摘要
为了获得能用于D-氨基酸工业化生产的D-氨基酰化酶(D-aminoacylase DAA),我们拟从Microbacterium natoriense菌属中分离出DAA,并对其特性进行了分析。通过多步层析分离技术从TNJL143-2菌株中纯化了DAA,应用SDS-PAGE和层析分离技术分析了DAA的纯度、分子量大小和聚体数;利用酶促反应与TLC(薄层层析)及DAO(D-氨基酸氧化酶)技术检测了DAA的催化底物的特异性、最适酶促反应温度和pH值、以及耐受热、酸、碱、高浓度底物以及金属离子和EDTA的能力;通过蛋白质序列分析仪分析了DAA的氨基酸序列;应用巢式PCR技术从菌体DNA中钓取了目的基因,并推定了蛋白开放阅读框架(ORF);对比了TNJL143-2菌株DAA与其它菌株DAA的同源性。结果显示获得的DAA为单聚体,其分子量为56kDa,对于N-乙酰-D-氨基酸底物具有高活性。催化反应的相对活性为46.4 U/mg、最适pH值为7.0、最适温度为50℃、以及在上述条件下反应1小时后仍具有85%残存酶活性。相对耐受Ca2~+、Mn~(2+)、Co~(2+)和Cd~(2+)和EDTA。该DAA的氨基酸序列与来自Alcaligenes xylosoxydans A-6、Alcalygenesfacelis DA1和paradoxus Iso1的DAA分别具有为25%、24%和26%的同源性。表明从Microbacterium natoriense TNJL143-2菌株中分离的DAA具有独特的结构和功能特性,以及潜在的应用价值。
A D-aminoacylase (DAA) was found in a novel Microbacterium natoriense TNJL143-2 strain that isolated from soil in Japan miyagi natrori. The enzyme was purified by multistep chromatography. The native molecular mass was 56kDa, which agree with predicted molecular mass 56 kDa and the enzyme appered to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 46.4 U/mg was finally obtained. The enzyme had an optimal pH and temperature of 7.0 and 50℃, respectively, after 1 hour heat treatment at 45℃at pH 7.0 85% activity remained. The DAA had higher hydrolyzing activity aganst N-acetyl-D-amino acid. After N-terminal and internal peptide sequending by LC/MS/MS, and designing the degenerate primers according to the N-terminal and internal peptide sequence. The gene was cloned and sequenced. The gene consisted of a 1485-bp ORF encoding a polypeptide of 495 amino acid. The M. natorines DAA showed a low amino acid similarity to Alcaligenes xylosoxydans A-6 (25%), Alcalygenes facelis DA1 (24%), and paradoxus Isol (26%).
引文
[1] KREBS, H. A. .CXCVII. Metabolism of amino-acids. 111. Deamination of amino-acids. Biochem. J., 1935, 29:1620-1644
[2] G.C. Barrett, Editor. Chemistry and Biochemistry of the Amino Acids, Chapman and Hall, 1985, p415-425
[3] Fisher GH, Pay an IL, Chou S-J, et al. Racemized D-aspartate in Alzheimer neurofibrillary tangles. Brain Res Bull, 1992a, 28:12713—127131
[4] Fisher GH, D'Aniello A, Vetere A, et al. Quantification of D-aspartic acid in normal and Alzheimer brains. Neurosci Lett, 1992b, 143(1-2):215—218
[5] D'AnielloA, Vetere A, Fisher GH, et al. Presence of D-alanine in proteins of normal and Alzheimer human brain. Brain Res, 1992, 592:44-48
[6] Roher AE, Lowenson JD, Clarke S, et al. Structural alterations in the peptide backbone of amyloid core protein may account for its deposition and stability in Alzheimer' s disease. J Biol Chem, 1993, 268:3072-2083
[7] Fisher GH, Petrucelli L, Gardner C, et al. Free D-amino acids in human cerebrospinal fluid of Alzheimer disease, multiple sclerosis, and healthy control subjects. Molec Chem Neuropathol, 1994, 23:115-124
[8] Martinez M, Frank A, Diez-Tejedor E, et al. Amino acid concentrations in cerebrospinal fluid and serum in Alzheimer's disease and vascular dementia. J Neural Transm, 1993, 6:1—9
[9] Dunlop DS, Neidle A, McHale D, et al. The presence of free D-aspartic acid in rodents and man. Biochemical and Biophysical Research Communications, 1986, 141:27—32
[10] Neidle A & Dunlop DS. Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat. Life Sciences, 1990, 46:1517—1522
[11] Hashimoto A, Nishikawa T, Oka T, et al. Endogenous D-serine in rat brain: N-methyl-D-aspartate receptor-related distribution and aging. J Neurochem, 1993, 60:783—786
[12] Hashimoto A, Oka T and Nishikawa T. Extracellular concentration of endogenous free D-serine in the rat brain as revealed by in vivo dialysis. Neurosci, 1995, 66:635—643
[13] Fujii N, Satoh K, Harada K, et al. Simultaneous stereoinversion and isomerization at specific aspartic acid residues in alpha A-crystallin from human lens. J Biochem, 1994, 116:663—669
[14] Huang, Y., Nishikawa, T., Satoh, K., et al. Urinary excretion of D-serine in human: comparison of different ages and species. Biol. Pharm. Bull, 1998, 21:156—162
[15] Nagata, Y., Masui, R., Akino, T., et al. The presence of free D-serine, D-alanine and D-proline in human plasma. Experientia, 1992, 48:986—988
[16] Johnson, B. J. Miller, G. G. Archeoloigcal application of amino acid racemization. Archaeometry, 1997, 39:265-287
[17] Fujii N, Tajima S, Tanaka N, et al. The presence of D-β-aspartic acid-containing peptides in elastic fibers of sun-damaged skin: a potent marker for ultraviolet-induced skin aging. Biochem Biophys Res Commun, 2002, 294:1047—1051
[18] Powell JT, Vine N, Crossman M. On the accumulation of D-aspartate in elastin and other proteins of the ageing aorta. Atherosclerosis, 1992, 97:201—208
[19] McFadden PN, Clarke S. Methylation at D-aspartyl residues in erythrocytes: possible step in the repair of aged membrane proteins. Proc Natl Acad Sci USA, 1982, 79:2460—2464
[20] Sakai, K., Obata, T., Ideta, K., Moriguchi, M. Purification and properties of D-aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4. J. Ferment. Bioeng, 1991, 71:79—82
[21] Ritz-Timme S, Laumeier I, Collins M. Age estimation based on aspartic acid racemization in elastin from the yellow ligaments. Int J Legal Med, 2003, 117: 96—101
[22] Hashimoto A, Nishikawa T, Oka T, et al. Widespread distribution of free D-aspartate in rat periphery. FEBS Letters, 1993, 331:4—8
[23] Hamase, K., H. Homma, Y. Takigawa, et al. Regional distribution and postnatal changes of D-amino acids in rat brain. Biochim. Biophys. Acta, 1997, 1334:214—222
[24] Kera, Y., Aoyama, H., Watanabe, N., et al. Distribution of D-aspartate oxidase and free D-glutamate and D-aspartate in chicken and pigeon tissues. Comp Biochem Physiol B Biochem Mol Biol, 1996, 115(1): 121-126
[25] Y. Nagata and K. Kubota. A trial to determine D-amino acids in tissue proteins of mice . Amino Acids, 1993, 4(1-2): 121-125
[26] Patrizia Spinelli, Euan R. Brown, Gabriele Ferrandino, et al. D-aspartic acid in the nervous system of Aplysia limacina: Possible role in neurotransmission. Journal of Cellular Physiology, 2005, 206(3): 672-681
[27] Okuma, E., Watanabe, K., Abe, H. Distribution of free D-amino acids in bivalve mollusks and the effects of physiological conditions on the levels of D- and L-alanine in-the tissues of hard clam. Meretrix lusoria. Fish. Sci, 1998, 64:606—611
[28] Preston, R. L. Occurrence of D-amino acids in higher organisms: a survey of the distribution of D-amino acids in marine vertebrates. Comp. Biochem. Physiol, 1987, 87B:55—62
[29] Fujita, E., Okuma, E., Abe, H., et al. Partial purification and properties of alanine racemase from the muscle of black tiger prawn Penaeus monodon. Fish. Sci, 1997, 63:440—445
[30] Sahl, H. G., Bierbaum, G. Lantibiotics. Biosynthesis and biological activities of uniquely modified peptides from Gram-positive bacteria. Annu Rev. Microbiol, 1998, 52:41-79
[31] Nagata, Y., Fujiwara T, Kawaguchi-Nagata K, et al. Occurrence of peptidyl D-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes. Biochim Biophys Acta, 1998, 1379: 76-82
[32] Fangmeier, N., Leistner, E. Conversion of D-lysine into L-lysine via L-pipecolic acid in Nicotiana glauca L. plants and cell-suspension cultures. J. 'Chem. Soc. Perkin Trans, 1981, 1769-1772
[33] Fukuda, M., Tokumura, A., Ogawa, T., et al. D-Alanine in germinating Pisum sativum seedlings. Phytochemistry, 1973, 12:2593-2595
[34] Noma, M., Nauwigewauk, M., Thymic, E., et al. Isolation and characterization of D-alanyl-D-alanine from tobacco leaves. Agric. Biol, 1973, 37:2439
[35] Manabe, H. Formation of dipeptides containing D-alanine in wild rice plants. Phytochemistry, 1992, 31:527-529
[36] Frahn, J. L., Illman, R. J. The occurrence of D-alanine and D-alanyl-D-alanine in Phalaris tuberosa. Phytochemistry, 1975, 14:1464—1465
[37] Bruckner, H., Westhauser, T. Chromatographic determination of D-amino acids as native constituents of vegetables and fruits. Chromatographia, 1994, 39: 419—426
[38] Gandolfi, I., Palla, G., Marchelli, A., et al. D-Alanine in fruit juices: a molecular marker of bacterial activity, heat treatments and shelf life. J. Food Sci, 1994, 59:152—154
[39] Tetsuya Miyamoto, Masae Sekine, Tetsuhiro Ogawa, et al. D-Amino acids detected in proteins synthesized in E. coli. Amino Acids. Published online 19 September 2009
[40] Dunlop DS, Neidle A. The origin and turnover of D-serine in brain. Biochem. Biophys Res Commun, 1997, 235:26-30
[41] Waxman,D.J. and Strominger, J. L. penicillin-binding proteins and the mechanism of action of β-lactam antibiotics Annu. Rev. Biochem, 1983, 52:825—869
[42] Kazuhisa Sekimizu, Jorge Larranaga, Hiroshi Hamamoto, et al. d-Glutamic Acid - Induced Muscle Contraction in the Silkworm, Bombyx mori. Journal of Biochemistry, 2005, 137(2): 199—203
[43] Wang H, Wolosker H, Morris JF, et al. Naturally occurring free D-aspartate is a nuclear component of cells in the mammalian hypothalamo-neurohypophyseal system. Neuroscience, 2002, 109:1—4
[44] D' Aniello A, Di Fiore MM, Fisher GH, et al. Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB Journal, 2000a, 14:699—714
[45] Wolosker H, D' Aniello A, Snyder SH. D-aspartate disposition in neuronal and endocrine tissues: ontogeny, biosynthesis and release. Neuroscience, 2000, 100:183—189
[46] Lee JA, Long Z, Nimura N, et al. Localization, transport, and uptake of D-aspartate in the rat adrenal and pituitary glands. Archives of Biochemistry and Biophysics, 2001, 385:242—249
[47] Enza Topo , Andrea Soricelli, Angela Di Maio, et al. Evidence for the involvement of d-aspartic acid in learning and memory of rat. Amino Acids. 10 October 2009 Published online
[48] K Kiran Kumar. Dental age estimation using amino acid racemization. SHORT COMMUNICATION, 2008, 19(2):172-174
[49]Ohtani,Susumu.Rate of Aspartic Acid Racemization in Bone.The American Journal of Forensic Medicine and Pathology,1998,19(3):284-287
[50]Yuichi Kaji,Tetsuro Oshika,Yutaka Takazawa,et al.Localization of D-β-Aspartic Acid-Containing Proteins in Human Eyes.Investigative Ophthalmology and Visual Science,2007,48:3923-3927
[51]Young G.A.,Kendall S.,Brownjohn A.M.D-Amino acids in chronic renal failure and the effects of dialysis and urinary losses.Amino Acids,1994,6(3):283-293
[52]Armstrong D.W.,Gasper M.P.,Lee S.H.,et al.Factors conirolling the level and determination of D-amino acids in the urine and plasma of laboratory rodents.Amino Acide,1993,5(2):299-315
[53]Wakayama,M.,K.Yoshimune,Y.Hirose,and M.Moriguchi.Production of d-amino acids by N-acyl-d-amino acid amidohydrolase and its structure and function.J.Mol.Catal.B,2003,23:71-85
[54]Julie Soutourina,Sylvain Blanquet,Pierre Plateau.Role of d-Cysteine Desulfhydrase in the Adaptation of Escherichia coli to d-Cysteine.The Journal of Biological Chemistry,2001,276:40864-40872
[55]陈建波,徐毅.D-型氨基酸的生物法制备.分子催化,2006,20(3):284-288
[56]K.Saigoh,K.Matsui and K.Takahashi,et al.The stereo-specific effect of D-serine ethylester and the D-cycloserine in ataxic mutant mice.Brain research,1998,808(1):42-7
[57]Tsai,G.,Yang,P.,Chung,L.C.,et al.D-Serine added to antipsychotics for the treatment of schizophrenia.Biol.Psychiatry,1998,44:1081-1089
[58]董占华.蛋氨酸的生产及应用.科技情报[吉林],1998,2:28-31
[59]封霞,袁静明.D-型氨基酸的生物法制各.微生物学通报,2003,30:95-98
[60]严传鸣,李翔,朱新荣等.2,6一二氯4-三氟甲基苯胺的合成.现代农药,2003,2(5):5-6
[61]方岩雄,王亚莉,熊绪杰等.手性氨基酸对映体的拆分.广东工业大学学报,2002,19(3):7-9
[62]Burkhard W.,Christoph S.,Ralf M.et al.Cloning,nucleotide sequence and expression of a hydantoinase and carbamoylase gene from hrthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from hrthrobacter aurescens DSM 3747.Appl Microbiol Biotech,2003,55(6):750-757
[63]Anja W.,Christoph S.,Ralf M.,et al.Organization of genes responsible for the stereospecific conversion of hydantoins to amino acids in Arthrobacter aurescens DSM 3747.Archives Microbiol,2003,176(3):187-196
[64]Umemura I.,Yanagiya K.,Komatsubara S.d-Alanine production from dl-alanine by Candida maltosa with asymmetric degrading activity.Appl Microbiol Biotechnol,1992,36(6):722-726
[65]Chibata,I.,T.Tosa,T.Sato,et al.Preparation and industrial application of immobilized aminoacylase, 1972, p383—389
[66] Wen-Lin Lai, Lien-Yang Chou, Chun-Yu Ting,et al. The Functional Role of the Binuclear Metal Center in D-Aminoacylase. J. Biol. Chem, 2004, 279(14): 13962—13967
[67] Yang, Y. B., Hsiao, K. M., Li, H., et al. Characterization of D-aminoacylase from Alcaligenes denitrificans DA181. Biosci. Biotechnol. Biochem, 1992, 56:1392—1395
[68] Yang, Y. B., Lin, C. S., Tseng, C. P., et al. Purification and characterization of D-aminoacyclase from Alcaligenes faecalis DAL Appl. Environ. Microbiol, 1991, 57:1259—1260
[69] Tsai, Y. C., Lin, C. S., Tseng, T.H., et al. Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids. Enzyme Microbiol. Technol, 1992, 14:384-389
[70] Moriguchi, M., Sakai, K., Miyamoto, Y., and Wakayama, M. Production, purification, and characterization of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. Biosci. Biotechnol. Biochem, 1993, 57:1149—1152
[71] Sugie, M. and Suzuki, H. Purification and properties of D-aminoacylase of Streptomyces olivaceus. Agric. Biol. Chem, 1978, 44:107—113
[72] Cheng-Sheng Hsu, Wen-Lin Lai, Wei-Wei Chang, et al. Structural-based mutational analysis of d-aminoacylase from Alcaligenes faecalis DAL Protein Sci, 2002, 11(11): 2545—2550
[73] Wakayama, M., Ashika, T., Miyamoto, Y., et al. Primary structure of N-acyl-D-glutamate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. J. Biochem, 1995a, 118:204—209
[74] Jian Liu, Toru Nakayama, Hisashi Hemmi, et al. Microbacterium natoriense sp. nov., a novel D-aminoacylase-producing bacterium isolated from soil in Natori, Japan. Int J Syst Evol Microbiol, 2005, 55:661-665
[75] S. Tokuyama. D-aminoacylase. US Patent, 2000, 6030823
[76] P. H. Lin, S. C. Su, Y. C. Tsai , et al. Identification and characterization of a new gene from Variovorax paradoxus Isol encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production. Eur. J. Biochem, 2002, 269: 4868—4878
[77] Shwu-Huey Liaw, Shen-Jia Chen, Tzu-Ping Ko, et al. Crystal Structure of d-Aminoacylase fromAlcaligenes faecalis DA1 A NOVEL SUBSET OF AMIDOHYDROLASES AND INSIGHTS INTO THE ENZYME MECHANISM. Journal of Biological Chemistry, 2003, 278:4957—4962.
[78] Wakayama, M., Yada, H., Kanda, S., et al. Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. Biosci. Biotechnol. Biochem, 2000, 64:1 — 8
[79] Yoshimune K, Ninomiya Y, Wakayama M, et al. Molecular chaperones facilitate the soluble expression of N-acyl-D-amino acid amidohydrolases in Escherichia coli.J Ind Microbiol Biotechnol, 2004, 31(9) :421-426
[80] M. Wakayama, T. Ashika, Y. Miyamoto, et al. Primary Structure of N-Acyl-D-Glutamate Amidohydrolase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6. J. Biochem, 1995, 118(1): 204-209
[81] ValleeBL, Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry , 1990, 29(24) :5647-5659
[82] Wakayama M, Yoshi mune K, Hirose Y, et al. Production of d-amino acids by N-acyl-d-amino acid amidohydrolase and its structure and function. Journal of Molecular Catalysis B: Enzymatic, 2003, 23(15):71-78
[83] Hsu CS, Lai WL, Chang WW, et al. Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1. Protein Sci, 2002, 11(11) :2545~2550
[84] Simmer, J P., Kelly, R. E, Rinker, A. G Jr., et al. Mammalian dihydroorotase: nucleotide sequence, peptide sequences, and evolution of the dihydroorotase domain of the multifunctional protein CAD, Proc. Natl Acad. Sci. U.S.A. 1990, 87(1) :174-178
[85] Lai, W. L., Chou, L. Y., Ting, C. Y., et al. The functional role of the binuclear metal center in D-aminoacylase. One-metal activation and second-metal inhibition. J. Biol. Chem, 2004, 279:13962-13967