葡萄糖异构酶的固定化及其性能研究
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摘要
本文以Tween-20和Span-60为复合分散剂,以烯丙基缩水甘油醚和甲基丙烯酸缩水甘油酯为功能性单体,N,N-亚甲基双丙烯酰胺为交联剂,采用反相悬浮聚合技术制备含环氧基团的聚合物载体GAMM,并将其应用于固定化葡萄糖异构酶,考察了固定化条件和异构化反应条件对固定化酶活性的影响以及固定化酶的使用性能。
研究结果表明,固定化酶最佳负载量为每克载体加入0.3 mL游离酶,最佳固定化温度为25℃,最佳固定化时间为24 h；固定化酶与游离酶具有相同的最适反应pH值(7.5),固定化酶在pH=5～7.5的条件下比较稳定,固定化酶比游离酶具有更宽的pH适用范围,固定化酶的最适反应温度为65℃；游离酶的Km为0.36 mol/L,最大反应速率为1.3×10-3mol/L·min,而固定化酶Km为1.16 mol/L,最大反应速率为11×10-3 mol/L·min,固定化酶的最大酶活为(?)50 U/g；固定化酶在重复使用18次以后,仍能保持90%的初始活性,在4℃条件下储存6周仍能保持97%的初始酶活,在80℃以下固定化酶比游离酶具有更好的热稳定性。
The polymer carrier GAMM with epoxy groups was synthesized by the inverse suspension polymerization, in which Tween-20 and Span-60 were used as the complex dispersants, and N, N'-methylene-bis (acryla-mide) (MBAA) as crosslinking agent, and ally glycidol ether (AGE) and glycidyl methacrylate (GMA) were used as reactive monomers. The carrier was used to immobilize glucose isomerase (GI), in which effects of immobilization conditions and isomerization reaction conditions on the activity of immobilized GI and the operating performance of immobilized GI were investigated.
The optimum immobilization conditions were as follows:the loading amount of free GI was 0.3 mL per gram carrier, the immobilization temperature was 25℃, and the immobilization time was 24 h; The optimum reaction pH values of free and immobilized GIs were both 7.5. Immobilized GI was more stable at pH value of 5-7.5 and had wider application pH range, The optimum reaction temperature was 65℃. As for free and immobilized GIs, the kinetic parameters were calculated as 0.36 and 1.16 mol/L for Km, and 1.3×10-3 and 1.1×10-3 mol/L·min for Vmax, respectively. The maximum activity of immobilized GI was 450 U/g (wet). The relative activity of immobilized GI was about 90% after recycled for 18 times and retained 97%of its initial activity after stored at 4℃for six weeks, immobilized GI had better thermal stability than free GI below 80℃.
研究结果表明,固定化酶最佳负载量为每克载体加入0.3 mL游离酶,最佳固定化温度为25℃,最佳固定化时间为24 h；固定化酶与游离酶具有相同的最适反应pH值(7.5),固定化酶在pH=5～7.5的条件下比较稳定,固定化酶比游离酶具有更宽的pH适用范围,固定化酶的最适反应温度为65℃；游离酶的Km为0.36 mol/L,最大反应速率为1.3×10-3mol/L·min,而固定化酶Km为1.16 mol/L,最大反应速率为11×10-3 mol/L·min,固定化酶的最大酶活为(?)50 U/g；固定化酶在重复使用18次以后,仍能保持90%的初始活性,在4℃条件下储存6周仍能保持97%的初始酶活,在80℃以下固定化酶比游离酶具有更好的热稳定性。
The polymer carrier GAMM with epoxy groups was synthesized by the inverse suspension polymerization, in which Tween-20 and Span-60 were used as the complex dispersants, and N, N'-methylene-bis (acryla-mide) (MBAA) as crosslinking agent, and ally glycidol ether (AGE) and glycidyl methacrylate (GMA) were used as reactive monomers. The carrier was used to immobilize glucose isomerase (GI), in which effects of immobilization conditions and isomerization reaction conditions on the activity of immobilized GI and the operating performance of immobilized GI were investigated.
The optimum immobilization conditions were as follows:the loading amount of free GI was 0.3 mL per gram carrier, the immobilization temperature was 25℃, and the immobilization time was 24 h; The optimum reaction pH values of free and immobilized GIs were both 7.5. Immobilized GI was more stable at pH value of 5-7.5 and had wider application pH range, The optimum reaction temperature was 65℃. As for free and immobilized GIs, the kinetic parameters were calculated as 0.36 and 1.16 mol/L for Km, and 1.3×10-3 and 1.1×10-3 mol/L·min for Vmax, respectively. The maximum activity of immobilized GI was 450 U/g (wet). The relative activity of immobilized GI was about 90% after recycled for 18 times and retained 97%of its initial activity after stored at 4℃for six weeks, immobilized GI had better thermal stability than free GI below 80℃.
引文
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[5]Nelson JM,Griffin EG:Adsorption of invertase J Am Chem Soc.1916,38:1109-1115
[6]Asif M. Abasaeed A.E. Modeling of Glucose Isomerization in a Fluidized Bed Immobilized Enzyme Bioreactor.Bioresour.Technol.1998,64:229-237
[7]Bravo V, Jurado E,Luzon G,Cruz N. Kinetics of Fructose-Glucose Isomerization with Sweetzyme Type A.Can.J.Chem.Eng.1998,76:778-786
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[9]Dehkordi A.M. Novel Type of Two-Impinging-Jets Reactor for Solid-Liquid Enzyme Reactions.AIChE J.2006,52:692-704
[10]Faqir N.M, Abu-Reesh I.M. Optimum Temperature Operation Mode for Glucose Isomerase Reactor Operating at Constant Glucose Conversion.Bioprocess Eng.1998,19: 11-18
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[13]Khorasheh F, Kheirolomoom A. Mireshghi,S.A.Application of an Optimization Algorithm for Estimating Intrinsic Kinetic of Immobilized Enzmye.J. Biosci.Bioeng.2002,94: 1-7
[14]Lee H.S, Hong J. Kinetics of Glucose Isomerization to Fructose by Immobilized Glucose Isomerase:Anomeric Reactivity of D-glucose in Kinetic Model.J.Biotechnol.2000,84: 145-150
[15]Messing R.A. Filbert A.M. An Immobilized Glucose Isomerase for the Continuous Conversion of Glucose to Fructose.J.Agric. Food Chem.1975,23:920-930
[16]Palazzi E, Convcrti A. Generalized Linearization of Kinetics of Glucose Isomerization to Fructose by Immobilized Glucose Isomerase. Biotechnol. Bioeng.1999,63:273-278
[17]Palazzi E. Converti A. Evaluation of Diffusional Resistances in the Process of Glucose Isomerization to Fructose by Immobilized Glucose Isomerase. Enzyme Microb. Technol. 2001,28:246-252
[18]Straatsma J, Vellenga K,de Willt H.G. J, Joosten G. E. H. Isomerization of Glucose to Fructose.2. Optimization of Reaction Conditions in the Production of High Fructose Syrup by Isomerization of Glucose Catalyzed by a Whole Cell Immobilized Glucose Isomerase Catalyst. Ind. Eng. Chem. Process Des. DeV.1983,22:356-363
[19]Toumi A, Engell S.Optimization-Based Control of a Reactive Simulated Moving Bed Process for Glucose Isomerization. Chem. Eng. Sci.2004,59:3777-3792
[20]Vilonen K.M, Vuolanto A, Jokela J. Leisola, M. S. A.Krause, A. O.I.Enhanced Glucose to Fructose Conversion in Acetone with Xylose Isomerase Stabilized by Crystallization and Cross-Linking.Biotechnol.Prog.2004,20:1555-1560
[21]Zhang Y, Hidajat K, Ray A.K. Optimal Design and Operation of SMB Bioreactor: Production of High Fructose Syrup by Isomerization of Glucose. Biochem.Eng.J.2004,21: 111-121
[22]朱国萍,称阳,宫春红,徐冲.葡萄糖异构酶的生物工程研究进展[J].Prog.Biochem.Biophys.2000,27(2):127-137
[23]Bhosale S.H, Rao M.B, Desphande V.V. Molecular and industrial aspects of glucose isomerase. Microbiological Reviews.1996,60:280-300.
[24]Misset O, Labout J, Wilms J, Quax W.J, Van Eekelen C.A.G, Busnach H.A. High fructose corn syrup. Biotechnological innovation in food processing. Oxford:Butterworth.
[25]Basciano H, Federico L, Adeli K. Fructose, insulin resistance, and metabolic dyslipidemia. Nutrition and Metabolism, (London).2005,2(1):5.
[26]Bray G.A, Nielsen S.J, Popkin B.M. Consumption of high-fructose corn syrup in beverages may play a role epidemic of obesity. American Journal of Clinical Nutrition. 2004,7(9):537-543.
[27]Lo C.Y, Li S, Wang Y, Tan D, Pan M.H, Sang S. Reactive dicarbonyl compounds and 5-hydroxymethyl-2-furfural in carbonated beverages containing high fructose corn syrup. Food Chem.2008,107:1099-1105
[28]Janket S.J, Manson J.E, Sesso H, Buring J.E, Liu S.A. prospective study of sugar intake and risk of type 2 diabetes in women. Diabetes Care.2003,26:1008-1015
[29]Sun S.Z, Empie M.W. Lack of findings for the association between obesity risk and usual sugar-sweetened beverage consumption in adults-A primary analysis of databases of CSFⅡ-1989-1991. CSFⅡ-1994-1998, NHANES Ⅲ. and combined NHANES 1999-Food and Chemical Toxicology.2002,45:1523-1536.
[30]Petrovska B, Winkelhausen E. Kuzmanova S. Ethanol and polyol production from glucose by Candida boidini NRLL Y-17213. Bulletin of the chemists and technologists of Macedonia.2000,19:57-63.
[31]Pazur J.H, Knull H.R. Cepure A.Glycoenzymes structure and properness of the forms of glucomylase from Aspergillus niger[J].Carbohydr Rev.1971,20:83-96
[32]Chen W.P, Anderson A.W. Purification, immobilization and some properties of glucose isomerase from Streptomyces flavogriseus. Appl.Environ Microbiol.1979,38:1111-1119.
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