摘要
扩张床吸附(EBA)技术是一种新型的生化分离技术，它集成了固液分离、浓缩和初期纯化于一步单元操作之中，可以直接从含有细胞和细胞碎片的发酵液或培养液中提取目标蛋白，而不必事先除去悬浮的固体颗粒。吸附剂基质是决定EBA技术能否成功应用的关键性因素，它首先必须具有合适的密度和粒径分布。
本文旨在通过“反相悬浮热再生”法制备一种纤维素-钛白粉复合微球，作为扩张床基质。使用环氧氯丙烷活化，然后与二乙胺反应，基质被衍生成一种阴离子吸附剂(Cell-Ti DEAHP)；另外，通过环氧氯丙烷交联后与氯乙酸反应，基质还被制成一种阳离子交换剂(cell-Ti CM)。考察了吸附剂在扩张床中的扩张行为、流体力学特性和蛋白质吸附能力。最后，所开发的阴离子吸附剂被应用于从细胞匀浆中提取脱卤酶，而阳离子交换剂则被用来从发酵液中提纯纳豆激酶。
全文共分为四个部分。第一部分集中综述了扩张床吸附剂基质的研究进展，包括基质所需要的理化性质、常见的扩张床基质和它们的制备方法。在分析和比较已有扩张床基质优缺点的基础上，作者提出了自己的研究思路，即采取再生纤维素和超细钛白粉分别作为反应性骨架材料和增重剂，复合制备出球形亲水性扩张床基质。此外，还建立了一系列表征所制备的扩张床基质和吸附剂的方法。
第二部分主要叙述纤维素-钛白粉复合扩张床基质和吸附剂的制备方法。详细研究了一些影响复合微球形成的因素，得到较优的工艺条件为：纤维素黄原酸酯粘胶粘度为5000～8000cSt，分散相为6:1(w／w)泵油和氯苯混合物，搅拌速度为350～400rpm。在此工艺条件下，复合基质具有规则的球形外观和与商业Streamline系列基质相当的粒径分布。研究表明，钛白粉加量的增加会使基质的密度线性增加，但对基质的孔结构影响不大，说明超细颗粒已被有效地包埋进再生纤维素骨架之中。由此制得了一种典型的复合基质(Cell-Ti)的理化性质如下：密度1.21g／mL、比表面积38.7m~2／mL、孔度83.7％和含水率69.5％。
此后，为了得到足够的环氧基团用于功能基化，详细考察了一些影响活化反应的因素，如环氧氯丙烷的用量、NaOH溶液的浓度、纤维素和钛白粉的含量等。当NaOH溶液浓度为2.5～3.0mol／L且环氧氯丙烷相对过量时，活化基质中的环氧基含量达220μmol／mL，由此产生的阴离子吸附剂的离子交换容量为0.20mmol Cl~-／mL。基质经过环氧氯丙烷交联后机械强度得到了很大的提高，用氯乙酸功能基化后的离子交换容量为0.22mmol Na~+／mL。扫描电镜照片显示复合基质具有大孔结构。
第三部分测试了所开发吸附剂的扩张床性能和蛋白质吸附能力。研究表明，Cell-Ti DEAHP的扩张行为遵循Richardson-Zaki关系式，在水流动相中的扩张指数为5.2，终点沉降速度约为1960cm／h。吸附剂起始装填高度的降低和流体相粘
摘要
度的增加会使扩张床中的轴向混合程度加剧。尽管如此，在实验范围内，所开发
的吸附剂在扩张床内的流动仍然可以近似为平推流。
研究发现，cell一TIDEAHP和cell一TicM的吸附等温线都遵从Lan脚uir等温
式。其中，cell一Ti DEAHP的平衡吸附容量Qm为61 mg/mL BsA，解离常数局
为0.11 mg/mL;而cell一Ti cM的Qm为98.7 mg/mLlysozyme，幻为0.349mg/担L。
测试了固定床和扩张床中不同初始浓度和吸附剂装填高度下的蛋白质穿透行为，
表明随着流速和初始浓度的增加，以及装填高度的下降，穿透曲线的斜率变缓;
从穿透行为来看，扩张床中的吸附效率与固定床中的相当。用Hall方程来评价和
预测扩张床中蛋白质的穿透行为，但需要事先测定相同初始浓度下的固定床穿透
曲线。研究证明，在一定的流速范围以内Hall模型计算值能与实验结果相符合。
第四部分是吸附剂的纯化应用实例。将阴离子吸附剂Cell一Ti DEAHP应用于
从细胞匀浆中提取脱卤酶，总收率为68%，纯化倍数达22倍;将阳离子吸附剂
cell一Ti cM应用于从发酵液中提取纳豆激酶，收率达89%，纯化倍数为5，6倍。
较之于传统的提取工艺，扩张床吸附技术节省了大量的操作时间和生产成本，充
分显示出其高效和集成化的特点。
Expanded bed adsorption (EBA) is a novel bioseparation technique, which integrates clarification, concentration and initial purification into a single unit operation. It enables proteins to be recovered directly from unclarified cultivations of microorganisms or cells and homogenates of disrupted cells, without the need for prior removal of suspended solids. Matrix is the principal "hardware" pillar supporting the successful application of EBA. The basic criteria of the matrices for EBA are formulated as being a sufficiently high density and an appropriate size distribution.
The purpose of this work is to develop a spherical TiO2-densified cellulose composite matrix for EBA, through the method of water-in-oil suspension thermal regeneration. After activated by epichlorohydrin and coupling with diethylamine, the matrix was derived to function as an anion exchanger (Cell-Ti DEAHP). The matrix was also crosslinked by epichlorohydrin and attached to monochloroacetic acid to produce a cation exchanger (Cell-Ti CM). The expansion behavior, hydrodynamic properties and protein adsorption capacities of two adsorbents were investigated. Finally, the anion exchanger was used to recover dehalogenase from unclarified cell homogenate, while the cation exchanger was introduced to purify nattokinase directly from fermentation broth.
The thesis is divided into four sections. The focus of Section 1 is the review of advances in matrices for EBA, including physicochemical properties required, matrices in use as well as their preparation methods. On the base of analyzing and comparing merits and drawbacks of the available matrices for EBA, the author put forward his study idea, which adopted the composite of regenerated cellulose and superfine TiO2, respectively as reactive matrix and densifier, to prepare a spherical hydrophilic matrix for EBA. Furthermore, a series of methods for analysis and characterization of the prepared matrix or adsorbent were established.
Section 2 involves the preparation of the composite matrix and its derivation to function as ion exchangers. Some factors influencing the forming of cellulose-TiO2 beads were investigated in details. The optimal preparing conditions were found to be as follows: the initial viscosity of cellulose xanthate ranging from 5,000 to 8,000 cSt, the mixture of pump oil and chlorobenzene with a 6:1 mass ratio as disperse phase, the mass ratio of disperse phase to water phase at 6:1, the mixing speed at 350 ~ 400 rpm. Under these conditions, the prepared matrix had regular sphericity and a similar size distribution to that of the commercial Streamline matrix. The study also shows that the increase of TiO2 content lead to the ascent of particle density but had few effects on the pore structure, confirming that superfine TiO2 had been successfully entrapped in the regenerated cellulose matrix. A typical matrix (Cell-Ti) with some physical properties as follows: wet density 1.21 g/cm3, specific surface area 38.7 m2/mL, po
rosity 83.7%, and water content 69.5%, was attained.
In order to hold more epoxy groups for subsequent ionization, several factors affecting the efficiency of activation reaction, such as the amount of epichlorohydrin, the concentration
of NaOH solution, and the contents of TiO2 and cellulose in matrix, were investigated emphatically. Reacted with excessive epichlorohydrin and dipped in 2.5-3.0 M NaOH solution, the content of epoxy groups in activated Cell-Ti was found to be up to 220 μ mol/mL. This content resulted in a high anion exchange capacity of 0.2 mmol Cl-/mL. Epichlorohydrin was also used to crosslink chemically the composite matrix, showing improved mechanical intensity. After coupling with monochloroacetic acid, the matrix was functioned as a cation exchange, whose exchange capacity was found to be 0.22 mmol Na+/mL. SEM photography shows that Cell-Ti had a macroporous structure.
In Section 3, the 5th chapter studies the expansion characteristics of the aimed adsorbents and their liquid mixing performance in an expanded bed, while the 6th chap
引文
1．贺林主编，解码生命-人类基因组计划和后基因组计划，科学出版社，北京，2000
2. Hieter P and Boguski M, Functional genomics: it is all how you read it, Science, 1997, 278 (5338): 601-602
3．孙彦，生物分离工程，化学工业出版社，北京，1998
4．俞俊棠，唐孝宣主编，生物工艺学(上)，华东化工学院出版社，上海，1991
5．严希康，生化分离技术，华东理工大学出版社，上海，1996
6．梅乐和，姚善泾，林东强，朱自强，生物分离过程研究的新趋势—高效集成化，化学工程，1999，27(5)：38—41
7．胡洪波，扩张床吸附过程的研究-安通溶栓酶的分离和纯化[博士学位论文]，浙江大学，杭州，1999
8．林东强，朱自强，姚善泾，梅乐和，生化分离过程的新探索—双水相分配及相关技术的集成化，化工学报，2000，51：1—6
9. McCormick D K, Expanded bed adsorption-the first new unit process operation in decades, Bio/Technology, 1993, 11: 1059-1059
10. Elias K, Affinity membranes: a 10-year review, J. Membr. Sci., 2000, 179:1-27
11. Amersham Pharmacia Biotech, Expanded bed adsorption-Principles and Methods, Code No. 18-1124-26, Uppsala, Sweden, 1998
12. Anspach F B, Curbelo D, Hartmann R, Garke G and Deckwer W-D, Expanded bed chromatography in primary protein purification, J. Chromatogr. A, 1999, 865: 129-144
13. Lee S M, The primary stages of protein recovery, J. Biotechnol., 1989, 11: 103-118
14. Datar R and Rosen C G, Centrifugal separation in the recovery of intracellular protein from E. coli, Chem. Eng. J., 1987, 34:49-56
15. Asenjo J A, Separation Processes in Biotechnology, Marcel Dekker, 1990
16. Datar R V, Cartwright T and Rosen C G, Process economics of animal cell and bacterial fermentations: a case study analysis of tissues plasminogen activator, Bio/Technology, 1993, 11:349-357
17. Fish N M and Lilly M D, The interactions between fermentation and protein recovery, Bio/Technology, 1984, 2: 623-627
18. Spalding B J, Downstream processing: key to slashing production costs 100 fold, Bio/Technology, 1991, 9:229-233
19. Thmmes J, Bader A, Halfar M, Karau A and Kula M-R, Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A coated adsorbent in expanded beds, J. Chromatogr A, 1996, 752:111-122
20. Nigam S C, Sakada A and Wang H Y, Bioseparation recovery from unclarified broths and homogenates using immobilised adsorbents, Biotechnol. Prog., 1988, 4: 166-172
21. Gorden N F, Tsujimura H and Cooney C L, Optimization and simulation of continuous affinity recycle extraction, Bioseparation, 1990, 1: 9-21
22. Kula M-R, Trends and future prospects of aqueous two-phase extraction, Bioseparation, 1990, 1:181-189
23. Brummelhuis H G J, Methods of Plasma Protein Fractionation, Academic Press, London, 1980
24. Barthels C R, Kleinman G, Korzon N J and Irish D B, A novel ion-exchange method for the isolation of streptomycin, Chem. Eng. Prog., 1958, 54: 49-52
25. Belter P A, Cunningham F L and Chen J W, Development of a recovery process for novobiocin, Biotechnol. Bioeng., 1973, 15:533-549
26. Gailliot F P, Gleason C, Wilson J J and Zwarick J, Fluidized bed adsorption for whole broth extraction, Biotechnol. Prog., 1990, 6:370-375
27. Koh Joon-Ho, Ion exchange in fluidized/expanded beds for recovery of L-phenylalanine [Doctor thesis], Purdue University, West Lafayette, 1995
28．佟晓冬，流化床吸附分离蛋白质的基础研究[博士学位论文]，天津大学，天津，2002
29. Buijs A and Wesselingh J A, Batch fluidized ion-exchange column for streams containing suspended particles, J. Chromatogr., 1980, 201: 319-327
30. Burns M A and Graves D J, Continuous affinity chromatography using a magnetically stabilized fluidized bed, Biotechnol. Prog., 1985, 1: 95-103
31. Nixon L, Koval C A, Xu L, Noble R D and Slaff G S, The effects of magnetic stabilization on the structure and performance of fluidized beds, Bioseparation, 1991, 2:217-230
32．官月平，朱星华，姜波，刘会洲，生物磁性分离研究进展(Ⅱ)-磁性分离装置和生物技术应用，化工学报(增刊)，2000，51：320-324
33. Draeger M N and Chase H A, Liquid fluidized beds for protein purification, Ⅰ. Chem. Eng., 1990, Symp. Ser. No. 118:12.1-12.12
34. Draeger M N and Chase H A, Liquid fluidized bed adsorption of proteins in the presence of cells, Bioseparation, 1991, 2:67-80
35. Chase H A and Draeger M N, Expanded bed adsorption of proteins using ion-exchangers, Sep. Sci. Technol., 1992, 27:2021-2039
36. Chase H A and Draeger M N, Affinity purification of proteins using expanded beds, J. Chromatogr., 1992, 597:129-145
37. Schmidt C, Expanded bed adsorption—A new way for industrial recovery of recombinant proteins, Poster presented at New Zealand Biotech Association Meeting, New Zealand, 1993
38. Chase H A, Purification of proteins by adsorption chromatography in expanded beds, Trends Biotechnol., 1994, 12:296—303
39. Levenspiel O, Chemical Reaction Engineering, 3rd Edition, Wiley, New York, 1999
40. Chang Y K and Chase H A, Development of operating conditions for protein purification using expanded bed techniques: the effect of the degree of bed expansion on adsorption performance, Biotechnol. Bioeng., 1996, 49:512—526
41. Hjorth R, Kampe S and Carlsson M, Analysis of some operating parameters of novel adsorbents for recovery of proteins in expanded bed adsorption, Bioseparation, 1995, 5:217-223
42. Batt B C, Yabannavar V M and Singh V, Expanded bed adsorption process for protein recovery from whole mammalian cell culture broth, Bioseparation, 1995, 5:41-52
43. Lindgren A, Johannson S and Nystrom L-E, Scale up of expanded bed adsorption, In: BED-
The 7th Bioprocess Engineering Symposium, Henon B eds., The American Society of Mechanical Engineering, 1993, 27-30
44. Ghose S, Chase H A and Hooker N T, Bed height monitoring and control for expanded bed chromatography, Bioprocess Eng., 2001, 23: 701-708
45. Thelen T V and Ramirez W F, Monitoring, modeling and control strategies for expanded-bed adsorption processes, Bioseparation, 1999, 8:11-31
46. Thelen T V and Ramirez W F, Bed-height dynamics of expanded beds, Chem. Eng. Sci., 1997, 52: 3333-3344
47. Chang Y K and Chase H A, Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption, Biotechnol. Bioeng., 1996, 49: 204-216
48. Hjorth R, Expanded bed adsorption: elution in expanded bed mode, Bioseparation, 1999, 8:1-9
49. Lihme A, Zafirakos E, Hansen M and Olander M, Simplified and more robust EBA processes by elution in expanded bed mode, Bioseparation, 1999, 8:93-97
50. Fee C J, Economics of wash strategies for expanded bed adsorption of proteins from milk with buoyancy-induced mixing, Chem. Eng. Process., 2001, 40:329-334
51. Owen R O and Chase H A, Direct purification of lysozyme using continuous counter-current expanded bed adsorption, J. Chromatogr. A, 1997, 757:41-49
52. Thmmes J, Halfar M, Lenz S and Kula M-R, Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption, Biotechnol. Bioeng., 1995, 45: 205-211
53. Ujam L B, Clemmitt R H and Chase H A, Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of E. coli and S. cerevisiae, Bioprocess Eng., 2100, 23:245-250
54. Ferreira G N M, Cabral J M S and Prazeres D M F, Anion exchange purification of plasmid DNA using expanded bed adsorption, Bioseparation, 2000, 9:1-6
55. Nandakumar M P, Tocaj A and Mattiasson B, Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis, Bioseparation, 1999, 8: 247-254
56. Mattiasson B and Nandakumar M P, Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column, Bioseparation, 1999, 8:237-245
57. Pllson E, Nandakumar M P, Mattiasson B and Larsson P-O, Miniaturised expanded-bed column with low dispersion suitable for fast flow-ELISA analyses, Biotechnol. Lett., 2000, 22: 245-250
58．金业涛，冯小黎，苏志国，扩张床吸附层析及其在生物化工中的应用，化工进展，1998，4：45—50
59．胡洪波，姚善泾，朱自强，膨胀床吸附层析及其在生化分离中的应用，化学工程，1999，27：137—143
60．刘坐镇，陈士安，邬行彦，扩张床吸附技术，离子交换与吸附，1999，15：279—288
61. Hjorth R, Expanded-bed adsorption in industrial bioprocessing: recent developments, Trends Biotechnol., 1997, 15:230-235
62. Willoughby N A, Kirschner T and Smith M P, Immobilized metal ion affinity chromatography
purification of alcohol dehydrogenase from baker's yeast using an expanded bed adsorption system, J. Chromatogr. A, 1999, 840:195-204
63. Pierce JJ, Fischer E J and Smith M P, Purification of a periplasmic enzyme by a stirred adsorbent, and by expanded and packed beds, Bioprocess Eng., 1999, 20:449-457
64. Noppe W, Haezebrouck P, Hanssens I and Curper M D, A simplified purification procedure of α-lactalbumin from milk using Ca~(2+)-dependent adsorption in hydrophobic expanded bed chromatography, Bioseparation, 1999, 8:153-158
65. Hansen M B, Lihme A, Spitali M and King D, Capture of human Fab fragments by expanded bed adsorption mode adsorbent, Bioseparation, 1999, 8: 189-193
66. Brobjer M, Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in E. coli, Bioseparation, 1999, 8:219-228
67. Clemmitt R H and Chase H A, Facilitated downstream processing of a Histidine-tagged protein from unclarified E. Coli homogenate using immobilized metal affinity expanded-bed adsorption, Biotechnol. Bioeng., 2000, 67: 206-216
68. Pai A, Gondkar S and Lali A, Enhanced performance of expanded bed chromatography on rigid superporous adsorbent matrix, J. Chromatogr. A, 2000, 867: 113-130
69. Gibert S, Bakalara N and Santarelli, Three-step chromatographic purification procedure for the production of a His-tag recombinant kinesin overexpressed in E. Coli, J. Chromatogr. B, 2000, 737:143-150
70. Clemmitt R H and Chase H A, Immobilised metal affinity chromatography of β-galactosidase from unclarified E. Coli homogenate using expanded bed adsorption, J. Chromatogr. A, 2000, 874:27-43
71. Garke G, Deckwer W-D and Anspach F B, Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of E. Coli, J. Chromatogr. B,2000, 737:25-38
72. Tong W-Y, Yao S-J and Zhu Z-Q, Separation characteristics of human epidermal growth factor in ion exchange chromatography with Streamline DEAE resin, Chem. Eng. Sci., 2001, 56: 6956-6965
73. Michel P, Torkkeli T, Karp M and Oker-Blom C, Expression and purification of polyhistidine-tagged firefly luciferase in insect cells-a potential altemative for process scale-up, J. Bioteehnol., 2001, 85:49-56
74. Nayak D P, Ponrathnam S and Rajan C R, Macroporous copolymer matrix Ⅳ. expanded bed adsorption application, J. Chromatogr. A, 2001, 922:63-76
75. Reichert U, Knieps E, Slusarczyk H, Kula M-R and Thommes J, Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption, J. Biochem. Biophys. Methods, 2001, 49:533-552
76. Blank G S, Zapata G, Fahrner R, Milton M, Yedinak C, Knudsen H and Schmelzer C, Expanded bed adsorption in the purification of monoclonal antibodies: a comparison of process alternatives, Bioseparation, 2001, 10:65-71
77. Giovannini R and Freitag R, Isolation of a recombinant antibody from cell culture supernatant: continuous annular versus batch and expanded-bed chromatography, Biotechnol. Bioeng.,
2001, 73:522-529
78. Ozyurt S, Kirdar B and Ulgen K O, Recovery of antithrombin Ⅲ from milk by expanded bed chromatography, J. Chromatogr. A, 2002, 944:203-210
79. Suh C W, Nah S J and Lee E K, Application of expanded bed adsorption chromatography to protein recovery from fusion protein cleavage, Abstract P. 43 from The 4th International Conference on Expanded Bed Adsorption, Florida, USA, 2002
80. Abdullah N and Chase H A, Recent developments in the use of IMAC in expanded bed purifications of recombinant proteins tagged with polyhistidine sequences, Abstract P. 46 from The 4th International Conference on Expanded Bed Adsorption, Florida, USA, 2002
81. Ujam L, Clemmitt R and Chase H A, Cell separation in expanded beds, Abstract P. 53 from The 4th International Conference on Expanded Bed Adsorption, Florida, USA, 2002
82. Lee Y S, Suh C W, Park S K and Lee E K, Purification of soluble hEGF from recombinant E. coli culture broth by using expanded bed adsorption chromatography, Abstract P. 58 from The 4th International Conference on Expanded Bed Adsorption, Florida, USA, 2002
83. Gonzalez Y, Ibarra N, Gomez H, Gonzalez M, Dorta L, Padilla S and Valdes R, Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography, J. Chromatogr. B, 2003, 784:183-187
84. Dieryck W, Noubhani A M, Coulon D and Santarelli X, Cloning, expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in E. coli, J. Chromatogr. B, 2003, 786: 153-159
85. Shiloach J, Santambien P, Trinh L, Schapman A and Boschetti E, Endostatin capture from Pichia pastoris culture in a fluidized bed from on-chip process optimization to application, J. Chromatogr. B, 2003, 790:327-336
86. Bamfield Frej A K, Hjorth R and Hammarstrom A, Pilot scale recovery of recombinant annexin V from unclarified E. coli homogenate using expanded bed adsorption, Biotechnol. Bioeng., 1994, 44:922-929
87. Barnfield Frej A K, Johansson H J, Johansson S and Leijon P, Expanded bed adsorption at production scale: scale-up verification, process example and sanitization of column and adsorbent, Bioprocess Eng., 1997, 16:57-63
88. Zapata G, Lindgren A, Barnfield Frej A K, Leijon P, Liten A and Mayes T L, Expanded bed adsorption chromatography purification of a monoclonal antibody, Abstract P.16 from Recovery of Biological Products Ⅷ, Tucson, USA, 1996
89. Tong X D and Sun Y, Nd-Fe-B alloy-densified agarose gel for expanded bed adsorption of proteins, J. Chromatogr. A, 2001, 943: 63-75
90. Lin D Q, Fern'andez-Lahore H M, Kula M-R and Thmmes J, Minimizing biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach, Bioseparation, 2001, 10: 7-19
91. Richardson J F and Zaki W N, Sedimentation and fluidization: part Ⅰ, Trans. Inst. Chem. Eng., 1954, 32:35-53
92. Chang Y K, McCreath G E and Chase H A, Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates, Biotechnol. Bioeng.,
1995, 48:355-366
93. Li Q, Grandmaison E W, Hsu C C, Taylor D and Goosen M F G, Interparticle and intraparticle mass transfer in chromatographic separation, Bioseparation, 1995, 5:189-202
94. Joshi J B, Solid-liquid fluidised beds: some design aspects, Chem. Eng. Res. Des., 1983, 61: 143-161
95. Mullick A, Griffith C M and Flickinger M C, Expanded and packed bed albumin adsorption on fluoride modified Zirconia, Biotechnol. Bioeng., 1998, 60:333-340
96. Griffith C M, Morris J, Robichaud M, Annen M J, McCormick A V and Flickinger M C, Fluidization characteristics of and protein adsorption on fluoride-modified porous Zirconium oxide particle, J. Chromatogr A, 1997, 776: 179-195
97. Voute N and Boschetti E, High dense beaded sorbents suitable for fluidized bed applications, Bioseparation, 1999, 8:115-120
98. Plsson E, Gustavsson P-E and Larsson P-O, Pellicular expanded bed matrix suitable for high flow rates, J. Chromatogr. A, 2000, 878:17-25
99. Finette G M S, Baharin B, Mao Q M and Hearn M T W, Optimization consideration for the purification of α_1-antitrypsin using silica-based ion-exchange adsorbents in packed and expanded beds, Biotechnol. Prog., 1998, 14:286-293
100. Gilchrist G R, Bums M T and Lyddiatt A, Solid phases for protein adsorption in liquid fluidized beds: comparison of commercial and custom-assembled particles, in: Separations for Biotechnology 3, Pyle D L eds., Royal Society of Chemistry, London, 1994, 186-192
101. McCreath G E, Chase H A and Owen R O, Expanded bed affinity chromatography of dehydrogenases from Baker's yeast using dye-ligand perfluoropolymer supports, Biotechnol. Bioeng., 1995, 48:341-354
102. AL-Dibouni M R and Garside J, Particle mixing and classification in liquid fluidised bed, Trans. Inst. Chem. Eng., 1979, 57: 94-103
103．雷引林，姚善泾，刘坐镇，朱自强，扩张床吸附基质研究进展，功能高分子学报，2002，15：219—224
104. Voute N, Bataille D, Girot P and Boschetti E, Characterization of very dense mineral oxide-gel composites for fluidized-bed adsorption of biomolecules, Bioseparation, 1999, 8:121-129
105. Kuga S, New cellulose gel for chromatography, J. Chromatogr., 1980, 195:221-230
106．雷引林，钱建华，姚善泾，刘坐镇，邬行彦，朱自强，一种大孔共聚物扩张床吸附剂基质的制备，第十届全国生物化工学术会议，2002，中国杭州，550—553
107．高洁，汤烈贵主编，纤维素科学，科学出版社，北京，1996
108．张力田，碳水化合物化学，轻工业出版社，北京，1988
109．杨之礼，苏茂尧，纤维素醚基础与应用，华南理工大学出版社，广州，1990
110．杨之礼，蒋听培，王庆瑞，邬国铭，纤维素与粘胶纤维，纺织工业出版社，北京，1985
111．印寿根，李朝兴，徐欢驰，李欣，何炳林，球状纤维素的制备及其应用，高分子通报，1996，(2)：100—104
112．唐爱民，梁文芷，纤维素的功能化，高分子通报，2000，(4)：1—9
113．朱伯儒，史作清，何炳林，球形纤维素吸附剂的制备和应用研究进展，精细化工，1996，13(3)：42—46
114. Stamberg J, Peska J, Dautzenberg H and Philipp B, Bead cellulose, in: Affinity Chromatography and Related Techniques, Gribnau T C J, Visser J and Nivard R J F eds., Elsevier, Amsterdam, 1982, 131-141
115. Peska J, Stamberg J and Pelzbauer Z, Regenerated cellulose in the bead form after treatments and their effects on the porous structure of cellulose, Cell. Chem. Technol., 1978, 21:419-428
116. Westman L and Lindstrom T, Swelling and mechanical properties of cellulose hydrogels Ⅰ. preparation, characterization, and swelling behavior, J. Appl. Polym. Sci., 1981, 26:2519—2532
117. Stamberg J, Bead cellulose, Separation and Purification Methods, 1988, 17:155—183
118. Peska J, Stamberg J and Hradil J, Chemical transformations of polymers ⅩⅨ. ion exchange derivatives of bead cellulose, Angew. Makromol. Chemic, 1980, 53:73-80
119. Boeden H-F, Pommerening K, Becker M, Rupprich C, Holtzhauer M, Loth F, Muller R and Bertram D, Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins, J. Chromatogr., 1991, 552:389-414
120．张中勤，宋清，球状再生纤维素离子交换剂的制备，离子交换与吸附，1998，14：23—30
121．曾庆冰，许家瑞，符若文，蛋白质分离色谱填料及其特性，功能高分子学报，2000，13：343—348
122. O'Neill J J and Reichardt E P, Cellulose pellets, U.S. Pat., 2,543,928, 1951
123. Yasui K, J.P. Pat., 73,34,082,
124. Determann H, Rehner H and Wieland T, Cellulose gel beads for chromatography, Makromol. Chem., 1968, 114:253-274
125. Klemm D, Philipp B, Heinze T, Heinze U and Wagenknecht W, eds., Comprehensive cellulose chemistry, Wiley-VCH, Chichester, 1998
126. Peska J, Stamberg J and Blace Z, Method for manufacturing of spherical cellulose particles, U.S. Pat., 4,055,510, 1977
127. Brown W, Partitioning in gel chromatography, Chem. Scr, 1972, 2:25-29
128．朱伯儒，史作清，何炳林，大孔球形纤维素离子交换剂的制备及其对蛋白质的吸附富集性能研究Ⅰ．大孔球形纤维素的制备研究，离子交换与吸附，1996，12：522—525
129．印寿根，李欣，徐欢驰，李朝兴，何炳林，珠状纤维素的粒径分布研究，离子交换与吸附，1997．13：127—132
130. Loth, Fritz, Fanter and Carola, Bead-shaped cellulose products for separating and carrier materials and their manufacture, U.S. Pat., 5,527,902, 1996
131．刘明华，李翔，刘卫国，张新申，一种新型球形纤维素吸附剂的研究，精细化工，2001，18：401—404
132．刘明华，刘卫国，张宏，张新申，刘邓斌，球形纤维素吸附剂对Cr~(3+)吸附和解吸的研究，化工环保，2001，21：1—5
133. Matejka Z, Bead cellulose anion exchange for ultra pure water, Effluent and Water Treatment Journal, 1984, 24:275-277
134. Gemeiner P, Benes M J and Stamberg J, Bead cellulose and its use in biochemistry and biotechnology, Chem. Papers, 1989, 43:805-848
135. Mislovicova D, Gemeiner P, Kuniak L and Zemek J, Affinity chromatography of rat liver
lactate dehydrogenase on the remazol derivative of bead cellulose, J. Chromatogr., 1980, 194: 95-99
136. Amicon, A Grace Co., Catalog/Membrane Filtration Chromatography, Publication No. 277E, Beverly, USA, 1993
137. Gemeiner P, Polakovic M, Mislovicova D and Stefuca V, Cellulose as a (bio)affinity carrier: properties, design and applications, J. Chromatogr. B, 1998, 715:245-271
138. Drobnik J, Labsky J, Kudlvasrova H, Saudek V and Svec F, The activation of hydroxy-groups of carriers with 4-nitrophenyl and N-hydroxysuccinimidyi chloroformates, Biotechnol. Bioeng., 1982, 24:487-493
139. Kminkova M, Proskova A and Kucera J, Immobilization of mold β-galactosidase, Collect. Czech. Chem. Commun., 1988, 10:568-573
140. Chen L F and Taso G T, Physical characteristics of porous cellulose beads as supporting material for immobilized enzymes, BiotechnoL Bioeng., 1976, 18:1507-1516
141. Baumann H, Linβen M and Keller R, Konzepte zur verbesserung der hmokompatibilitt von cellulose, Das Papie, 1990, 43:674-681
142. Arai M, J.P. Pat., 90,75,340
143．孔德领，代军，陈长治，俞耀庭，球形纤维素固定化DNA制备免疫吸附剂，高等学校化学学报，2000，21：1848—1851
144．付利，杨志兴，纳豆激酶的研究和应用，生物工程进展，1995，15(5)：46—49
145. Sumi H, Hamada H, Tsushima H, Mihara H and Muraki H, A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto: a typical and popular soybean food in the Japanese diet, Experiential, 1987, 43:1110-1111
146. Nakamura T, Yamagata Y and Ichishima E, Nucleotide sequence of the subtilisin NAT gene, aprn, of Bacillus subtilis (natto), Biosci. Biotech. Biochem., 1992, 56: 1869-1871
147. Fujita M, Nomura K, Hong K, Ito Y, Asada A and Nishimuro S, Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan, Biochem. Biophy. Res. Commun., 1993, 197:1340-1347
148. Fujita M, Hong K, Ito Y, Fujii R, Kariya K and Nishimuro S, Thrombolytic effect of nattokinaseon a chemically induced thrombosis model in rat, Biol. Pharm. Bull., 1995, 18: 1387-1391
149. Sumi H, Hamada H, Nakanishi K and Hiratani H, Enchancement of the fibrinolytic activity in plasma by oral administration of nattokinase, Acta Haematol., 1990, 84: 139-143
150. Fujita M, Hong K, Ito Y, Misawa S, Takeuchi N, Kariya K and Nishimuro S, Transport of nattokinase across the rat intestinal tract, Biol. Pharm. Bull., 1995,18:1194-1196
151．邹和昌，溶栓剂的发展及研究，中国药学杂志，1997，32：263—267
152．“八五”国家攻关课题组(85-915-02-01)，急性心肌梗死溶栓治疗对比研究，中华心血管病杂志，1994，22：17—19
153．链激酶临床试验协作组，急性心肌梗塞链激酶静脉溶栓疗法的多中心试验，中华心血管病杂志，1994，22：403—405
154．徐仲，赵晓东，郑稚莺，宋玉庆，纳豆激酶的纯化及活力测定，生物技术，1997，7(4)：16—18
155. Jensen H L, Decomposition of chlorosubstituted aliphatic acids by soil bacteria, Can. J.
Microbiol., 1957, 3:151-164
156. Slater J H, Bull A T and hardman D J, Microbial dehalogenation of halogenated alkanoic acids, alcohols and alkanes, Adv. Microbial Physiol., 1997, 38: 133-176
157. Leigh J A, Skinner A J and Cooper R A, Partial purification, Stereospecificity and stochiometry of three Dehalogenases from a Rhizobium species, Fed. Eur. Microbiol. Soc. Microbiol. Lett., 1988, 49:353-356
158. Omori T and Alexander M, Bacterial and spontaneous dehalogenation of organic compounds, Appl. Environ. Microbiol., 1978, 35:512-516
159. Allpress J D and Gowland P C, Dehalogenases: environmental defence mechanism and model of enzyme evolution, Biochem. Edu., 1998, 26: 267-276
160. Taylor S C, S-2-Chloropropanoic acid by biotransformation, in: Opportunities in Biotransformations, Pickett J A, Copping L G, Bucke C, Martin R E and Bunch A Weds., Society of Chemical Industry, New York, 1990: 170-176
161. Cambou B and Klibanov A M, Lipase-catalyzed production of optically active acids via asymmetric hydrolysis of esters, Appl. Biochem. and Biotechnol., 1984, 9: 255-260
162．离子交换树脂含水量测定方法，GB／T5757-86，1988
163．离子交换树脂湿真密度测定方法，GB／T8330-87，1988
164．李欣，李朝兴，何炳林，磁性珠状纤维素的性能表征，离子交换与吸附，1997，13：496—502
165．陈敏恒，丛德滋，方图南编，化工原理，化学工业出版社，北京，1985
166. Zhang L-N, Zhou J-P, Yang G and Chen J-H, Preparative fractionation of polysaccharides by columns packed regenerated cellulose gels, J. Chromatogr. A, 1998, 816:131-136
167．复旦大学编，物理化学实验(上)，人民教育出版社，北京，1979
168. Kaewprasit C, Hequet E, Abidi N and Gourlot J P, Quality measurements-application of methylene blue adsorption to cotton fiber specific surface area measurement: part Ⅰ. methodology, J. Cotton Sci., 1998, 2: 164-173
169．何炳林，黄文强编，离子交换与吸附树脂，上海科技教育出版社，上海，1992
170．张志贤，张瑞镐编，有机官能团定量分析，化学工业出版社，北京，1990
171．阳离子交换树脂交换容量测定方法，GB／T8144-87，1988
172．师治贤，王俊德编著，生物大分子的液相色谱分离和制备，科学出版社，北京，1999
173. Skidmore G L, Horstmann B J and Chase H A, Modeling single-component protein adsorption to the cation exchanger Sepharose F F, J. Chromatogr., 1990, 498:113-128
174. Hall K R, Eagelton L C, Acrivos A and Vermeulen T, Pore- and solid-diffusion kinetics in fixing-bed adsorption under constant-pattern conditions, I&EC Fundamentals, 1966, 5: 212-223
175．李建武，萧能赓，余瑞元，袁明秀，陈丽蓉，陈雅蕙，陈来同合编，生物化学实验原理和方法，北京大学出版社，北京，1994
176. Astrup T and Mullertz S, The fibrin plate method for estimating fibrinolytic activity, Arch. Biochem. Biophy., 1952, 40:346-351
177. Motosugi K, Esaki N and Soda K, Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp., J. Bacteriol., 1982, 150:522-527
178．潘祖仁主编，高分子化学，化学工业出版社，北京，1986
179．杨之礼，王庆瑞，邬国铭编，粘胶纤维工艺学(第二版)，纺织工业出版社，北京，1988
180．李欣，李朝兴，何炳林，磁性珠状纤维素制备工艺研究，离子交换与吸附，1997，13：378—384
181．朱伯儒，史作清，何炳林，大孔球形纤维素离子交换剂的制备及其对蛋白质的吸附富集性能研究Ⅱ．球形纤维素的交联活化及功能基化研究，离子交换与吸附，1997：13，37—42
182. Sundberg L and Porath J, Preparation of adsorbents for biospecific affinity chromatography Ⅰ. attachment of group-containing ligands to insoluble polymers by means of bifunctional oxiranes, J. Chromatogr., 1974, 90:87-98
183. Lei Y-L, Liu Z-Z, Liu Q-F and Wu X-Y, Synthesis of a macroporous hydrophilic ternary copolymer and its application in boronate-affinity separation, React. Funct. Polym., 2001, 48: 159-167
184. De Luca L, Hetlenbrioich D, Titchener-Hooker N J and Chase H A, A study of the expansion characteristics and transient behavior of expanded beds of adsorbent particles suitable for bioseparations, Bioseparation, 1994, 4:311-318
185. Dasari G, Prince I and Hearn M T W, High performance liquid chromatography of amino acids, peptides and proteins: Physical characterization of fluidized-bed behavior of chromatographic packing materials, J. Chromatogr., 1993, 631: 115-124
186. Karau A, Benken C, Thmmes J and Kula M R, The influence of particle size distribution and operating conditions on the adsorption performance in fluidized beds, Biotechnol. Bioeng., 1997, 55:54-64
187. Carlos C R and Richardson J F, Solid movement in liquid fluidized beds Ⅱ. Measurements of axial mixing coefficients, Chem. Eng. Sci., 1968, 23: 825-831
188. Asif M, Kalogerakis N and Behie L A, On the constancy of axial dispersion coefficients in liquid fluidized beds, Chem. Eng. J., 1992, 49:17-26
189. Dreager N M and Chase H A, Liquid fluidized beds for protein purification, Trans. Inst. Chem. Eng., 1991,69:45-53
190. Krishnaswamy P R and Shemilt L W, Correlation for axial mixing in liquid-fluidized beds, Can. J. Chem. Eng., 1972, 50:419-420
191. Tang W T and Fan L S, Axial liquid mixing in liquid solid and particles, Chem. Eng. Sci., 1999, 45: 542-551
192. Thmmes J, Weiher M, Karau A and Kula M, Hydrodynamics and performance in fluidized bed adsorption, Biotechnol. Bioeng., 1995, 48:376-374
193. Webster G H and Perona J J, The effect of taper angle on the hydrodynamics of a tapered liquid-solid fluidized bed, AIChE Symp. Ser., 1990, 86: 104-113
194. Davidson J F and Harrison D, eds., Fluidization, Academic press, London, 1971
195. Thmmes J, Fluidized bed adsorption as a primary recovery step in protein purification, in: Advances in Biochemical Engineering/Biotechnology 58, Scheper T eds., Springer, Berlin, 1997
196. Bonnerjea J, Oh S, Hoare M and Dunnill P, Protein purification-the right step at the right time, Bio/Technology, 1986, 4:954-954
197. Wright P R and Glasser B J, Modeling mass and hydrodynamics in fluidized-bed adsorption of proteins, AIChE J., 2001, 47:474-488
198. Lin D Q and Thmmes J, Process design in expanded bed adsorption-A methodological approach, Heinrich-Heine Düsseldorf Universitt, 2001(Internal report)
199. Chase H A, Prediction of the performance of preparative affinity chromatography, J. Chromatogr., 1984, 297:179-202
200. Tsou H S and Graham E E, Prediction of adsorption and desorption of protein on dextran based ion-exchange resin, AIChE J., 1985, 31: 1959-1966
201. Horstmann B J and Chase H A, Modelling the affinity adsorption of immunoglobulin G to protein A immobilised to agarose matrices, Chem. Eng. Res. Des., 1989, 67:243-254
202. Yamamoto S and Sano Y, Short-cut method for predicting the productivity of affinity chromatography, J. Chromatogr., 1992, 597:173-179
203. Nelson P A and Galloway T R, Particle-to-fluid heat and mass transfer in multiparticle systems at low Reynolds numbers, AIChE J., 1975, 10:605-611
204. Thmmes J, Zur Flieβbettadsorption als Primrschritt in der Proteinaufarbeitung[Doctor thesis], Heinrich-Heine DüsseldorfUniverstitt, Habilitationsschrift, 1999,转引自文献[198]
205．俞丽华，层析法分离纯化若干种酶的研究[硕士学位论文]，浙江大学，杭州，2001
206. Chang Y K and Chase H A, Expanded bed adsorption for the direct extraction of proteins, in: Separations for Biotechnology 3, Pyle D L eds., Royal Society of Chemistry, London, 1994, 106-112
207．胡升，新型溶栓酶-纳豆激酶的基础研究[硕士学位论文]，浙江大学，杭州，2003
208．胡升，梅乐和，姚善泾，响应面法优化纳豆激酶液体发酵，食品与发酵工业，2003，29：13—17
209. Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I and Lee S, Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp strain CK 11-4 screened from Chungkook-Jang, Appl. Environ. Microbiol., 1996, 62:2482-2488
210．杨志兴，张淑梅，张云湖，赵克强，董桂华，张凤琴，赤爱胜蚯蚓纤溶酶的研究Ⅱ．药理学作用，生物技术，1992，2(1)：15—18
211. Hu H-B, Yao S-J, Mei L-H, Zhu Z-Q and Hur B-K, Partial purification of nattokinase from Bacillus subtilis by expanded bed adsorption, Biotechnol. Lett., 2000, 22:1383-1387
212．齐楠，α-氯丙酸脱卤酶产生菌的选育和R型酶的分离[硕士学位论文]，浙江大学，杭州，2003
213. Strotmann U and Roschenthaler R, A method for screening bacteria: aerobically degrading chlorinated short-chain hydrocarbons, Curr. Microbiol., 1987, 15:159-163
214. Fernandez-Lahore H M, Geilenkirchen S, Boldt K, Nagel A, Kula M-R and Thmmes J, The influence of cell adsorbent interactions on protein adsorption in expanded beds, J. Chromatogr. A, 2000, 873:195-208
215. Smith J M, Harrison K and Colby J, Purification and characterization of D-2-haloacid dehalogenase from Pseudomonasputida strain A J1/23, J. Gen. Microbiol., 1990, 136:881-886
216. Keuning S, Janssen D B and Witholt B, Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, J. Bacteriol., 1985, 163: 635-639