Pseudomonas sp. BA2 by entrapment in calcium alginate and its application for the production of -alanine, Enzyme and Microbial Technology, 1996,19(3): 176～180.
69Bódalo A, Bastida J, Gómez JL, Gómez E, Alcaraz I, Asanza ML. Stabilization studies of aminoacylase-producing Pseudomonas sp. BA2 immobilized in calcium alginate gel. Enzyme and Microbial Technology, 1997,21 (1):64～69.
70张龙翔等编著．生化实验方法和技术(第二版)．北京：高等教育出版社．1997．7，pp163～165．
71李建武等编著．生物化学实验原理和方法．北京：北京大学出版社．1994．9，pp174～176．
72徐克勋主编．精细化工原料及中间体手册．化学工业出版社．1997．
73国家药典委员会编．中华人民共和国药典(2000年版二部)．北京：化学工业出版社．2000．1，pp161．
74国家药典委员会编．中华人民共和国药典(2000年版二部)．北京：化学工业出版社．2000．1，pp364-365．
75Dictionary of Organic Compounds. 6th Ed. Chapman&Hall. 1996. pp5190
76禹邦超，刘德立编著．应用酶学导论．武汉：华中师范大学出版社．1994．12，pp233～234．
77Chibata I, Tetsuya Tosa, Tadashi Sato. Production of L-aminoAcids by aminoacylase absorbed on DEAE-Sephadex. Methods in Enzymology, Academic Press, 1976,44(51):746～759.
78徐东，曹竹安．国产大孔树脂固定氨基酰化酶．清华大学学报(自然科学版)，
1990，30(6)：33～38．
79韩际宏，姜坪．氨基酰化酶的的固定化及其对D，L-蛋氨酸的光学拆分．离子交换与吸附，1993，9(2)：107～113．
80李建武等编著．生物化学实验原理和方法．北京：北京大学出版社．1994．9，pp171～173．
81王淑豪等．米曲霉3042的液态培养特性及其产氨基酰化酶的特性研究．食品科学，2000，20(7)：9～13．
82Scardi V. Immobilization of enzymes and microbial cells in gelatin. Methods of Enzymology, New York, Academic Press. 1987, 135, pp293～299.