Please use this identifier to cite or link to this item:
標題: 以酵素法生產海藻糖與製程純化之研究
Enzymatic process for the Production and Purification of Trehalose
作者: 吳宗達
Wu, Tsung-Ta
關鍵字: 海藻糖的純化;trehalose synthesis;固定化海藻糖合成酶;固定化澱粉葡萄糖化酶;固定化葡萄糖氧化酶;trehalose purification;immobilized trehalose synthase;immobilized glucoamylase;immobilized glucose oxidase
出版社: 化學工程學系所
引用: [1] Richards AB, Krakowka S, Dexter LB, Schmid H, Wolterbeek APM, Waalkens-Berendsen DH, Shigoyuki A, Kurimoto M. Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food and Chemical Toxicology 2002;40:871-898. [2] Ohtake S, Wang YJ. Trehalose: current use and future applications. Journal of Pharmaceutical Sciences 2011;100:2020-2053. [3] Elbein AD. The metabolism of [alpha],[alpha]-trehalose. Advances in carbohydrate chemistry 1974;30:227-256. [4] Harding TS. History of trehalose, its discovery and methods of preparation. Sugar 1923;25:476-478. [5] Koch EM, Koch FC. The presence of trehalose in yeast. Science 1925;61:570-572. [6] Donnamaria MC, Howard EI, Grigera JR. Interaction of water with [small alpha],[small alpha]-trehalose in solution: molecular dynamics simulation approach. Journal of the Chemical Society, Faraday Transactions 1994;90:2731-2735. [7] Wyatt GR, Kalf GF. The chemistry of insect hemolymph. II. Trehalose and other carbohydrates. Journal of General Physiology 1957;40:833-847. [8] Trehalose as stabilizer and tableting excipient. US 4762857. [9] Chen J, Kimura Y, Adachi S. Surface activities of monoacyl trehaloses in aqueous solution. LWT - Food Science and Technology 2007;40:412-417. [10] Higashiyama T. Novel functions and applications of trehalose. Pure and Applied Chemistry 2002;74:1263-1269. [11] Tanaka K. Development of Trehaose and its properties. Food Industry 2009;52:45-51. [12] Tanaka M, Machida Y, Nukina N. A novel therapeutic strategy for polyglutamine diseases by stabilizing aggregation-prone proteins with small molecules. J Mol Med 2005;83:343-352. [13] Tanaka M, Machida Y, Niu S, Ikeda T, Jana NR, Doi H, Kurosawa M, Nekooki M, Nukina N. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nature Medicine 2004;10:148-154. [14] Liu R, Barkhordarian H, Emadi S, Park CB, Sierks MR. Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobiology of Disease 2005;20:74-81. [15] Schiraldi C, Di Lernia I, De Rosa M. Trehalose production: exploiting novel approaches. Trends in Biotechnology 2002;20:420-425. [16] Matula M, Mitchell M, Elbein AD. Partial purification and properties of a highly specific trehalose phosphate phosphatase from Mycobacterium smegmatis. Journal of Bacteriology 1971;107:217-222. [17] Maruta K, Nakada T, Kubota M, Chaen H, Sugimoto T, Kurimoto M, Tsujisaka Y. Formation of trehalose from maltooligosaccharides by a novel enzymatic system. Bioscience, Biotechnology, and Biochemistry 1995;59:1829-1834. [18] Di Lernia I, Morana A, Ottombrino A, Fusco S, Rossi M, De Rosa M. Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch. Extremophiles 1998;2:409-416. [19] Nishimoto T, Nakano M, Ikegami S, Chaen H, Fukuda S, Sugimoto T, Kurimoto M, Tsujisaka Y. Existence of a novel enzyme converting maltose into trehalose. Bioscience, Biotechnology, and Biochemistry 1995;59:2189-2190. [20] Shaw JF, Sheu JR. Production of high-maltose syrup and high-protein flour from rice by an enzymatic method. Bioscience, Biotechnology, and Biochemistry 1992;56:1071-1073. [21] Nishimoto T, Nakano M, Nakada T, Chaen H, Sfukud A, Sugimoto T, Kurimoto M, Tsujisaka Y. Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp.R48. Bioscience, Biotechnology, and Biochemistry 1996;60:640-644. [22] Ohguchi M, Kubota N, Wada T, Yoshinaga K, Uritani M, Yagisawa M, Ohishi K, Yamagishi M, Ohta T, Ishikawa K. Purification and properties of trehalose-synthesizing enzyme from Pseudomonas sp. F1. Journal of Fermentation and Bioengineering 1997;84:358-360. [23] Koh S, Shin HJ, Kim JS, Lee DS, Lee SY. Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus. Biotechnology Letters 1998;20:757-761. [24] Koh S, Kim J, Shin HJ, Lee D, Bae J, Kim D, Lee DS. Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase. Carbohydrate Research 2003;338:1339-1343. [25] Chen YS, Lee GC, Shaw JF. Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. Journal of Agricultural and Food Chemistry 2006;54:7098-7104. [26] Lee JS, Hai T, Pape H, Kim TJ, Suh JW. Three trehalose synthetic pathways in the acarbose-producing Actinoplanes sp. SN223/29 and evidence for the TreY role in biosynthesis of component C. Applied Microbiology Biotechnology. 2008;80:767-778. [27] Xiuli W, Hongbiao D, Ming Y, Yu Q. Gene cloning, expression, and characterization of a novel trehalose synthase from Arthrobacter aurescens. Applied Microbiology Biotechnology. 2009;83:477-482. [28] Kim TK, Jang JH, Cho HY, Lee HS, Kim YW. Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032. Food Science and Biotechnology 2010;19:565-569. [29] Filipkowski P, Panek A, Agnieszka F, Pietrow O, Synowiecki J. Expression of Deinococcus geothermalis trehalose synthase gene in Escherichia coli and its enzymatic properties. African Journal of Biotechnology 2012;13131-13139. [30] Yue M, Wu XL, Gong WN, Ding HB. Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei. Microbial Cell Factories 2009;8:1-7. [31] Zhu YM, Wei DS, Zhang J, Wang YF, Xu HY, Xing L, Li MC. Overexpression and characterization of a thermostable trehalose synthase from Meiothermus ruber. Extremophiles 2010;14:1-8. [32] Pan YT, Koroth Edavana V, Jourdian WJ, Edmondson R, Carroll JD, Pastuszak I, Elbein AD. Trehalose synthase of Mycobacterium smegmatis. European Journal of Biochemistry 2004;271:4259-4269. [33] Ma Y, Xue L, Sun D-W. Characteristics of trehalose synthase from permeablized Pseudomonas putida cells and its application in converting maltose into trehalose. Journal of Food Engineering 2006;77:342-347. [34] Gao Y, Xi Y, Lu X-L, Zheng H, Hu B, Liu X-Y, Jiao B-H. Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005. World Journal of Microbiology and Biotechnology 2013;1-12. [35] Yan J, Qiao Y, Hu J, Ding H. Cloning, expression and characterization of a trehalose synthase gene from Rhodococcus opacus. Protein J 2013;32:223-229. [36] Nishimoto T, Nakada T, Chaen H, Fukuda S, Sugimoto T, Kurimoto M, Tsujisaka Y. Purification and charaterization of a thermostable trehalose synthase from Thermus aquaticus. Bioscience, Biotechnology, and Biochemistry 1996;60:835-839. [37] Liang J, Huang R, Huang Y, Wang X, Du L, Wei Y. Cloning, expression, properties, and functional amino acid residues of new trehalose synthase from Thermomonospora curvata DSM 43183. Journal of Molecular Catalysis B: Enzymatic 2013;90:26-32. [38] Wang JH, Tsai MY, Chen JJ, Lee GC, Shaw JF. Role of the C-terminal domain of Thermus thermophilus trehalose synthase in the thermophilicity, thermostability, and efficient production of trehalose. Journal of Agricultural and Food Chemistry 2007;55:3435-3443. [39] Wang Y, Zhang J, Wang W, Liu Y, Xing L, Li M. Effects of the N-terminal and C-terminal domains of Meiothermus ruber CBS-01 trehalose synthase on thermostability and activity. Extremophiles 2012;16:377-385. [40] Cho YJ, Park OJ, Shin HJ. Immobilization of thermostable trehalose synthase for the production of trehalose. Enzyme and Microbial Technology 2006;39:108-113. [41] Kim HJ, Kim AR, Jeon SJ. Immobilization on chitosan of a thermophilic trehalose synthase from Thermus thermophilus HJ6. Journal of Microbiology and Biotechnology 2010;20:513-517. [42] Panek A, Pietrow O, Synowiecki J, Filipkowski P. Immobilization on magnetic nanoparticles of the recombinant trehalose synthase from Deinococcus geothermalis. Food and Bioproducts Processing 2013. In press. [43] Ho LF, Li SY, Lin SC, Hsu WH. Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents. Process Biochemistry 2004;39:1573-1581. [44] Tsai SY, Lin SC, Suen SY, Hsu WH. Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents. Process Biochemistry 2006;41:2058-2067. [45] Puri M, Kaur A, Singh RS, Schwarz WH, Kaur A. One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis. Process Biochemistry 2010;45:451-456. [46] Sulkowski E. The saga of IMAC and MIT. BioEssays 1989;10:170-175. [47] Scope R. editor. Protein purification: principles and practice. 2nd ed. New York: pringer-Verlag; 1987. p. 109. [48] Whistler RL, Durso DF. Chromatographic separation of sugars on charcoal1. Journal of the American Chemical Society 1950;72:677-679. [49] Hendrix DL, Lee Jr RE, Baust JG, James H. Separation of carbohydrates and polyols by a radially compressed high-performance liquid chromatographic silica column modified with tetrathylenepentamine. Journal of Chromatography A 1981;210:45-53. [50] Cataldi TRI, Campa C, Angelotti M, Bufo SA. Isocratic separations of closely-related mono- and disaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection using dilute alkaline spiked with barium acetate. Journal of Chromatography A 1999;855:539-550. [51] James JA, Lee BH. Glucoamylase: microbial sources, industrial applications and moleculase biology — a review. Journal of Food Biochemistry 1997;21:1-52. [52] Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B. Glucoamylase: structure/function relationships, and protein engineering. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 2000;1543:275-293. [53] Sierks MR, Ford C, Reilly PJ, Svensson B. Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori Protein Engineering 1990;3:193-198. [54] Casey JP. High fructose corn Syrup. A case history of innovation. Starch - Starke 1977;29:196-204. [55] Saha BC, Zeikus JG. Microbial glucoamylases: biochemical and biotechnological features. Starch-Starke 1989;41:57-64. [56] Celebi SS, Tsai GJ, Tsao GT. Characterization of glucoamylase immobilized on celite Applied Biochemistry and Biotechnology 1991;27:163-171. [57] Bahar T, Celebi SS. Characterization of glucoamylase immobilized on magnetic poly(styrene) particles. Enzyme and Microbial Technology 1998;23:301-304. [58] Silva RN, Asquieri ER, Fernandes KF. Immobilization of Aspergillus niger glucoamylase onto a polyaniline polymer. Process Biochemistry 2005;40:1155-1159. [59] Tanriseven A, Olcer Z. A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin. Biochemical Engineering Journal 2008;39:430-434. [60] Tardioli PW, Vieira MF, Vieira AMS, Zanin GM, Betancor L, Mateo C, Fernandez-Lorente G, Guisan JM. Immobilization–stabilization of glucoamylase: Chemical modification of the enzyme surface followed by covalent attachment on highly activated glyoxyl-agarose supports. Process Biochemistry 2011;46:409-412. [61] Chen G, Ma Y, Su P, Fang B. Direct binding glucoamylase onto carboxyl-functioned magnetic nanoparticles. Biochemical Engineering Journal 2012;67:120-125. [62] Wu TT, Lin SC, Shaw JF. Integrated process for the purification and immobilization of recombinant trehalose synthase for trehalose production. Process Biochemistry 2011;46:1481-1485. [63] Sparham SJ, Huehns ER. The separation of human globin chains by ion-exchange chromatography on Cm-Sepharose Cl-6B. Hemoglobin 1979;3:13-20. [64] Yeoh HH, Tan TK, Chua SL, Lim G. Properties of β-glucosidase purified from Aspergillus niger. Mircen Journal 1988;4:425-430. [65] Xiao R, Tanida M, Takao S. Purification and characteristics of two exoinulinases from Chrysosporium pannorum. Journal of Fermentation and Bioengineering 1989;67:331-334. [66] James Brockbank W, Lynn KR. Purification and preliminary characterization of two asclepains from the latex of Asclepias syriaca L. Biochimica et Biophysica Acta (BBA) - Protein Structure 1979;578:13-22. [67] Nakayama K, Saito T, Fukui T, Shirakura Y, Tomita K. Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1985;827:63-72. [68] Katchalski-Katzir E, Kraemer DM. EupergitR C, a carrier for immobilization of enzymes of industrial potential. Journal of Molecular Catalysis B: Enzymatic 2000;10:157-176. [69] Mateo C, Torres R, Fernandez-Lorente G, Ortiz C, Fuentes M, Hidalgo A, Lopez-Gallego F, Abian O, Palomo JM, Betancor L, Pessela BCC, Guisan JM, Fernandez-Lafuente R. Epoxy-amino groups:  a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules 2003;4:772-777. [70] Wheatley JB, Schmidt Jr DE. Salt-induced immobilization of affinity ligands onto epoxide-activated supports. Journal of Chromatography A 1999;849:1-12. [71] Mateo C, Fernandez-Lorente G, Abian O, Fernandez-Lafuente R, Guisan JM. Multifunctional epoxy supports:  a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 2000;1:739-745. [72] Yu RC, Hang YD. Purification and characterization of a glucoamylase from Rhizopus oryzae. Food Chemistry 1991;40:301-308. [73] Chang SW, Liu PT, Hsu LC, Chen CS, Shaw JF. Integrated biocatalytic process for trehalose production and separation from rice hydrolysate using a bioreactor system. Food Chemistry 2012;134:1745-1753. [74] Bankar SB, Bule MV, Singhal RS, Ananthanarayan L. Glucose oxidase -- an overview. Biotechnology Advances 2009;27:489-501. [75] Witt S, Wohlfahrt G, Schomburg D, Hecht HJ, Kalisz HM. Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of b-D-glucose. Biochemical Journal 2000;347:553-559. [76] Anette I, Jens AN. Antioxidative effect of glucose oxidase and catalase in Mayonnaises of different oxidative susceptibility. I. Product trials. LWT-Food Science and Technology 1997;30:841-846. [77] Afseth J, Rolla G. Clinical experiments with a toothpaste containing amyloglucosidase and glucose oxidase. Caries Research 1983;17:472-475. [78] Kleppe K. The effect of Hydrogen peroxide on glucose oxidase from Aspergillus niger. Biochemistry 1966;5:139-143. [79] Bao J, Furumoto K, Fukunaga K, Nakao K. A kinetic study on air oxidation of glucose catalyzed by immobilized glucose oxidase for production of calcium gluconate. Biochemical Engineering Journal 2001;8:91-102. [80] Bao J, Furumoto K, Yoshimoto M, Fukunaga K, Nakao K. Competitive inhibition by hydrogen peroxide produced in glucose oxidation catalyzed by glucose oxidase. Biochemical Engineering Journal 2003;13:69-72. [81] Wilson R, Turner APF. Glucose oxidase: an ideal enzyme. Biosensors and Bioelectronics 1992;7:165-185. [82] Mislovičova D, Michalkova E, Vikartovska A. Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures. Process Biochemistry 2007;42:704-709. [83] Mislovičova D, Turjan J, Vikartovska A, Patoprsty V. Removal of d-glucose from a mixture with d-mannose using immobilized glucose oxidase. Journal of Molecular Catalysis B: Enzymatic 2009;60:45-49. [84] Kyoko KT, Sueko H, Hideko N, Satoshi N. Properties of Aspergillus niger catalase. Journal of Biochemistry 1982;92:1449-1456. [85] Wu TT, Lin SC, Shaw JF. Enzymatic processes for the purification of trehalose. Biotechnology Progress 2013;29:83-90.
海藻糖為一種多功能的非還原雙醣,具低卡路里與新風味之優點,已逐漸成為可取代蔗糖與麥芽糖的食品調味劑。因海藻糖具有保濕性與穩定蛋白質之功能,又可做為化妝品保濕劑與食品抗凍劑。雖然海藻糖廣泛存在於自然界生物中,但其含量稀少無法直接取得。文獻提及合成海藻糖的方法有數種,僅有酵素合成法較符合生產成本,其中以海藻糖合成酶直接催化麥芽糖轉成海藻糖的製程較具有工業化量產之潛力。源自Picrophilus torridus海藻糖合成酶轉殖於大腸桿菌後,可經由大腸桿菌大規模生產海藻糖合成酶。因海藻糖合成酶在進行基因重組時,植入了poly-His tag的基因序列,其基因重組海藻糖合成酶的poly-His tag可與含有金屬離子的螯合吸附基材擔體進行配位共價鍵固定化反應。比較六種金屬離子的金屬螯合吸附材之擔體可知,雖然Cu(II)-loaded adsorbent吸附蛋白質的能力最高,蛋白質承載量為5.39 ± 0.03 mg/g gel,但其吸附海藻糖合成酶的專一性卻不如其他種金屬離子。然而,Co(II)-loaded adsorbent吸附海藻糖合成酶呈現出高度的專一性,其純化倍率為Cu(II)-loaded adsorbent的四倍以上。海藻糖合成酶經由固定化反應後,其最適反應的pH值、反應溫度與穩定性與自由態海藻糖合成酶相較並無顯著差異。固定化海藻糖合成酶於最適的反應條件之下進行麥芽糖的轉換反應6 h後,可獲得64%的海藻糖轉換率。固定化海藻糖合成酶經由24次重複操作後,尚有80%的殘存活性,經由蛋白質活性分析可知,酵素活性的下降係由海藻糖合成酶的脫附所致。然而,此製程中尚有26 mM麥芽糖與20 mM葡萄糖的存在,需進行麥芽糖與葡萄糖的移除或分離,增加海藻糖之純度。
為了簡化海藻糖的純化製程,本研究接續發展一套結合固定化澱粉葡萄糖化酶水解麥芽糖與固定化葡萄糖化酶氧化葡萄糖的製程,有效地將殘存的麥芽糖與葡萄糖轉換成葡萄糖酸。在固定化澱粉葡萄糖化酶的部分,因CM Sepharose的蛋白質承載量(49.35 ± 1.43 mg/g gel)與固定化酵素的酵素總活性(1141.89 ± 31.89 U/g gel)高於EupergitR C,故選用CM Sepharose做為澱粉葡萄糖化酶之固定化擔體。澱粉葡萄糖化酶經由固定化反應後,其最適反應pH值和溫度與自由態澱粉葡萄糖化酶相同,無顯著差異。當固定化澱粉葡萄糖化酶於最適的反應條件之下進行麥芽糖的水解作用,反應40 min後即可完全地將殘餘麥芽糖水解轉換成葡萄糖。在固定化澱粉葡萄糖化酶的穩定性方面,經過80個批次反應後,酵素活性仍含有92%的殘存活性;根據此趨勢推算,固定化澱粉葡萄糖化酶可於操作110個批次反應後,尚有80%的的殘存活性,此結果說明固定化澱粉葡萄糖化酶具有高度的穩定性。在固定化葡萄糖氧化酶的部分,使用環氧基聚丙烯酯Immobead 150做為葡萄糖氧化酶之擔體。當葡萄糖氧化酶進行共價鍵固定化反應時,提升離子濃度有助於酵素承載量的增加。葡萄糖氧化酶經由固定化反應後,其最適反應的pH值、反應溫度以及穩定性與自由態葡萄糖氧化酶相較並無顯著差異。固定化葡萄糖氧化酶於最適的反應條件之下,反應90 min後可完全地將殘存的葡萄糖轉換成gluconolactone,再水解成葡萄糖酸。在操作穩定性的部分,固定化葡萄糖氧化酶於第12個批次反應後活性驟然下降,推測應為過氧化氫引起的不可逆之抑制作用所致。最後再以陰離子交換樹酯,移除海藻糖液體中的葡萄糖酸,獲得高純度的海藻糖。
其他識別: U0005-0608201317050900
Appears in Collections:化學工程學系所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.