摘要
糖化酶，全名葡萄糖淀粉酶（Glucoamylase），是一种具有外切酶活性的酸性单链糖苷水解酶。它可以从淀粉或低聚糖的非还原末端水解α-1,4-糖苷键生成葡萄糖。糖化酶被广泛地应用于淀粉糖、食品、医药、酿造等工业生产中，是我国产量最大、应用范围最广的酶制剂。
目前，工业用糖化酶存在的缺点之一是热稳定性差。淀粉在高温液化后必须经过一个降温过程才能添加糖化酶进行糖化；此外，长时间的糖化过程也会造成糖化酶活力的损失，大大增加了生产成本。与此同时，关于糖化酶热稳定机制的研究还不完善，热稳定糖化酶的开发尚处于研究阶段。为了推动这一问题的解决，我们以一株经诱变获得的糖化酶高产菌株Aspergillus niger B-30为材料，对其产生的糖化酶进行了分离纯化及诸酶学性质分析，并对温度诱导糖化酶去折叠机制进行了详细研究。本文中也阐述了添加稳定剂提高糖化酶的热稳定性及其稳定机制，尝试了对纯化后糖化酶的固定化，并对固定化条件及固定化酶学性质进行探讨和分析。
首先，对A. niger B-30产生的糖化酶进行纯化和酶学性质分析。通过硫酸铵沉淀、DEAE FF离子交换层析和Superdex G-75凝胶过滤层析对A. niger B-30液体发酵产生的糖化酶进行分离纯化，获得两个不同的单一的糖化酶组分，分别命名为GAM-1和GAM-2。经SDS-PAGE检测其分子量分别为97.2kDa和78.3kDa。同时，通过MALDI-TOF-MS检测其分子量分别为80.5kDa和70.4kDa。GAM-1和GAM-2均为糖蛋白，其碳水化合物含量分别为10.4%和13.4%。GAM-1和GAM-2的最适反应pH和最适反应温度相同，分别为4.0-4.6和70℃。两糖化酶均有较好的热稳定性和pH稳定性，且GAM-2优于GAM-1。GAM-1和GAM-2酶学性质研究显示二者均适于淀粉质原料的糖化过程。
其次，本论文对温度诱导糖化酶去折叠机制进行了探讨。通过圆二色谱技术、荧光光谱技术、紫外吸收光谱、动态光散射等技术以及Native-PAGE等分析方法系统地研究了温度对GAM-1和GAM-2构象的影响。结果表明二者去折叠机制相似，即温度诱导GAM-1和GAM-2去折叠时，α-螺旋含量逐渐减少，β-折叠、β-转角和无规则卷曲含量逐渐增加；疏水基团外露且伴随着蛋白质的聚集；在加热过程中，GAM-2的结构比GAM-1更稳定是其表现出较高热稳定性的重要原因。
本论文还进行了稳定剂对糖化酶热稳定性影响及热稳定机制的研究。为了稳定糖化酶构象，使其在高温下维持活力，研究中向糖化酶反应体系内分别加入1M山梨醇和海藻糖，发现稳定剂在低温时不会明显提高酶活力，只有在高温反应时，才具有稳定作用。为解析山梨醇和海藻糖使糖化酶热稳定性提高的机制，我们向GAM-1和GAM-2中分别加入1M山梨醇和海藻糖后，在75℃和80℃下测定了两糖化酶的热动力学参数，结果显示热失活常数明显减小，半衰期增加，热失活自由能△G增大。同时，我们又通过光谱学方法解析了其热稳定机制。结果表明25℃时，加入稳定剂对其构象无显著影响，而在高温时可以很大程度上阻止糖化酶二级结构、三级结构的变化以及热聚集，维持其天然构象的稳定，使其在高温下保持较高的酶活力，且海藻糖对糖化酶蛋白的稳定能力高于山梨醇。
最后，对糖化酶进行了固定化研究。我们以两种性质优良的树脂SepabeadsEC-OD和Sepabeads EC-HA为载体探索了GAM-1的最适固定化条件。结果发现两种树脂固定化条件一致：最适固定化酶浓度为1mg/mL；最适固定化温度为25℃；最适固定化pH为4.6；最适固定化离子强度为25mM； SepabeadsEC-HA交联糖化酶需要的最适戊二醛浓度为2%。我们通过激光粒度分析发现两固定化酶EC-OD-GA和EC-HA-GA分散度较好，平均粒径分别为259.9±41.10μm和229.2±44.04μm；通过扫描电镜观察表面形态发现，GAM-1成功固定到两树脂上。固定化酶性质分析的结果表明，两固定化糖化酶的最适温度与游离酶相同，均为70℃；但与游离酶相比固定化酶在高温下活力更高，且EC-HA-GA高于EC-OD-GA；与游离酶相比，EC-OD-GA的最适pH稍偏酸性，而EC-HA-GA没发生变化；两固定化酶的热稳定性和pH稳定性也均有提高，且EC-HA-GA的热稳定性高于EC-OD-GA；GAM-1通过两种树脂固定化后，均有较好的循环使用性，在重复使用8次后，EC-HA-GA和EC-OD-GA分别可保持接近60%和28%的残留活性；与游离酶相比，两固定化酶的储藏稳定性均有较大提升。
Glucoamylase is an acidic glucoside hydrolase with exo-acting enzymeactivity. Glucoamylase could catalyze the hydrolysis of α-1,4glycosidic linkagesfrom the non-reducing ends of starch and oligosaccharide to produce glucose.Glucoamylase is an industrially important biocatalyst and has the most extensiveuses in the manufacture of starch sugar, food, medicine and brewing, and it hasthe largest production in China.
At present, the glucoamylase has the disadvantage of low thermal stability.Hence, the starch after liquefying at high temperature has to be cooled tocontinue the following saccharification process by glucoamylase. In addition, thelong-time saccharification also causes greater loss of glucoamylase, which hasgreatly increased the cost of production. However, the mechanism for the thermalstability of glucoamylase is not clear, and meanwhile the exploitation ofglucoamylase with high thermostability is still on the experimental level. Tosolve this problem, the glucoamylases from a mutant strain Aspergillus nigerB-30were purified, and the properties and unfold mechanism induced bytemperature of these two enzymes were also characterized. Meanwhile, thethermal stability of glucoamylases was improved by adding stabilizer, and themechanism was also eluciadted. Finally, the glucoamylase were immobilized,and the properties of the immobilized glucoamylase were analyzed.
First, the glucoamylases from A. niger B-30were purified and the propertiesof these enzymes were also characterized. The two different glucoamylasesGAM-1and GAM-2were purified by ammonium sulfate precipitation, DEAE Fast Flow and Superdex G-75gel filtration columns from the fermentation brothof A. niger B-30. The molecular weight values of GAM-1and GAM-2weredetermined as97.2kDa and78.3kDa by SDS-PAGE, while the correspondingvalues of GAM-1and GAM-2were determined to be80.5kDa and70.4kDa byMALDI-TOF MS, respectively. Both the enzymes were glycosylated, with10.4%and13.4%carbohydrate content. The optimal pH and temperature of these twoenzymes were4.0-4.6and70℃. Both the two glucoamylases displayed highthermal stability and pH stability, and the GAM-2was more stable than GAM-1.Thus, GAM-1and GAM-2are suitable for starch saccharification due to theirhigh catalytic acitivty and stability.
Secondly, the folding and unfolding mechanisms of glucoamylases inducedby temperature were studied. The effect of temperature on the conformation ofGAM-1and GAM-2was analyzed by the method of circular dichroism spectrum,fluorescence spectrum, ultraviolet absorption spectrum, dynamic light scatteringand Native-PAGE. The results suggested that both GAM-1and GAM-2displayedsimilar unfolding mechanism. During the process of GAM-1and GAM-2, theα-helix content decreased, and β-fold, β-turn and random coil contents increased,and hydrophobic groups exposed with the aggregation of the protein. Thestructure of GAM-2was more stable during heating, which was the probablereason for that GAM-2showed higher thermal stability.
Then the effect of stabilizer on the thermal stability and the mechanism werestudied.1M sorbitol and mycose were respectively added into glucoamylasereaction mixture to stabilize the conformation to maintain the activity at hightemperature. The results suggested that the activity was not obviously increasedat low temperature. On the contrary, the glucoamylase with stabilizer remainedhigher activity at high temperature compared to the negative control. To analyze the thermal stability mechanism of the stabilizer, the thermodynamics data with1M sorbitol and mycose was calculated at75℃and80℃, respectively. Theresults indicated that the kinetics data of heat inactivation obviously reduced afterthe adding of stabilizers, and the half-life and△G increased. The glucoamylaseconformation was not influenced by the stabilizer at25℃, however, at hightemperature the stabilizer could restrain the changing of the secondary andtertiary structure and aggregating of glucoamylase to maintain the naturalconformation, which would lead to higher activity, and the mycose was moreefficient than sorbitol.
Finally, the glucoamylase was immobilized on two resins Sepabeads EC-ODand Sepabeads EC-HA, and the properties of the immobilized glucoamylase wereanalyzed. The results suggested that the optimal conditions of immobilizing theGAM-1onto these two supports were uniform. The optimal concentration ofenzyme, temperature, pH and ionic strength were determined as1mg/mL,25℃,4.6and25mM, respectively. The optimal concentration of glutaraldehyde usedto cross-link Sepabeads EC-HA was2%. The two immobilized enzymeEC-OD-GA and EC-HA-GA exhibited a uniform disperse. Particle sizes of thetwo immobilized enzymes were259.9±41.10and229.2±44.04μm by laserscattering analysis, respectively. The surface morphologies of the carriers and theimmobilized enzyme were studied using SEM analysis. The results provided adirect evidence of the successful adsorption of the enzyme on the resins. Theproperties analysis of the immobilized glucoamylase suggested that the optimaltemperature of immobilized and free enzyme were both70℃, but the activity ofimmobilized enzyme was higher than free enzyme at high tempreture.Furthermore, the activity of the EC-OD-GA was higher than EC-HA-GA.Compared with free GAM-1, the optimal pH of the EC-OD-GA is more acidic, and the optimal pH of EC-HA-GA did not change. Both the thermostability andpH stability of immobilized enzyme were improved, and the EC-HA-GA wasmore stable than EC-OD-GA. After immobilization using two supports, theGAM-1showed a good ability to be recycled. After eight times repeated use, theEC-HA-GA and EC-OD-GA could maintained60%and28%relative activity,respectively. Compared with the free enzyme, the storage stability of theimmobilized enzyme was largely improved.
引文
[1] Norouzian D, Akbarzadeh A, Scharer JM, et al. Fungal glucoamylases[J].Biotechnol Adv.,2006,24:80–85.
[2] Cardona F, Goti A, Brandi A, et al. Molecular dynamics simulation of thecomplexes of glucoamylase Ⅱ from Aspergillus awamori var.X100withdeoxynojiromycin and lentiginosine[J]. J Mol Model,1997,3(1):249.
[3]姚婷婷.糖化酶生产菌的遗传改良[D].无锡：江南大学.2006.
[4] Pavezzi F C, Carneiro A A J, Martins D A, et al. Influence of different substateson the production of a mutant thermostable glucoamylase in submergedfermentation[J]. Appl Bicohem Biotechnol,2011,163:14-24.
[5] Bourne Y, Henrissat B. Glycoside hydrolases and glycosyltransferases: familiesand functional modules[J]. Curr Opin Struct Biol,2001,11:593-600.
[6] Reilly PJ. Glucoamylase.2006, In Whitaker JR, Voragen AGJ, Wong DWS, eds.Handbook of Enzymology (pp.727–738). New York: Marcel Dekker, Inc.
[7] Bender H. A bacterial glucoamylase degrading cyclodextrins, partial purificationand properties of the enzyme from a Flavobacterium species[J]. Eur J Biochem,1981,115:287-291.
[8] Srivastava RAK. Studies on extracelluar and intracellular purified amylases froma thermophilic Bacillus stearothermophilus[J]. Enzyme Microb Technol,1984,6:422-426.
[9] Oren A. A thermophilic amylogicosidase from Halobacterium sodomense, ahalophilic bacterium from the Dead Sea[J]. Curr Microbiol.1983,8:225-230.
[10] Katkocin D M, Word N S, Yang S S. Thermostable glucoamylase and methodfor its production.1985, US Patent4536477.
[11] Ohnishi H, Sakai H, Ohta T. Purification and some properties of a glucoamylasefrom Clostridium sp. G0005[J]. Agric Biol Chem,1991,55:1901-1902.
[12] Feng P H, Berensmeier S, Buchholz K, el at. Production, purification, andcharacterization of Thermoanaerobacerium thrmosaccharolyticum glucoamylae.Statch/Starke[J].2002,54:328-337.
[13] Stamford T L, Stamford N P, Coelho L C, et al. Production and characterizationof a thermostable glucoamylase from Streptosporangium sp. Endophyte ofmaize leaves[J]. Bioresour Technol.2002,83:105-109.
[14] Kim M S, Park J T, Kim Y W, et al. Properties of a novel thermostableglucoamyase from the hyperthermophilic archaeon Sulfollbus solfataricus inrelation to starch processing[J]. Appl Environ Microbiol,2004,70:3933-3940.
[15] Gill R K, Kaur J. A thermostable glucoamylase from a thermophilic Bacillus sp.:characterization and thermostability[J]. J Ind Microbiol Biotechmol,2004,11:540-543.
[16] Dock C, Hess M, Antranikian G. A thermoactive glucoamylae withbiotechnological relevance from the thermoacidophilic EuryarchaeonThermoplasma acidophilum[J]. Appl Microbiol Biotechnol,2008,78:105-114.
[17] Zheng Y Y, Xue Y F, Zhang Y L, et al. Cloning, expression, and characterizationof a thermostable glucoamylase from Thremoanaerobacter tengcongensisMB4[J]. Appl Microbiol Biotechnol,2010,87:225-233.
[18] Miyoshi K, Teruhito K, Haruyuki Iefuji, et al. Isolation and characterization ofglucoamylae from a wastewater treatment yease Hansenula fabianii J640, andconstruction of expression vector[J]. Appl Microbiol Biotechnol,2011,90:981-987.
[19] Sevcik J, Hostinova E, Solovicova A, et al. Strcuture of the complex of a yeastglucoamylase with acarbose reveals the presence of a raw statch binding site oncatalytic domain[J]. FEBS J,2006,273:2171-2171.
[20] Bul D M, Kunze I, Horstamann C, et al. Expression of the Arxula adeninivoransglucoamylase gene in Kluyveromyces lactis[J]. Appl Microbiol Biotechnol.1996b,45:102-106.
[21] Sturtevant J, Dixon F, Wadsworth E, et al. Identification and cloning of GCA1,a gene that encodes a cell surface glucoamylase from Candida albicans[J].Medical Mycol,1999,37:357-366.
[22] Alteriis E, Silvestro G, Poletto M, et al. Kluyveromyces lactis cells entrapped inCa-alginate beads for the continuous production of a heterologousglucoamylase[J]. J Biotechnol,2004,109:83-92.
[23] Li H, Chi Z, Duan X, et al. Glucoamylase production by the marine yeastAureobasidium pullulans N13d and hydrolysis of potato starch granules by theenzyme. Process Biochem[J],2007a,42:462-465.
[24] Negi S, Banerjee R. Characterization of amylase and protease produced byAspergillus awamori in a single bioreactor[J]. Food Research Intermational2009,42:443-448.
[25] Christensen T, Frandsen T P, Kaarsholm N C, et al. Physicochemicalcharacterization of the two active site muatants Trp52→Phe and Asp55→Val ofglucoamylase from Aspergillus niger[J]. Biochim Biophys Acta,2002,1601:163-171
[26] Silva T M, Maller A, Lima A R, et al. Properties of a purified thermostableglucoamylase from Aspergillus niveus[J]. J Ind Microbiol Biotechnol,2009,36:1439-1446.
[27] Lin S C, Liu W T, Liu S H, et al. Role of the linker region in the expression ofRhizopus oryzae glucoamylase[J]. BMC Biochemistry,2007,8:9(1-12).
[28] Sun H, Ge X, Zhang W. Production of a novel raw-starch-digestingglucoamylase by Penicillium sp. X-1under solid-state fermentation and its usein direct hydrolysis of raw statch[J]. World J Microbiol Biotechnol,2007,23:603-613.
[29] Soni K S, Gupa K J. A solid state fermentation based bacterial α-amylase andfungal glucoamylase system and its suitability for the hydrolysis of wheatstarch[J]. Process Biochem,2003,39:185-192.
[30] Pacheco-Chavez R A, Carvalho J C M, Tavares L C, et al. Production ofα-amylase and glucoamylase by a new isolate of Trichoderma sp. usingsorghum starch as a carbon source[J]. Eng Life Sci,2004,4:369-372.
[31] Feng B, Hu W, Ma B P, et al. Purification, characterization, and substratespecificity of a glucoamylase with steroidal saponin-rhamnosidase activity fromCurvularia lunata[J]. Appl Microbiol Biotechnol,2007a,76:1329-1338.
[32] Bhatti H N, Rashid M H, Nawaz R, et al. Optimization of media for enhancedglucoamylase production in solid-state fermentation by Fuasrium solani[J].Food Chem,2007a,45:51-56.
[33] Bhatti H N, Rashid M H, Nawaz R, et al. Purification and characterization of anovel glucoamylase from Fusarium solani[J]. Food Chem,2007b,103:338-343.
[34] Rey M W, Brown K M, Golightly E J, et al. Cloning, hererologous expression,and characterization of Thielavia terrestri glucoamylase[J]. Biotechnol ApplBiochem,2003,111:153-166.
[35] Kumar P, Satyanaryana T. Overproduction of glucoamylase by a deregulatedmutant of a thermophilic mold Thermomucor indicae-seudaticae[J]. ApplBiochem Biotechnol,2009,158:113-125.
[36] Chen J, Zhang Y Q, Zhao C Q, et al. Cloning of a gene encoding thermostaleglucoamylase from Chaetomium thermophilum and its expression in Pichiapastoris[J]. J Appl Microbiol,2007,103:2277-2284.
[37] Cereia M, Terenzi H F, Jorge J A, et al. Glucoamylase activity from thethermophilic fungus Scytalidium thermophilum: biochemical and regulatoryproperties[J]. J Basic Microbiol,2000,40:83-92.
[38] Nielsen B R, Lehmbeck J, Frandsen T P. Cloning, heterologous expression, andenzymatic characterization of a thermostable glucoamylae from Talaromycesemersonii[J]. Protein Expr Purif,2002,26:1-8.
[39] Tachibama S, Yasuda, M. Purification and characterization of hererogeneousglucoamylase from Monascus purpureus[J]. Biosci Biotech Bioch,2007,10:408-415.
[40] Michelin M, Ruller R, Ward R J, et al. Purification and biochemicalcharacterization of a thermostable extracellular glucoamylase produced by thethermotolerantfungus Paecilomyces variotii[J]. J Ind Microbiol Biotechnol,2008,35:17-25.
[41] Reilly PJ. Protein engineering of glucoamylase to improve industrialperformance-a review[J]. Starch/Starke,1999,51:269–274.
[42] Gibbs PA, Seviour RJ, Schmid F. Growth of filamentous fungi in submergedculture: problems and possible solutions[J]. Crit Rev Biotechnol,2000,20:17–48.
[43] Silveira ST, Oliveira MS, Costa JAV, et al. Optimization of glucoamylaseproduction by Aspergillus niger in solid-state fermentation[J]. Appl BiochemBiotechnol,2006,128:131–139.
[44] Mohamed L, Zakaria M, Ali A, et al. Optimization of growth and extracellularglucoamylase production by Candida famata isolate[J]. African J Biotechnol,2007,6:2590–2595.
[45] Wang Q, Wang X, Wang X, et al. Glucoamylase production from food waste byAspergillus niger under submerged fermentation[J]. Process Biochem,2008,43:280–286.
[46] Kumar P, Satyanarayana T. Production of thermostable and neutralglucoamylase using immobilized Thermomucor indicae-seudaticae[J]. World JMicrobiol Biotechnol,2007a,23:509–517.
[47] Negi S, Banerjee R. Optimization of extraction and purification ofglucoamylase produced by Aspergillus awamori in solid-state fermentation[J].Biotechnol Biopros E,2009,14:60-66.
[48] Minami NM, Kilikian BV. Separation and purification of glucoamylase inaqueous two-phase system by a two-step extraction[J]. J Chromatogr (B),1998,771:309–312.
[49] Mondal K, Sharma A, Gupta MN. Macroaffinity ligand-facilitated three phasepartitioning for purification of glucoamylase and pullulanase using alginate[J].Protein Expr Purif,2003,28:190–195.
[50] Nelson N. A photometric adaptation of the somogyi method for thedetermination of glucose[J]. J Bio Chem,1944,153:375-380.
[51] Somogy M. Notes on sugar determination[J]. J Bio Chem,1952,195:19-23.
[52] Miller G L. Use of dinitrosalicylic acid reagent for determination of reducingsugar[J]. Anal Chem,1959,31:426-428.
[53]中华人民共和国国家标准GB8267-2006食品添加剂糖化酶制剂[S].
[54]刘洋，唐芳荣，相宏宇，等.小体系高通量比色测定糖化酶新方法[J].中国酿造，2012,140:140-143.
[55] Takahashi T, Inokuchi N, Irie M. Purification and characterization of aglucoamylase from Aspergillus saitoi[J]. J Biochem (Tokyo),1981,89:125–134.
[56] Semimaru T, Goto M, Furudawa K, et al. Functional analysis of the threonineand serine-rich Gp-1domain of glucoamylase I from A. awamori varkawachi[J]. Appl Environ Microbiol,1995,61:2885–2890.
[57] Jafari-Aghdam J, Khajeh K, Ranjbar B, et al. Deglycosylation of glucoamylasefrom Aspergillus niger: effects on structure, activity and stability[J]. BiochimBiophys Acta,2005,1750:61–68
[58] Svensson B, Larsen K, Svendsen I, Boel E. The complete amino acid sequenceof the glycoprotein, glucoamylase G1, from Aspergillus niger[J]. Carlsberg ResCommun,1983,48:529–544.
[59] Gunnarsson A, Svensson B, Nilson B, et al. Structural studies on theO-glycosidically linked carbohydrate chains of glucoamylase G1fromAspergillus niger[J]. Eur J Biochem,1984,145:463–467.
[60] Aleshin AE, Golubev A, Firsov LM, et al. Crystal structure of glucoamylasefrom Aspergillus awamori var. X100–2.2resolution[J]. J Biol Chem,1992,267:19291–19298.
[61] Jensen B, Olsen J. Amylases and their inudstrial potential. In Johri BN,Satyanarayana T, Olsen J, eds. Thermophilic Moulds in Biotechnology (pp.115–137).1999, The Netherlands: Kluwer Academic Publishers.
[62] Boel E, Hjort I, Svensson B, et al. Glucoamylases G1and G2from Aspergillusniger are synthesized from two different but closely related mRNAs[J]. EMBOJ,19843:1097–1102.
[63] Nunberg JH, Meade JH, Cole G, et al. Molecular cloning and characterizationof the glucoamylase gene of Aspergillus awamori[J]. Mol Cell Biol,1984,4:2306–2315.
[64] Takahashi T, Tsuchida Y, Irie M. Isolation of two inactive fragments ofRhizopus sp. glucoamylase: relationship among three forms of the enzyme andthe isolated fragments[J]. J Biochem (Tokyo),1982,92:1623–1633.
[65] Pretorius I S, Lambrechts M G, Marmur J. The glucoamylase multigene familyin Saccharomyces cerevisiae var. diastaticus: an overview[J]. Crit Rev BiochemMol Biol,1991,26:53–76.
[66] Chen J, Li D C, Zhang Y Q, et al. Purification and characterization of athermostable glucoamylase from Chaetomium thermophilum[J]. J Gen ApplMicrobiol,2005,51:175–181.
[67] Marin-Navarro J, Polaina J. Glucoamylases: structural and biotechnologicalaspects[J]. Appl Microbiol Biotechnol,2011,89:1267-1273.
[68] Lee J, Partzel M. Structure of the catalytic domain of glucoamylase fromAspergillus niger[J]. Acta Crystallogr F,2011, F67:188-192.
[69] Sauer J, Sigurskjold B W, Christensen U, et al. Glucoamylase:structure/function relationships, and protein engineering[J]. Biochim BiophysActa,2000,1543:275–293.
[70] Kuamr P, Satyanarayana T. Microbial glucoamylases: characteristics andapplications[J]. Crit Rev Biotechnol,2009,29:225-255.
[71] Synowiecki J. The use of starch processing enzymes in the food industry. InPolaina J, Mac Cabe AP, eds. Industrial Enzymes, Structure, Function andApplications (pp.19–34).2007, The Netherlands: Springer.
[72] Vainio A E I, Lantto R, Parkkinen E E M, et al. Production of Hormoconisresinae glucoamylase P by a stable industrial strain of Saccharomycescerevisiae[J]. Appl Microbiol Biotechnol,1994,41:53–57.
[73] Kumar P, Satyanarayana T. Potential applications of microbial enzymes inimproving quality and shelf life of bakery products. In Koutinas A, Pandey A,Larroche C, eds. Current Topics on Bioprocesses in Food Industry (pp.132–142),2008, New Delhi, India: Asiatech Publishers.
[74] Fukuda T, Kato-Murai M, Kuroda K, et al. Improvement in enzymatic desizingof starched cotton cloth using yeast codisplaying glucoamylase andcellulose-binding domain[J]. Appl Microbiol Biotechnol,2008,77:1225–1232.
[75] Kelly S M, Jess T J, Price N C. How to study proteins by circular dichroism[J].Biochim Biophys Acta,2005,1751:119–139.
[76]刘跃.芽孢杆菌产β-甘露聚糖酶的纯化及其稳定性研究[D].天津：天津大学.2008.
[77] Yamamote T, Fukui N, Hori A, et al. Circular dichroism and fluorescencespectroscopy studies of the effect of cyclodextrins on the thermal stability ofchicken egg white lysozyme in aqueous solution[J]. J Mol Struct,2006,782:60-66.
[78] Mundi S, Aluko R E. Effects of NaCl and pH on the stuctrual conformations ofkidney bean vicilin[J]. Food Chem,2013,139:624-630.
[79]王守业，徐小龙，刘清亮，等.荧光光谱在蛋白质分子构象研究中的应用[J].化学进展,2001,7:257-260.
[80] Li L, Muriel S. Study of the acid and thermal stability of β-lactoglobulin-ligand complexes using fluorescence quenching[J]. Food Chem,2012,132:2023-2029.
[81] Bakkialakshmi S, Shanthi B, Bhuvanapriya T. Study of fluorescence quenchingof Barley α-amylase[J]. Spec Acta A,2012,90:12-17.
[82] Shen, C L, Scott, G L, Merchant, F. Light-scattering analysis of fibril growthfrom the amino-terminal fragment β (1-28) of β-Amyloid Peptide[J].Biophysical Journal,1993,65:2383–2395.
[83]李绍新.动太光散射技术在生物大分子测量上的应用[D].广州：华南师范大学.2007.
[84] Shiba K, Niidome T, Katoh E, et al. Polydispersity as a parameter for indicatingthe thermal stability of proteins by dynamic light scattering[J]. Anal Sci,2010,26:659-663.
[85] Su R G, Qi W, He Z, et al. Multilevel structural nature and interactions of bovineserum albumin during heat-induced aggregation process[J]. Food Hydrocolloid,2008,22:995-1005.
[86] Iyer P V, Ananthanarayan, L. Enzyme stability and stabilization-Aqueous andnon-aqueous environment[J]. Process Biochem,2008,43:1019-1032.
[87] Lee J, Lin E W, Lau U Y, et al. Trehalose glycopolymers as excipients forprotein stabilization[J]. Biomacromolecules,2013,14:2561-2569.
[88] Andrioli W J, Silva T M, Silva V B, et al. The fungal metabolite eugenitin asadditive for Aspergillus niveus glucoamylase activation[J]. J Mol CatalB-Enzym,2012,74:156-161.
[89] Andrioli W J, Damasio A R L, Silva T M, et al. Endo-xylanase GH11activationby the fungal metabolite eugenitin[J]. Biotechnol Lett,2013,34:1478-1492.
[90] Usha R, Ramam S S, Subramanian V, et al. Role of polyols (erythritol, xylitoland sorbitol) on the structural stabilization of collagen[J]. Chemical PhysicsLetters,2006,430:391-396.
[91] Timasheff S N. Protein–solvent preferential interactions, protein hydration, andmodulation of biochemical reaction by solvent components[J]. Proc Natl AcadSci USA,2002,99:9721–9726.
[92] Bolen D W, Baskakov I V. The osmophobic effect: natural selection of athermodynamic force in protein folding[J]. J Mol Biol,2001,310:955–963.
[93] Wang A, Bolen D W. A naturally occurring protective system in urea-rich cells:mechanism of osmolyte protection of proteins against urea denaturation[J].Biochemistry,1997,36:9101–9108.
[94] Caves M S, Derham B K, Jezek J, et al. Thermal inactivation of uricase (urateoxidase): mechanism and effect of additives[J]. Biochemistry,2013,52:497-507.
[95] Chanansattru W, Decker E A, Mcclements D J. Influence of glycerol andsorbitol on thermally induced droplet aggregation in oil-in-water emulsionsstabilized by β-lactoglobulin[J]. Food Hydrocolloid,2009,23:995-1005.
[96] Sathish H A, Kumar P R, Prakash V. Mechanism of solvent induced thermalstabilization of papain[J]. Int J Biol Macromol,2007,41:383-390.
[97] Ohtake S, Wang Y J. Trehalose: current use and future applications[J]. JPharmacol Sci,2011,100:2020-2049.
[98] Timasheff S N. Conrtol of protein stability and reactions by weakly interactingcosolvents: the simplicity of the complicated[J]. Adv Protein Chem,1998,51:355-432.
[99] Slade L. Beyond water activity: recent advances based on an alternativeapproach to the assessment of food quality and safety[J]. Crit Rev Food SciNutr,1991,30:115-360.
[100] Crowe J H. Anhydrobiosis: an unsolved proplem[J]. The American Naturalist,1971,105:563-573.
[101] Hedoux A, Paccou L, Achir S, et al. Mechanism of protein stabilization bytrehalose during freeze-drying analyzed by in situ micro-ramanspectroscopy[J]. J Pharmacol Sci,2013,102:2484-2494.
[102]李杰.海藻糖在碳酸酐酶II的去折叠和再折叠过程中的保护作用[D].杭州:浙江大学.2007.
[103] Grubhofer N, Schleith L. Modifizierte ionenuascher als spezifischeadsobrentien[J]. Naturwissenschaften,1953,40:508.
[104] Sundaram P V, Kendal P E. Enzyme Engineering, Plenum Perss，NewYorkand London.1973,2nd ed: P449-452.
[105] Trevan M D. Immobilized Enzymes: an introduction and application inbiotechnology. John Wiley and Sons, Chichester, NewYork, Brisbane, Toronto,1980,1st ed.
[106] Adlercreutz P. Immobilisation and application of lipases in organic meidia[J].Chem Sov Rev,2013,42:6406-6436.
[107] Zhao L F, Zheng L Y. Resolution of2-octanol via immobilized Pseudomonassp lipase in organic medium[J]. Biocatal Biotransfor,2011,29:47-53.
[108] Kharrat N, Ali Y B, Marzouk S, et al. Immobilization of Rhizopus oryzaelipase on silica aerogels by adsorption: comparion with the free enzyme[J].Process Biochem,2011,46:1083-1089.
[109] Bernfeld P, Wan J. Antigens and enzymes made insoluble by entrapping theminto lattice of synthetic polymers[J]. Science,1963,142:678-679.
[110] Ungurean M, Paul C, Peter F. Cellulase immobilized by sol-gel entrapment forefficient hydrolysis of cellulose[J]. Bioprocess Biosyst Eng,2013,36:1327-1338.
[111] Wang Y, Guan Y, Yang Y, et al. Enhancing the stability of immobilizedcatalase on Activated Carbon with gelatin encapsulation[J]. J Appl Polym Sci,2013,1458-1502.
[112] Damnjanpvic J, Zuza M G, Savanovic J K, et al. Covalently immobilizedlipase catalyzing high-yielding optimized geranyl butyrate synthesis in abatch and fluidized bed reactor[J]. J Mol Catal B-Enzym,2012,75:50-59.
[113] Schoffer J N, Klein M P, Rodrigues R C, et al. Continuous production ofβ-cyclodextrin from starch by highly stablecyclodextrin glycosyltransferaseimmobilized on chitosan[J]. Carbohyd Polym,2013,98:1311-1316.
[114] Nwagu T N, Aoyahi H, Okolo B N, et al. Immobilization of a saccharifyingraw starch hydrolyzing enzyme on functionalized and non-functionalizedsepabeads[J]. J Mol Catal B-Enzym,2012,78:1-8.
[115] Gupta P, Dutt K, Misra S,et al. Characterizaiton of cross-linked immobilizedlipase from thermophilic mould Thermomyces lanuginose usinggluraraldehyde[J]. Bioresource Technol,2009,100:4074-4076.
[116]王静，贾英民，邹磊，等.固定化酶生产低乳糖牛乳的研究[J].现代食品科技，2008，24:791-7951.
[117] Anjank B, Parimal C, Utpal R, et al. A biosensorbased on co immobilized L-glutamate oxidase and L-glutamate d ehydrogenase for analysis of m onosodium glutam ate in food [J]. Biosens Bioelectron,2006,21:1968-19721.
[118] Negi S, Gupal S, Banerjee R. Extraction and purification of glucoamylase andprotease produced by Aspergillus awamori in a single-stage fermentation[J].Food Technol Biotechnol,2011,49:310-350.
[119] Riaz M, Rashid M H, Sawyer L, et al. Physiochemical properties and kineticsof glucoamylase produced from deoxy-D-glucose resistant mutant ofAspergillus niger for soluble starch hydrolysis[J]. Food Chem,2012,130:24-30.
[120] Wallis G L F, Swift R J, Hemming F W, et al. Glucoamylase overexpressionand secretion in Aspergillus niger: analysis of glycosylation[J]. BiochimBiophys Acta,1999,1472:576-586.
[121] Thorsen T S, Johnsen A H, Josefsen K, et al. Identification andcharacterization of glucoamylase from the fungus Thermomyceslanuginosus[J]. Biochim Biophys Acta,2006,1764:671-676.
[122] Vita C D D, Fontana A. Limited proteolysis of thermolysin by subtilisin,isolation and characterization of a partially active enzyme derivative[J].Biochemistry,1985,24:1798-1806.
[123] Wong Y H, Tayyab S. Protein stabilizing potential of simulated honey sugarcocktail under various denaturation conditions[J]. Process Biochem,2012,47:1933-1943.
[124] Zaroog M S, Tayyab S. Formation of molten globule-like state during aciddenaturation of Aspergillus niger glucoamylase[J]. Process Biochem,2012,47:775-784.
[125] Kamiyama T, Satoh M, Tateishi T, et al. Effects of modified β-cyclodextrin onthermal stability and conformation of lysozyme[J]. Thermochim Acta,2012,532:10-14.
[126] Negi S, Banerjee R. Study of conformational changes in glucoamylase ofAspergillus awamori nakazawa in presence of denaturants throughCD-spectroscopy[J]. Bioresource Technol,2010,101:7577-7580.
[127] Fu D Y, Li C, Lu J K, et al. Relative between thermal inactivation andconformational change of Yarrowia lipolytica lipase and the effect ofadditives on enzyme stability[J]. J Mol Catal B-Enzym,2010,66:136-141.
[128] Karabacak M, Sinba L, Prasad O, et al. An experimental and theoreticalinvestigation of Acenaphthene-5-boronic acid: Conformational study, NBOand NLO anaylsis, molecular structure and FT-IR, FT-Raman, NMR and UVspectra[J]. Spec Acta A,2013,115:754-766.
[129] Jqrgensen A D, Nqhr J, Kastrup J S, et al. Small angle X-ray studies revealthat Aspergillus niger glucoamylase has a defined extended conformation andcan form dimers in solution[J]. J Biol Chem,2008,283:14772-14780.
[130] Johnson, W. C. Analyzing protein circular dichroism spectra for accuratesecondary structures[J]. Proteins—Structure Function and Genetics,1999,35,307–312.
[131] Whitmore, L.,&Wallace, B. A. DICHROWEB, an online server for proteinsecondary structure analyses from circular dichroism spectroscopic data[J].Nucleic Acids Research,2004,32, W668–W673.
[132] Zhang X Y, Cao T L, Tian X D, et al. Effect of microwave irradiation on thestructure of glucoamylase[J]. Process Biochem,2012,47:2323-2328.
[133] Arntfield S D, Ismond M A H, Murray E D. Use of intrinsic fluorescence tofollow the denaturation of vicilin, a storage protein from Vicia faba.International[J]. Journal of Protein and Peptide Research,1987,29:9–20.
[134] Pica A, Krauss I R, Castellano I, et al. Exploring the unfolding mechanism ofγ-glutamyltranspeptidases: The case of the thermophilic enzyme fromGeobacillus thermodenitrificans[J]. Biochim Biophys Acta,2012,1824:571-577.
[135] Venkatesu P, Lee M J, Lin H M. Thermodynamic characterization of theosmolyte effect on protein stability and the effect of GdnHCl on the proteindenatured state[J]. J Phys Chem B,2009,111:9045–9056
[136] Kumar V, Sharma V K, Kalonia D S. Effect of polyols on polyethylene glycol(PEG)-induced precipitation of proteins: Impact on solubility, stability andconformation[J]. Int J Pharmaceut,2009,366:38-43.
[137] Kumar A, Attri P, Venkatesu P. Effect of polyols on the native structure ofα-chymotrypsin:Acomparable study[J]. ThermochimActa,2012,536:55-62.
[138] Palaniappan L, Velusamy V. Impact of cosolvent (glucose) on the stabilizationof ovalbumin[J]. Food Hydrocolloid,2013,30:217-223.
[139] Esposito A, Comez L, Cinelli S, et al. Influence of glycerol on the structureand thermal stability of lysozyme: A dynamic light scattering and circulardichroism study[J]. J Phys Chem B,2009,113:16420–16424.
[140] Chi M C, Wu T J, Chen H L, et al. Sorbitol counteracts temperature andchemical-induced denaturation of a recombinant α-amylase from alkaliphilicBacillus sp. TS-23[J]. J Ind Microbiol Biotechnol,2012,39:1779-1788.
[141] Azizi A, Ranjbar B, Khajeh K, et al. Effect of trehalose and sorbitol on theactivity and structure of Pseudomonas cepacia lipase: Spectroscopicinsight[J]. Int J Biol Macromol,2011,49:652-656.
[142] Devaraneni P K, Mishra N, Bhat R. Polyol osmolytes stabilize native-likecooperative intermediate state of yeast hexokinase A at low pH[J]. Biochimie,2012,94:947-952.
[143] Dong X Y, Liu J H, Liu F F, et al. Self-interaction of native and denaturedlysozyme in the presence of osmolytes, L-arginine and guanidinehydrochloride[J]. Biochem Eng J,2009,43:321-326.
[144]李彬春.嗜热脂肪酶NC-Loop对催化活性-稳定性和趋异进化的作用[D].长春：吉林大学.2012.
[145]王越.渗透压补偿性溶质ECTONINE对酶稳定性影响的研究[D].大连：大连海事大学.2011.
[146] Ragoonanan V, Aksan A. Protein stabilization[J]. Transfus Med Hemoth,2007,34:246-252.
[147] Gangadhara, Kumar P R, Prakash V. The stabilizing effects of polyols andsugars on porcine pancreatic lipase[J]. J Am Oil Soc,2009,86:773-781.
[148] Gangadhara, Kumar P R, Prakash V. Influence of polyols on the stability andkinetic parameters of invertase from Candida utilis: correlation with theconformational stability and activity[J]. Protein J,2008,27:440-449.
[149] Nazari-Robati M, Khajeh K, Aminian M, et al. Co-solvent mediated thermalstabilization of chondroitinase ABC I form proteus vulgaris[J]. Int J BiolMacromol,2012,50:487-492.
[150] Hernandez K, Fernandez-Lafuente R. Control of protein immobilization:coupling immobilization and site-directed mutagenesis to improve biocatalystor biosensor performance[J]. Enzyme Microb Tech,2011,48:107-122.
[151] Ashly P C, Joseph M J, Mohanan P V. Activity of diastase α-amylaseimmobilized on polyanilines (PANIs)[J]. Food Chem,2011,127:1808-1813.
[152] Hilterhaus L, Minow B, Muller J, et al. Parctial application of differentenzymes immobilized on sepabeads[J]. Bioprocess Biosyst Eng,2008,31:163-171.
[153] Prlainovic N Z, Knezevic-Jugovic K, et al. Immobilization of lipase fromCandida rugosa on Spedabeads: the effect of lipase oxidation by periodates[J].Bioprocess Biosyst Eng,2011,34:803-810.
[154] Bayraktar H, Serilmez M, Karkas T, et al. Immobilizaiton and stabilization ofα-galactosidase on Sepabeads EC-EA and EC-HA[J]. Int J Biol Macromol,2011,49:855-860.
[155] Li G Q, Li Q S. Thermophilic eaterase from the archaeon Archaeoglobusfulgidus physically immobilized on hydrophobic macroporous resin: A novelbiocatalyst for polyester synthesis[J]. Biotechnol Bioproc E,2011,16:1201-1207.
[156] Wang J Z, Zhao, G H, Li Y F, et al. Reversible immobilization ofglucoamylase onto magnetic chitosan nanocarriers[J]. Appl MicrobiolBiotechnol,2013,97:681-692.
[157] Silva R N, Asquieri E R, Fernandes K F, et al. Immobilization of Aspergillusniger glucoamylase onto a polyaniline polymer[J]. Process Biochem,2005,40:1155-1159.
[158] Panek A, Pietrow O, Synowecki J. Characterization of glucoamylaseimmobilized on magnetic nanoparticles[J]. Starch-Starke,2012,64,1003-1008.
[159] Zhao G H, Li Y F, Wang J Z, et al. Reversible immobilization of glucoamylaseonto magnetic carbon nanotubes functionalized with dendrimer[J]. ApplMicrobiol Biotechnol,2011,91:591-601.
[160]赵丽芳.脂肪酶的固定化及其催化性能的研究[D].长春：吉林大学.2008.
[161] Bai Y X, Li Y F, Wang M T. Study on synthesis of a hydrophilic bead carriercontaining epoxy groups and its properties for glucoamylaseimmobilization[J]. Enzyme Microb Tech,2006,39:540-547.
[162]吴卓夫.硅基介质固定化酶的研究[D].长春：吉林大学.2009.
[163] Zhao G H, Wang J Z, Li Y F, et al. Reversible immobilization of glucoamylaseonto metal-ligand functionalized magnetic[J]. Biochem Eng J,2012,68:159-166.
[164] Nwagu, T W, Okolo B N, Aoyagi H. Stabilization of a raw starch digestingamylase from Aspergillus carbonarius via immobilization on activated andnon-activated agarose gel[J]. World J Microb Biot,2012,28:335-345.
[165] Okutucu B, Celem E B, Onal S. Immobilizaiton of α-galactosidase ongalactose-containing polymeric beads[J]. Enzyme Microb Tech,2010,46:200-205.
[166] Khan M J, Husain Q, Ansari S A. Polyaniline-assisted silver nanoparticles: anovel support for the immobilization of α-amylase[J]. Appl MicrobiolBiotechnol,2013,97:1513-1522.
[167] Dib I, Nidetzky B. The stabilizing effects of immobilization in D-amino acidoxidase from Trigonopsis variabilis[J]. BMC biotechnol,2008,8:72-82.
[168] Torres P, Batista-Viera F. Improved biocatalysts based on Bacillus cirulansβ-galactosidase immobilized onto epoxy-activated acrylic supports:Applications in whey processing[J]. J Mol Catal B-Enzym,2012,83:57-64.
[169] Singh V, Singh D. Polyvinyl alcohol-silica nanohybrids: an efficient carriermatrix for amylase immobilization[J]. Process Chem,2013,48:96-102.
[170] Singh V, Ahmad S. Synthesis and characterization of carboxymethylcellulose-silver nanoparticle (AgNp)-silica hybrid for amylaseimmobilization[J]. Cellulose,2012,19:1759-1769.
