新型生物催化剂固定化方法的初步研究
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摘要
本论文的研究目的就是针对现今流行的固定化方法已无法完全满足当今生物催化剂发展的需要这一现状，试图以脂肪酶作为待固定化的生物催化剂，通过絮凝技术对之进行固定化并与膜生物反应器耦合应用，来寻求一种高效、通用且成本低廉的全新的固定化方法。
本文通过研究絮凝过程的最佳工艺路线和主要操作参数，膜生物反应器的最佳形式来考察不同转化体系下膜生物反应器与催化剂絮凝后所形成的固定化体系相结合的组合反应体系对相应底物的转化情况，并与传统的反应体系如直接转化法、固定化细胞法及新工艺反应、分离耦合法相比较，分析其应用的前景，看是否有实际的应用价值（即最终是否能用于工业生产）。
一直以来，膜生物反应器都是科学研究和生产领域内的热点，而各种新型的固定化技术和固定化载体材料更是层出不穷。絮凝固定化技术作为一种既古老，又新颖的固定化技术，正逐渐引起人们的重视。早在二十世纪七十年代末，国外就已有人把这种方法用于微生物细胞的固定化，但是目前该技术在我国还属空白，而用于酶的絮凝固定化技术国内外至今仍未见报道。本文的第一章就综述了近年来国内外与本论文有关的技术的相关进展及它们在各个领域中的应用，并由此引出本研究的课题，即需求一种高效、低廉、普适性强的固定化技术。
本文的第二章对絮凝固定化技术的具体问题作出了探讨，由此确定了絮凝固定化技术的操作参数和操作过程，并对所得到的固定化酶的活力与游离酶作了对比，并对结果做出了一定的解释。
本文的第三章对固定化酶用于天然油脂的水解反应进行了研究，并把所制得的固定化酶同新型的仿生膜生物反应器耦合起来，共同应用于天然油脂的水解反应，对所得到的结果进行了分析和比较，并做出了初步的说明。
本文的第四章对本文的研究做出了初步的结论，并提出了未来发展的方向，希望能为今后从事此项研究的人员提供参考。
For prevalent immobilization methods cannot satisfy the needs of biocatalyst's development, so, in this paper, we tried to find a novel immobilization method which is of high efficiency , widely used and low cost ,and couple it with membrane bio-reactors(MBR). During this subject, we choosed lipase as the experimental biocatalyst for immobilization.
By studying the optimum technology progress, operation parameters of flocculation and the best MBR form, under different conditions, we investigated the conversion of combined systems, which were composed of MBR and immobilized systems prepared by flocculating catalysts. As compared with traditional reaction systems ,such as direct transformation, immobilized organism, and intergration of reaction and separation, we analysed the new method's facets on application and practical apply values.
Continuously, MBR is the focus of scientific study and production field. Various novel immobilization methods and carrier for immobilization have been developed. Flocculation technology, traditional and novel, has been paid more and more attention. Since 1970s, this method has been employed on the immobilization of micro-oganism abroad, while which is not investigate in China. Furthermore, there are still no reports on immobilized enzymes with flocculation.
In Chapter 1,the progress and applications of immobilization in various fields are supplied, which apparently included the development of the modern immobilization methods and their advantages and disadvantages. Based on above, we tried to search some method which is high efficient, economic and widely practicable.
Chapter 2 is comprised by the research of parameters on flocculation immobilization which influenced its effects, operation parameters and progresses. Finally, we give some explains about the results.
In Chapter 3 we researched the hydrolysis of natural oil catalysted by immobilized enzyme, and coupled it with new bio-mimic membrane bioreactor, which was applied in hydrolysis of natural oil. Moreover, the results were analysed and contrusted with the data in some literatures.
At last Chapter, rudimentary conclusion was given .Supported by what we have studed ,trends of immobilization technique were posed. Which maybe give some reference to those who would carry out similar study.
本文通过研究絮凝过程的最佳工艺路线和主要操作参数，膜生物反应器的最佳形式来考察不同转化体系下膜生物反应器与催化剂絮凝后所形成的固定化体系相结合的组合反应体系对相应底物的转化情况，并与传统的反应体系如直接转化法、固定化细胞法及新工艺反应、分离耦合法相比较，分析其应用的前景，看是否有实际的应用价值（即最终是否能用于工业生产）。
一直以来，膜生物反应器都是科学研究和生产领域内的热点，而各种新型的固定化技术和固定化载体材料更是层出不穷。絮凝固定化技术作为一种既古老，又新颖的固定化技术，正逐渐引起人们的重视。早在二十世纪七十年代末，国外就已有人把这种方法用于微生物细胞的固定化，但是目前该技术在我国还属空白，而用于酶的絮凝固定化技术国内外至今仍未见报道。本文的第一章就综述了近年来国内外与本论文有关的技术的相关进展及它们在各个领域中的应用，并由此引出本研究的课题，即需求一种高效、低廉、普适性强的固定化技术。
本文的第二章对絮凝固定化技术的具体问题作出了探讨，由此确定了絮凝固定化技术的操作参数和操作过程，并对所得到的固定化酶的活力与游离酶作了对比，并对结果做出了一定的解释。
本文的第三章对固定化酶用于天然油脂的水解反应进行了研究，并把所制得的固定化酶同新型的仿生膜生物反应器耦合起来，共同应用于天然油脂的水解反应，对所得到的结果进行了分析和比较，并做出了初步的说明。
本文的第四章对本文的研究做出了初步的结论，并提出了未来发展的方向，希望能为今后从事此项研究的人员提供参考。
For prevalent immobilization methods cannot satisfy the needs of biocatalyst's development, so, in this paper, we tried to find a novel immobilization method which is of high efficiency , widely used and low cost ,and couple it with membrane bio-reactors(MBR). During this subject, we choosed lipase as the experimental biocatalyst for immobilization.
By studying the optimum technology progress, operation parameters of flocculation and the best MBR form, under different conditions, we investigated the conversion of combined systems, which were composed of MBR and immobilized systems prepared by flocculating catalysts. As compared with traditional reaction systems ,such as direct transformation, immobilized organism, and intergration of reaction and separation, we analysed the new method's facets on application and practical apply values.
Continuously, MBR is the focus of scientific study and production field. Various novel immobilization methods and carrier for immobilization have been developed. Flocculation technology, traditional and novel, has been paid more and more attention. Since 1970s, this method has been employed on the immobilization of micro-oganism abroad, while which is not investigate in China. Furthermore, there are still no reports on immobilized enzymes with flocculation.
In Chapter 1,the progress and applications of immobilization in various fields are supplied, which apparently included the development of the modern immobilization methods and their advantages and disadvantages. Based on above, we tried to search some method which is high efficient, economic and widely practicable.
Chapter 2 is comprised by the research of parameters on flocculation immobilization which influenced its effects, operation parameters and progresses. Finally, we give some explains about the results.
In Chapter 3 we researched the hydrolysis of natural oil catalysted by immobilized enzyme, and coupled it with new bio-mimic membrane bioreactor, which was applied in hydrolysis of natural oil. Moreover, the results were analysed and contrusted with the data in some literatures.
At last Chapter, rudimentary conclusion was given .Supported by what we have studed ,trends of immobilization technique were posed. Which maybe give some reference to those who would carry out similar study.
引文
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[2] 将中华，张津辉主编，化学工业出版社，生物分子固定化技术及应用，1998
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[9] 彭立凤，刘新喜等，膜科学与技术，21（2），2001，44-49
[10] 阎建民，马润宇，化工进展，5，1999，35-38
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[12] Keese C R,Griaever I., microcarries.Science, 219, 1983,1448-1449
[13] Tatsuya O,Masamichi K,Shyji T,et al.,J Ferment Bioeng, 74(1), 1992,27-31
[14] Aubert N,Reach G,Serune H.Reversible ,J Biocomed Mater Rea,
21, 1987,585-601
[15] Bailey JE,Cho YK.,Biotechnol.Bioeng.,25,1983,1923-1925
[16] Arcia MY.,Alaeddinoglu NG.,Hasirei V.,Enzyme Mocrob.Technol.,
22,1998,152-157
[17] Joo-Tack Oh,Jung-Hyun Kim,Enzyme and Microbial Technology,
27,2000,356-361
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[19] 白凤武，生物工程进展，20（2），2000，32-36
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[21] 陆茂林，施大林等，食品与发酵工业，23(3),1997,23-28
[22] 刘传斌，白凤武等，微生物学报，41(3)，2001，367-371
[23] 胡勇有，高健，环境科学进展，7(4)，1999，24-29
[24] Srivatsa V.Rao,Kimberly W.Anderson,Leonidas G.Bachas,Biotechnol. And Bioeng.,65(4), 1999,389-396
[25] XiaoLin Huang，Geroge L.Catignani,Harlod E.Swaisgood,J.Agri.Food Chem.,43,
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[26] Shing- Yi Suen,Yi-Da Tsai,Separation Science and Technology,35(1),2000,69-87
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[32] J.A.Teixeira,R.Oliveira,Z.Azeredo,et al.,Colloids and Surfaces B:Biointerfaces,
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[34] R.艾克斯著：絮凝技术，1981年，原子能出版社
[35] 姚重华编著：混凝剂与絮凝剂，1991年，中国环境科学出版社
[36] 马青山，贾瑟等编著：絮凝化学和絮凝剂，1988，中国环境科学出版社
[37] Patent No.EP0574050;Pub.Date 1993.12.15;Inventor Koster frans;Webbers Jos;
[38] Yosry A.Attia编著：Flocculation in Biotechnology and Separation System,1987
[39] Patent No. US5552316;Pub.Date 1996.09.03;Inventor Savage Christopher M;
[40] Patent No.WO0112778;Pub.Date 2001.02.22;Inventor Wer Steven;Hughes Jonathan;Moran Paul James;
[41] Patent No.Wo9858072;Pub.Date 1998.12.23;Inventor Wer Steven;Hughes Jonathan;Moran Paul James;
[42] Patent No. US5571720;Pub.Date 1996.11.5;Inventor:Grandics Peter; Szathmary Susan;
[43] Patent No. US3725312;Pub.Date 1973.04.03;Inventor Panzer H;Rabinowitz R;
[44] 曾宪放，中国医药工业杂志，23（7），1992
[45] Sarad R.Parekh,Munir Cheryan, Enzyme microb.technol, vol16,104-109,1994
[46] Seung-Goo lee, Hak-sung kim, Biotechnology and Bioengineering,vol42, 737-746,1993
[47] Tae-jong Kim, Yun-dong Lee, Hak-sung Kim, Biotechnology and Bioengineeri-ng, vol41, 88-94,1993
[48] Yi Fenf Shi,Feng Xie Jin,Yi Ying Wu,Process Biochemistry,32(5),387-390,1997
[49] Patent Number: JP7123972 ,1995-05-16 , inventor, Yamauchi Fumio
[50] EP0480400, A3,1992-04-15 ,inventor, Amiot Bruce,Banas Martin H, Reichler Aliens,Waniger Scott;
[51] 马士洪等，生物工程学报，8（1），77-81，1992
[52] Miyawaki O et al,J.Chem.Eng.Jpn,15,224,1982
[53] Gekas VC,Enzyme Microb Technol ,vol8,450,1986
[54] K.Bagi,L.M.Simon,B.Szajanl,Enzyme and Microbial Technology,
1997,20:531-535
[55] A.M.Kilbanov,Acc.Chem.Res.,1990,23:114-120
[56] M.Inagaki,J.Hiratake,T.Nishioka,et al.,J.Am.Chem.Soc.,1991,113;9360-9361
[57] S.M.Roberts,N.J.Turner,J.Biotechnol,1992,22,227-244
[58] R.J.Kazlauskas,TIBTECH,1994,12,464-477
[59] M.Norin,J.Boutelje,et al.,Appl.Microbiol.Biotechnol.,1988,28,527-530
[60] M.D.Virto,I.Agud,S.Montero,et al.,Enzyme and Microbial Technology,1994,16:
61-65
[61] P.Tobback,M.Hendrickx,Z.Weng,et al.The use of immobilized enzymes in t hermal processing In:Advances in Food Engineering(Singh,R.P.and Wirakarta Kusumah,M.A.,F.ds.).CRC Press,London,1992,561-575
[62] H.Weetall,Covalent coupling methods for inorganic support materials.In:Metho
-ds in Enzymology :Immobilized Enzymes Vol.44(Mosbach,K.,Ed.).Academic Press,New York,1976,134-148
[63] M.Arroyo,J.M.Montero,J.V.Sinisterra,J. Molecular Catalysis,1993,83,261-271
[64] M.Murray,D.Rooney,M.Van.Neikerket al.,Process Biochem.,1997,32(6):479-486
[65] 曹国民，冷一欣，盛梅等，石油化工高等学校学报，1997，10(2)，35-37
[66] M.T.Reetz,A.Zonta,J.Simpelkamp,Biotechnol.Bioeng.,1996,49:427-434
[67] 陈秀琳，郑毅，王绍钧等，药物生物技术，1998，6(1)，20-23
[68] 张龙翔，张庭芳，李令媛主编，生化实验技术和方法，高等教育出版社，1981.11第一版
[69] 顾季译，德，G·迪克曼，工业油化学基础—天然油技术综论，中国轻工业出版社，1995，9，7-9
[70] L.H.Posorske,J.Am.Oil Chem.Soc.1984,61(11),:1758-1760
[71] L.H.Posorske,J.Am.Oil Chem.Soc.,1984,61(11):1958-1960
[72] C.Brady,L.Metcalfe,D.Slaboszewski,et al.,J.Am.Oil Chem.Soc.,1988,65
(6):917-921
[2] 将中华，张津辉主编，化学工业出版社，生物分子固定化技术及应用，1998
[3] Brinker C.J.,Smith D.M.,Deshpande R.et al.,Catal.Today,14,1992,155-163
[4] Youichi Kurokawa,Keiichiro Suzuki,Yuko Tamai,Biotechnol. and Bioeng.,
59(5),1998, 651-656
[5] Inama L.,Dire S.,Carturan G.,et al.,J.Biotechnol.,30,1993,197-210
[6] Reetz M.T.,Zonta A.,Simpelkamp J.,Biotechnol. Bioeng.,49,1996,527-
[7] Mantred T.Reetz,Regina Wenkel,David Avnir,Synthesis,6,2000,781-783
[8] 曹国民，赵庆祥等，中国环境科学，21（3），2001，207-211
[9] 彭立凤，刘新喜等，膜科学与技术，21（2），2001，44-49
[10] 阎建民，马润宇，化工进展，5，1999，35-38
[11] Wazel V., Nature,1967,216-64
[12] Keese C R,Griaever I., microcarries.Science, 219, 1983,1448-1449
[13] Tatsuya O,Masamichi K,Shyji T,et al.,J Ferment Bioeng, 74(1), 1992,27-31
[14] Aubert N,Reach G,Serune H.Reversible ,J Biocomed Mater Rea,
21, 1987,585-601
[15] Bailey JE,Cho YK.,Biotechnol.Bioeng.,25,1983,1923-1925
[16] Arcia MY.,Alaeddinoglu NG.,Hasirei V.,Enzyme Mocrob.Technol.,
22,1998,152-157
[17] Joo-Tack Oh,Jung-Hyun Kim,Enzyme and Microbial Technology,
27,2000,356-361
[18] Atkinson B. and Mavittuna,F.,Biochem.Eng.&Biotech.Handbook,2th,1991,768
[19] 白凤武，生物工程进展，20（2），2000，32-36
[20] Renner W.A.,Jordan M.,Eppenberger H.M.,Biotech.Bioeng.,41,1993,188
[21] 陆茂林，施大林等，食品与发酵工业，23(3),1997,23-28
[22] 刘传斌，白凤武等，微生物学报，41(3)，2001，367-371
[23] 胡勇有，高健，环境科学进展，7(4)，1999，24-29
[24] Srivatsa V.Rao,Kimberly W.Anderson,Leonidas G.Bachas,Biotechnol. And Bioeng.,65(4), 1999,389-396
[25] XiaoLin Huang，Geroge L.Catignani,Harlod E.Swaisgood,J.Agri.Food Chem.,43,
1995,895-901
[26] Shing- Yi Suen,Yi-Da Tsai,Separation Science and Technology,35(1),2000,69-87
[27] Yosry A.Attia，Flocculation in Biotechnology and Separation System,1987
[28] Patent No. US4060456;Pub.Date 1977.11.29;Inventor Long Margarete
[29] Patent No. US3989596;Pub.Date 1976.11.02;Inventor Long Margarete
[30] 邱广亮, 邱广明等，广州化工，2000，28(8)，24-27
[31] 万武光，魏文铃等，工业微生物，1999，29（2）,10-15
[32] J.A.Teixeira,R.Oliveira,Z.Azeredo,et al.,Colloids and Surfaces B:Biointerfaces,
1995,5:193-207
[33] 梁为民编著：凝聚与絮凝，1987年，冶金工业出版社
[34] R.艾克斯著：絮凝技术，1981年，原子能出版社
[35] 姚重华编著：混凝剂与絮凝剂，1991年，中国环境科学出版社
[36] 马青山，贾瑟等编著：絮凝化学和絮凝剂，1988，中国环境科学出版社
[37] Patent No.EP0574050;Pub.Date 1993.12.15;Inventor Koster frans;Webbers Jos;
[38] Yosry A.Attia编著：Flocculation in Biotechnology and Separation System,1987
[39] Patent No. US5552316;Pub.Date 1996.09.03;Inventor Savage Christopher M;
[40] Patent No.WO0112778;Pub.Date 2001.02.22;Inventor Wer Steven;Hughes Jonathan;Moran Paul James;
[41] Patent No.Wo9858072;Pub.Date 1998.12.23;Inventor Wer Steven;Hughes Jonathan;Moran Paul James;
[42] Patent No. US5571720;Pub.Date 1996.11.5;Inventor:Grandics Peter; Szathmary Susan;
[43] Patent No. US3725312;Pub.Date 1973.04.03;Inventor Panzer H;Rabinowitz R;
[44] 曾宪放，中国医药工业杂志，23（7），1992
[45] Sarad R.Parekh,Munir Cheryan, Enzyme microb.technol, vol16,104-109,1994
[46] Seung-Goo lee, Hak-sung kim, Biotechnology and Bioengineering,vol42, 737-746,1993
[47] Tae-jong Kim, Yun-dong Lee, Hak-sung Kim, Biotechnology and Bioengineeri-ng, vol41, 88-94,1993
[48] Yi Fenf Shi,Feng Xie Jin,Yi Ying Wu,Process Biochemistry,32(5),387-390,1997
[49] Patent Number: JP7123972 ,1995-05-16 , inventor, Yamauchi Fumio
[50] EP0480400, A3,1992-04-15 ,inventor, Amiot Bruce,Banas Martin H, Reichler Aliens,Waniger Scott;
[51] 马士洪等，生物工程学报，8（1），77-81，1992
[52] Miyawaki O et al,J.Chem.Eng.Jpn,15,224,1982
[53] Gekas VC,Enzyme Microb Technol ,vol8,450,1986
[54] K.Bagi,L.M.Simon,B.Szajanl,Enzyme and Microbial Technology,
1997,20:531-535
[55] A.M.Kilbanov,Acc.Chem.Res.,1990,23:114-120
[56] M.Inagaki,J.Hiratake,T.Nishioka,et al.,J.Am.Chem.Soc.,1991,113;9360-9361
[57] S.M.Roberts,N.J.Turner,J.Biotechnol,1992,22,227-244
[58] R.J.Kazlauskas,TIBTECH,1994,12,464-477
[59] M.Norin,J.Boutelje,et al.,Appl.Microbiol.Biotechnol.,1988,28,527-530
[60] M.D.Virto,I.Agud,S.Montero,et al.,Enzyme and Microbial Technology,1994,16:
61-65
[61] P.Tobback,M.Hendrickx,Z.Weng,et al.The use of immobilized enzymes in t hermal processing In:Advances in Food Engineering(Singh,R.P.and Wirakarta Kusumah,M.A.,F.ds.).CRC Press,London,1992,561-575
[62] H.Weetall,Covalent coupling methods for inorganic support materials.In:Metho
-ds in Enzymology :Immobilized Enzymes Vol.44(Mosbach,K.,Ed.).Academic Press,New York,1976,134-148
[63] M.Arroyo,J.M.Montero,J.V.Sinisterra,J. Molecular Catalysis,1993,83,261-271
[64] M.Murray,D.Rooney,M.Van.Neikerket al.,Process Biochem.,1997,32(6):479-486
[65] 曹国民，冷一欣，盛梅等，石油化工高等学校学报，1997，10(2)，35-37
[66] M.T.Reetz,A.Zonta,J.Simpelkamp,Biotechnol.Bioeng.,1996,49:427-434
[67] 陈秀琳，郑毅，王绍钧等，药物生物技术，1998，6(1)，20-23
[68] 张龙翔，张庭芳，李令媛主编，生化实验技术和方法，高等教育出版社，1981.11第一版
[69] 顾季译，德，G·迪克曼，工业油化学基础—天然油技术综论，中国轻工业出版社，1995，9，7-9
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