去氧核醣核苷三磷酸 (dNTP) 是生物體合成去氧核醣核酸必要的原料，也是分子生物學技術如聚合酶鏈鎖反應中常用的反應物。由於生物體dNTP含量不高，不易萃取，因此市面上販售的dNTP主要以化學合成方式製造，但通常產率不高且須使用毒性溶劑。本研究目的為建立在體外以酵素合成dNTP的系統，以解決這些問題。此生產過程以去氧核醣核苷單磷酸為起始反應物，先產生去氧核醣核苷二磷酸再進而生成dNTP。我們從嗜熱菌Thermus thermophilus HB8中選殖四個核苷酸激酶與一個核苷二磷酸激酶，分別在大腸桿菌中大量表現重組蛋白質。隨後並對此些酵素進行功能性與動力學特性分析。結果顯示此五種酵素在pH 7.0到pH 8.0且溫度介於75度至80度時表現出最佳活性。此外，在75度環境下處理三小時都可保有80%以上活性，即使處理24小時之後仍能保有50%以上活性，顯示這五種酵素具有極佳的熱穩定性。在動力學試驗測試中也發現此五種酵素在其最佳反應環境下都表現出較嗜溫性菌還高的催化效率。我們組合這些酵素進行初步dNTP生產，並將所得到的產物用於聚合酶鏈鎖反應進行檢測，結果顯示與市面上販售的dNTP無明顯差異。此一系統將有潛力應用於工業上，以乾淨有效的方式生產dNTP。

Deoxynucleoside triphosphates (dNTP) are essential biological materials for synthesizing DNA. The compounds are also used in molecular biotechnology such as the polymerase chain reaction (PCR) and other DNA polymerase-based applications. Nowadays, dNTPs are commercially produced by chemical synthesis due to their low concentrations in living organisms. However, the chemical synthesis method utilizes toxic solvents and the yield is low. To solve these problems, this study attempt to establish an in vitro enzymatic method for complete biosynthesis of dNTP. The process involves two stages of enzymatic phosphorylation procedures. The deoxynucleoside monophosphates (dNMPs) are used in the first step. The products of the first and second stages are deoxynucleoside diphosphates (dNDP) and dNTP, respectively. Five genes from the Thermus thermophilus HB8, which encode four kinds of deoxynucleoside monophosphate kinases (NMKs) and one nucleoside diphosphate kinase (NDK), were cloned and expressed in high quality in E. coli BL21 (DE3) and NovaBlue (DE3). We have performed functional and kinetic property analysis of NMKs and NDK. The results showed that the five enzymes exhibited optimum activity at the pH value of 7.0 to 8.0 and at the temperature between 75OC to 80 OC. Furthermore, all of them displayed excellent thermal stability. Approximate 80% and 50% of enzyme activities were retained after incubation at 75 OC for 3 hr and 24hr, respectively. The kinetic property analysis showed that five enzymes have higher catalytic efficiency under optimum environment as compared to the mesophilic bacteria. Later, we combined these crude enzymes preliminarily to produce dNTP. The PCR performed using the enzymes-synthesized dNTP showed the same efficiency compared to the commercial one. Finally, this system provides a clean and better method of preparation that has potential for commercialization.

中文摘要………………………………………………………………………………I
英文摘要…………………………………………………………………………….. II
致謝…………………………………………………………………………………..III
縮寫字對照表……………………………………………………………………..…IV
目錄…………………………………………………………………………………VII
表目錄………………………………………………………………………………..IX
圖目錄………………………………………………………………………………...X
壹、 前言………………………………………………………………………………1
貳、 材料與實驗方法…………………………………………………………………8
2.1. 菌株與生長環境…………………………………………………………....8
2.2. 基因體DNA萃取………………………………………………………..…8
2.3. 聚合酶鏈鎖反應……………………………………………………………9
2.4. 接合作用…………………………………………………………………....9
2.5. 勝任細胞製備………………………………………………………………9
2.6. 熱休克轉型法……………………………………………………………..10
2.7. 小量質體DNA純化………………………………………...………….....10
2.8. 質體構築…………………………………………………….……...……..11
2.9. 蛋白質表現並利用His6-tag親和性管柱純化………...…….……...……12
2.10. 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳……………………….….............13
2.11. 酵素最佳活性測定- pH的影響...………………………………....……...14
2.12. 酵素最佳活性測定-反應溫度的影響…………………………………….15
2.13. 酵素熱穩定性測試………………………………………………………..15
2.14. 酵素動力學特性分析………………………………………....…………..16
2.15. 純化之NMKs與NDK合成dNTP……………………………..……….17
2.16. 以高效液相層析儀進行dNTP生合成之定性分析……………………...17
參、 結果…………………………………………………………………....………..19
3.1. 分析T. thermophilus HB8的NMKs與NDK之胺基酸特性分………...19
3.2. 構築表現載體與重組蛋白質之表現純化………………………………..20
3.3. NMKs重組蛋白質之最適反應環境分析……………………...………...21
3.4. NMKs重組蛋白質之熱穩定性測試…………………………...………...22
3.5. NMKs重組蛋白質之酵素動力學特性分析…………………………......23
3.6. 建立新穎的dNTP生產系統進行dNTP生產與使用………………..….24
3.7. 利用高效液相層析儀進行dNTP之生合成之定性及定量分析………...24
肆、 討論……………………………………………………………………………..26
伍、 參考文獻…………………………………………………………………….….33

Agarwal KC, Parks RE, Jr. (1972) Adenosine triphosphate-guanosine 5'-phosphate phosphotransferase. IV. Isozymes in human erythrocytes and Sarcoma 180 ascites cells. Molecular pharmacology 8: 128-138

Argos P, Rossmann MG, Grau UM, Zuber H, Frank G, Tratschin JD (1979) Thermal-Stability and Protein-Structure. Biochemistry 18: 5698-5703

Bae E, Phillips GN, Jr. (2006) Roles of static and dynamic domains in stability and catalysis of adenylate kinase. Proceedings of the National Academy of Sciences of the United States of America 103: 2132-2137

Bao J, Bruque GA, Ryu DDY (2005) Biosynthesis of deoxynucleoside triphosphates, dCTP and dTTP: reaction mechanism and kinetics. Enzyme Microb Tech 36: 350-356

Bao J, Ryu DD (2005) Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP. Biotechnology and bioengineering 89: 485-491

Bao J, Ryu DD (2006) Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphates. Biotechnology and bioengineering 93: 572-580

Bao J, Ryu DD (2007) Total biosynthesis of deoxynucleoside triphosphates using deoxynucleoside monophosphate kinases for PCR application. Biotechnology and bioengineering 98: 1-11

Blaszczyk J, Li Y, Yan H, Ji X (2001) Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes. Journal of molecular biology 307: 247-257

Boehme RE (1984) Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl) guanine monophosphate by guanylate kinase isozymes. The Journal of biological chemistry 259: 12346-12349

Bohm G, Jaenicke R (1994) Relevance of Sequence Statistics for the Properties of Extremophilic Proteins. Int J Pept Prot Res 43: 97-106

Bollum FJ, Vanpotter R (1958) Incorporation of Thymidine into Deoxyribonucleic Acid by Enzymes from Rat Tissues. Journal of Biological Chemistry 233: 478-482

Briozzo P, Golinelli-Pimpaneau B, Gilles AM, Gaucher JF, Burlacu-Miron S, Sakamoto H, Janin J, Barzu O (1998) Structures of escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity. Structure 6: 1517-1527

Cai G, Zhu SC, Yang S, Zhao GP, Jiang WH (2004) Cloning, overexpression, and characterization of a novel thermostable penicillin g acylase from Achromobacter xylosoxidans: Probing the molecular basis for its high thermostability. Appl Environ Microbiol 70: 2764-2770

Chambers RW, Khorana HG (1957) Nucleoside Polyphosphates .5. Syntheses of Guanosine 5'-Diphosphates and Triphosphates. Journal of the American Chemical Society 79: 3752-3755

Chambers RW, Moffatt JG, Khorana HG (1957) Nucleoside Polyphosphates .4. A New Synthesis of Guanosine 5'-Phosphate. Journal of the American Chemical Society 79: 3747-3752

Chaudhary SK, Jeyakanthan J, Sekar K (2013) Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of thymidylate kinase (TTHA1607) from Thermus thermophilus HB8. Acta crystallographica Section F, Structural biology and crystallization communications 69: 118-121

Cihlar T, Ray AS (2010) Nucleoside and nucleotide HIV reverse transcriptase inhibitors: 25 years after zidovudine. Antiviral research 85: 39-58

Colomb MG, Cheruy A, Vignais PV (1972) Nucleoside diphosphokinase from beef heart cytosol. I. Physical and kinetic properties. Biochemistry 11: 3370-3378

Colomb MG, Cheruy A, Vignais PV (1974) Adenosine-Diphosphate as a Regulatory Ligand in Beef Heart Cytosol Nucleoside Diphosphokinase. Biochemistry 13: 2269-2277

Coppock DL, Pardee AB (1987) Control of thymidine kinase mRNA during the cell cycle. Molecular and cellular biology 7: 2925-2932

de Marco A, Casatta E, Savaresi S, Geerlof A (2004) Recombinant proteins fused to thermostable partners can be purified by heat incubation. J Biotechnol 107: 125-133

Dzeja P, Terzic A (2009) Adenylate kinase and AMP signaling networks: metabolic monitoring, signal communication and body energy sensing. International journal of molecular sciences 10: 1729-1772

Dzeja PP, Zeleznikar RJ, Goldberg ND (1998) Adenylate kinase: kinetic behavior in intact cells indicates it is integral to multiple cellular processes. Molecular and cellular biochemistry 184: 169-182

Egner U, Tomasselli AG, Schulz GE (1987) Structure of the complex of yeast adenylate kinase with the inhibitor P1,P5-di (adenosine-5'-) pentaphosphate at 2.6 A resolution. Journal of molecular biology 195: 649-658

Erlich HA, Gelfand D, Sninsky JJ (1991) Recent advances in the polymerase chain reaction. Science 252: 1643-1651

Galmarini CM, Jordheim L, Dumontet C (2003) Pyrimidine nucleoside analogs in cancer treatment. Expert review of anticancer therapy 3: 717-728

Garces E, Cleland WW (1969) Kinetic studies of yeast nucleoside diphosphate kinase. Biochemistry 8: 633-640

Gavel OY, Bursakov SA, Di Rocco G, Trincao J, Pickering IJ, George GN, Calvete JJ, Shnyrov VL, Brondino CD, Pereira AS, Lampreia J, Tavares P, Moura JJG, Moura I (2008) A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774. J Inorg Biochem 102: 1380-1395

Gershenson A, Schauerte JA, Giver L, Arnold FH (2000) Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases. Biochemistry 39: 4658-4665

Gill S, Thomas RR, Goldberg RM (2003) Review article: colorectal cancer chemotherapy. Alimentary pharmacology & therapeutics 18: 683-692

Grabarse W, Vaupel M, Vorholt JA, Shima S, Thauer RK, Wittershagen A, Bourenkov G, Bartunik HD, Ermler U (1999) The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri. Struct Fold Des 7: 1257-1268

Guo H. (2000) Preparation of deoxynucleoside triphosphate, involves converting DNMP into DNTP through the enzyme reaction, then purifying by chromatography, and precipitating and separating in the form of sodium salt or barium salt. BEIJING TAIGONG BIOLOGICAL TECHNOLOGY CO, China.

Hanson PI, Whiteheart SW (2005) AAA+ proteins: Have engine, will work. Nat Rev Mol Cell Bio 6: 519-529

Hashimoto Y, Yano T, Kuramitsu S, Kagamiyama H (2001) Disruption of Thermus thermophilus genes by homologous recombination using a thermostable kanamycin-resistant marker. FEBS letters 506: 231-234

Henne A, Bruggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H, Johann A, Lienard T, Gohl O, Martinez-Arias R, Jacobi C, Starkuviene V, Schlenczeck S, Dencker S, Huber R, Klenk HP, Kramer W, Merkl R, Gottschalk G, Fritz HJ (2004) The genome sequence of the extreme thermophile Thermus thermophilus. Nature biotechnology 22: 547-553

Hess D, Kruger K, Knappik A, Palm P, Hensel R (1995) Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus. European journal of biochemistry / FEBS 233: 227-237

Hible G, Renault L, Schaeffer F, Christova P, Zoe Radulescu A, Evrin C, Gilles AM, Cherfils J (2005) Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase. Journal of molecular biology 352: 1044-1059

Ichikawa E, Kato K (2001) Sugar-modified nucleosides in past 10 years, a review. Current medicinal chemistry 8: 385-423

Ingwall JS, Shen W (2010) On energy circuits in the failing myocardium. European journal of heart failure 12: 1268-1270

Jaenicke R (1991) Protein stability and molecular adaptation to extreme conditions. European journal of biochemistry / FEBS 202: 715-728

Jaenicke R, Bohm G (1998) The stability of proteins in extreme environments. Curr Opin Struc Biol 8: 738-748

Janssen E, Kuiper J, Hodgson D, Zingman LV, Alekseev AE, Terzic A, Wieringa B (2004) Two structurally distinct and spatially compartmentalized adenylate kinases are expressed from the AK1 gene in mouse brain. Molecular and cellular biochemistry 256-257: 59-72

Jordan A, Reichard P (1998) Ribonucleotide reductases. Annual review of biochemistry 67: 71-98

Kakuda TN (2000) Pharmacology of nucleoside and nucleotide reverse transcriptase inhibitor-induced mitochondrial toxicity. Clinical therapeutics 22: 685-708

Kochanowski N, Blanchard F, Cacan R, Chirat F, Guedon E, Marc A, Goergen JL (2006) Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC. Analytical biochemistry 348: 243-251

Kosinska U, Carnrot C, Sandrini MP, Clausen AR, Wang L, Piskur J, Eriksson S, Eklund H (2007) Structural studies of thymidine kinases from Bacillus anthracis and Bacillus cereus provide insights into quaternary structure and conformational changes upon substrate binding. Febs J 274: 727-737

Ladner WE, Whitesides GM (1985) Enzymatic-Synthesis of Deoxy-Atp Using DNA as Starting Material. Journal of Organic Chemistry 50: 1076-1079

Lambeth DO, Mehus JG, Ivey MA, Milavetz BI (1997) Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon. The Journal of biological chemistry 272: 24604-24611

Lascu I, Gonin P (2000) The catalytic mechanism of nucleoside diphosphate kinases. Journal of bioenergetics and biomembranes 32: 237-246

Lee LS, Cheng YC (1976) Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia. The Journal of biological chemistry 251: 2600-2604

Leone A, Flatow U, King CR, Sandeen MA, Margulies IM, Liotta LA, Steeg PS (1991) Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23-transfected melanoma cells. Cell 65: 25-35

Lin TS, Schinazi RF, Prusoff WH (1987) Potent and selective in vitro activity of 3'-deoxythymidin-2'-ene (3'-deoxy-2',3'-didehydrothymidine) against human immunodeficiency virus. Biochemical pharmacology 36: 2713-2718

Liou JY, Dutschman GE, Lam W, Jiang Z, Cheng YC (2002) Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates. Cancer research 62: 1624-1631

Luo JY, Bergstrom DE, Barany F (1996) Improving the fidelity of Thermus thermophilus DNA ligase. Nucleic Acids Res 24: 3071-3078

Lutz S, Lichter J, Liu L (2007) Exploiting temperature-dependent substrate promiscuity for nucleoside analogue activation by thymidine kinase from Thermotoga maritima. Journal of the American Chemical Society 129: 8714-8715

Matsunaga Y, Fujisaki H, Terada T, Furuta T, Moritsugu K, Kidera A (2012) Minimum free energy path of ligand-induced transition in adenylate kinase. PLoS computational biology 8: e1002555

Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN, Gallo RC, Bolognesi D, Barry DW, Broder S (1985) 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proceedings of the National Academy of Sciences of the United States of America 82: 7096-7100

Morera S, LeBras G, Lascu I, Lacombe ML, Veron M, Janin J (1994) Refined X-ray structure of Dictyostelium discoideum nucleoside diphosphate kinase at 1.8 A resolution. Journal of molecular biology 243: 873-890

Moure VR, Razzera G, Araujo LM, Oliveira MAS, Gerhardt ECM, Muller-Santos M, Almeida F, Pedrosa FO, Valente AP, Souza EM, Huergo LF (2012) Heat stability of Proteobacterial P-II protein facilitate purification using a single chromatography step. Protein expression and purification 81: 83-88

Mrabet NT, Vandenbroeck A, Vandenbrande I, Stanssens P, Laroche Y, Lambeir AM, Matthijssens G, Jenkins J, Chiadmi M, Vantilbeurgh H, Rey F, Janin J, Quax WJ, Lasters I, Demaeyer M, Wodak SJ (1992) Arginine Residues as Stabilizing Elements in Proteins. Biochemistry 31: 2239-2253

Muller CW, Schlauderer GJ, Reinstein J, Schulz GE (1996) Adenylate kinase motions during catalysis: an energetic counterweight balancing substrate binding. Structure 4: 147-156

Muller CW, Schulz GE (1988) Structure of the complex of adenylate kinase from Escherichia coli with the inhibitor P1,P5-di (adenosine-5'-) pentaphosphate. Journal of molecular biology 202: 909-912

Noda L, Kuby SA (1957) Adenosine triphosphate-adenosine monophosphate transphosphorylase (myokinase). I. Isolation of the crystalline enzyme from rabbit skeletal muscle. The Journal of biological chemistry 226: 541-549

Oeschger MP, Bessman MJ (1966) Purification and properties of guanylate kinase from Escherichia coli. The Journal of biological chemistry 241: 5452-5460

Ohtani N, Tomita M, Itaya M (2010) An Extreme Thermophile, Thermus thermophilus, Is a Polyploid Bacterium. Journal of bacteriology 192: 5499-5505

Ohtani N, Tomita M, Itaya M (2012) The third plasmid pVV8 from Thermus thermophilus HB8: isolation, characterization, and sequence determination. Extremophiles : life under extreme conditions 16: 237-244

Okazaki R, Kornberg A (1964) Deoxythymidine Kinase of Escherichia Coli. I. Purification and Some Properties of the Enzyme. The Journal of biological chemistry 239: 269-274

Olichon A, Schweizer D, Muyldermans S, de Marco A (2007) Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains. Bmc Biotechnol 7

Oshima T, Imahori K (1974) Description of Thermus-Thermophilus (Yoshida and Oshima) Comb-Nov, a Nonsporulating Thermophilic Bacterium from a Japanese Thermal Spa. Int J Syst Bacteriol 24: 102-112

Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004) Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends in biotechnology 22: 253-260

Peabody DS (1989) Translation initiation at non-AUG triplets in mammalian cells. The Journal of biological chemistry 264: 5031-5035

Pontarin G, Gallinaro L, Ferraro P, Reichard P, Bianchi V (2003) Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools. Proceedings of the National Academy of Sciences of the United States of America 100: 12159-12164

Postel EH, Berberich SJ, Flint SJ, Ferrone CA (1993) Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science 261: 478-480

Ryan RP, Fouhy Y, Lucey JF, Dow JM (2006) Cyclic di-GMP signaling in bacteria: recent advances and new puzzles. Journal of bacteriology 188: 8327-8334

Schulz GE, Elzinga M, Marx F, Schrimer RH (1974) Three dimensional structure of adenyl kinase. Nature 250: 120-123

Schulz GE, Schiltz E, Tomasselli AG, Frank R, Brune M, Wittinghofer A, Schirmer RH (1986) Structural relationships in the adenylate kinase family. European journal of biochemistry / FEBS 161: 127-132

Seagrave J, Reyes P (1985) Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure. Analytical biochemistry 149: 169-176

Seagrave J, Reyes P (1987) Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: a kinetic analysis of the reaction mechanism. Archives of biochemistry and biophysics 254: 518-525

Segura-Pena D, Lichter J, Trani M, Konrad M, Lavie A, Lutz S (2007) Quaternary structure change as a mechanism for the regulation of thymidine kinase 1-like enzymes. Structure 15: 1555-1566

Sekulic N, Shuvalova L, Spangenberg O, Konrad M, Lavie A (2002) Structural characterization of the closed conformation of mouse guanylate kinase. The Journal of biological chemistry 277: 30236-30243

Smith M, Khorana HG (1958) Nucleoside Polyphosphates .6. An Improved and General Method for the Synthesis of Ribonucleoside and Deoxyribonucleoside 5'-Triphosphates. Journal of the American Chemical Society 80: 1141-1145

Sokalingam S, Raghunathan G, Soundrarajan N, Lee SG (2012) A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein. Plos One 7

Stolworthy TS, Black ME (2001) The mouse guanylate kinase double mutant E72Q/D103N is a functional adenylate kinase. Protein engineering 14: 903-909

Stubbe J (1998) Ribonucleotide reductases in the twenty-first century. Proceedings of the National Academy of Sciences of the United States of America 95: 2723-2724

Takeishi S, Nakagawa N, Maoka N, Kihara M, Moriguchi M, Masui R, Kuramitsu S (2003) Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Thermus thermophilus HB8. Acta crystallographica Section D, Biological crystallography 59: 1843-1845

Tan ZW, Liu J, Zhang XF, Meng FG, Zhang YZ (2010) [Expression, purification and enzymatic characterization of adenylate kinase of Thermus thermophilus HB27 in Escherichia coli]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University 30: 1-6

Tanabe T, Yamada M, Noma T, Kajii T, Nakazawa A (1993) Tissue-specific and developmentally regulated expression of the genes encoding adenylate kinase isozymes. Journal of biochemistry 113: 200-207

Thoma R, Hennig M, Sterner R, Kirschner K (2000) Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima. Struct Fold Des 8: 265-276

Tomasselli AG, Schirmer RH, Noda LH (1979) Mitochondrial GTP-AMP phosphotransferase. 1. Purification and properties. European journal of biochemistry / FEBS 93: 257-262

Tong J, Cao WG, Barany F (1999) Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucleic Acids Res 27: 788-794

Turunen O, Vuorio M, Fenel F, Leisola M (2002) Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein engineering 15: 141-145

Verma NK, Singh B (2013) Insight from the structural molecular model of cytidylate kinase from Mycobacterium tuberculosis. Bioinformation 9: 680-684

Vieille C, Epting KL, Kelly RM, Zeikus JG (2001) Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase. European Journal of Biochemistry 268: 6291-6301

Vieille C, Krishnamurthy H, Hyun HH, Savchenko A, Yan H, Zeikus JG (2003) Thermotoga neapolitana adenylate kinase is highly active at 30 degrees C. Biochem J 372: 577-585

Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiology and molecular biology reviews : MMBR 65: 1-43

Voorhorst WGB, Rik IL, Luesink EJ, Devos WM (1995) Characterization of the Celb Gene Coding for Beta-Glucosidase from the Hyperthermophilic Archaeon Pyrococcus-Furiosus and Its Expression and Site-Directed Mutation in Escherichia-Coli. Journal of bacteriology 177: 7105-7111

Walinder O (1968) Identification of a phosphate-incorporating protein from bovine liver as nucleoside diphosphate kinase and isolation of 1-32P-phosphohistidine, 3-32P-phosphohistidine, and N-epsilon-32P-phospholysine from erythrocytic nucleoside diphosphate kinase, incubated with adenosine triphosphate-32P. The Journal of biological chemistry 243: 3947-3952

Walker EJ, Dow JW (1982) Location and properties of two isoenzymes of cardiac adenylate kinase. The Biochemical journal 203: 361-369

Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. The EMBO journal 1: 945-951

Williams RL, Oren DA, Munoz-Dorado J, Inouye S, Inouye M, Arnold E (1993) Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution. Journal of molecular biology 234: 1230-1247

Zavodszky P, Kardos J, Svingor A, Petsko GA (1998) Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proceedings of the National Academy of Sciences of the United States of America 95: 7406-7411

Zhang K. (2003) Production process of deoxynucleoside triphosphate. China, Vol. CN1398977.