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研究生:呂仁貴
研究生(外文):LU JEN KUEI
論文名稱:饋料批式培養Agrobacteriumradiobacter用以轉化生產D-氨基酸
論文名稱(外文):Fed-batch fermentation of Agrobacterium radiobacter for D-amino acid production
指導教授:李振綱李振綱引用關係
指導教授(外文):C.K. Lee
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:84
中文關鍵詞:饋料批式發酵D型氨基酸
外文關鍵詞:Agrobacterium radiobacterFed-batch fermentationhydantoinaseamidohydrolase
相關次數:
  • 被引用被引用:2
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中文摘要
D-對位-氫氧苯基甘氨酸( D-p-hydroxyphenylglycine ; D-pHPG )是一種D型氨基酸,此種氨基酸可與6-aminopenicillanic acid(6-APA)反應合成半合成抗生素Amoxicillin,以酵素法製造D-pHPG可分為兩個步驟:首先將D-對位-氫氧苯基乙內醯 ( D-p-hydroxyphenylhydantoin ;D-pHPH )經由D-乙內醯水解(D-hydantoinase)水解,生成中間產物N-氨甲醯基-D-對位氫氧苯基甘氨酸(N-carbamoyl-D-p-hydroxyphenylglycine ;D-CpHPG ),D-CpHPG再經由第二步驟N-氨甲醯基-D-氨基酸氨基水解(N-carbamoyl-D-amino acid amidohydrolase)水解生成D-pHPG。
Agrobacterium radiobacter菌體同時含有D-hydantoinase及amidohydrolase兩種酵素,可直接將pHPH轉化為D-pHPG。本論文的目的是希望利用饋料批式發酵獲得大量高活性的A. radiobacter菌體,用以生產D-pHPG。首先以實驗設計法找出最佳之培養基組成用以培養A. radiobaacter,並以pHPH與大豆水解液做為誘導劑,分別研究此兩種誘導劑對於此兩種酵素之誘導效果,再以先決饋料模式進行饋料批式高密度培養A. radiobacter,得到最大菌體濃度為19.48g DCW(dry cell weight)/l菌體,比活性為0.279 µmole/hr.mg cell。
此外為了增加菌體反應的穩定性,將培養所得之菌體以聚乙烯亞胺凝聚,再加入戊二醛交聯,最後再加入chitosan增加交聯程度以此法做簡單的菌體固定化。在高基質濃度 4%(w/v)pHPH下固定化菌體可將pHPH轉化為D-pHPG,重覆反應10次後pHPG轉化率仍可達58.5%。
Abstract
D-p-hydroxyphenylglycine (D-pHPG) is a D-amino acid which can be used to prepare semisynthetic antibiotics Amoxicillin. D-pHPG can be produced by two enzymatic steps. The substrate D-p-hydroxyphenylhydantoin (pHPH) is stereo-specifically hydrolysed by D-hydantoinase to the N-carbamoyl-D-p-hydroxyphenylglycine(D-CpHPG). CpHPG can be further converted to D-pHPG by a second enzymatic step catalysed by an N-carbamyl-amino acid amidohydrolase.The bacterium Agrobacterium radiobacter can produce both D-hydantoinase and the N-carbamoyl-amino-acid amidohydrolase.
The aim of this thesis is to develope an optimized complex medium for A. radiobacter and to evaluate pHPH and soy bean hydrolysate as inducers for the both enzymes. Fed-batch fermentation using a pre-determined feeding profiles was carried out for high cell density fermentation of A. radiobacter . The higest cell concentration obtained was 19.48g DCW/L with specific activity of 0.279 µmole/hr.mg cell .
In order to reuse the D-hydantoinase and amidohydrolase of the A. radiobacter cell a simple cell immobilization method was proposed. The cells suspension was floculated with PEI and further cross-linked with glutaraldehyde and chitosan After ten repeated batch reaction ,the immobilyed cell still can convert 4% (w/v)pHPH into D-pHPG with 58.5% conversion.
目 錄
中文摘要……………………………………………..……………… I
英文摘要……………………………………………….…………...III
誌謝…………………………………………………………………IV
目錄…………………………………………………………………V
圖目錄………………………………………………….…………...IX
表目錄…………………………………………….…. …………….XI
第一章 緒論…………………………………………………………1
1.1研究背景與目的………………………………………...1
1.2研究內容簡介…………………………………………...4
第二章 文獻回顧……………………………………………………6
2.1 D-乙內醯水解及N-氨甲醯基-氨基酸氨基水解………………………………………………………..6
2.2 D-對位-氫氧苯基甘氨酸之生產製程………………..10
2.2.1兩步酵素法生產D-對位-氫氧苯基甘氨酸……10
2.2.2酵素-化學法生產D-對位-氫氧苯基甘氨酸……12
2.3培養基與誘導劑對菌體轉化生產D-pHPG活性的影 響………………………………………………………14
2.4以饋料批式達到高細胞密度之培養………………………17
2.4.1饋料批式發酵原理……………………………………17
2.4.2適用饋料批式的培養製程……………………………19
2.4.3葡萄糖效應(glucose effect)……………..……………20
2.4.4饋料批式達到高細胞密度之培養……………………21 2.5菌體固定化…………………………………………………24
第三章 實驗材料、設備與方法………………………………………25
3.1實驗試藥……………………………………………………25
3.1.1發酵試藥……………………………………………...25
3.1.2酵素反應試藥…………………………………………25
3.1.3固定化菌體試藥………………………………………26
3.1.4葡萄糖分析試藥………………………………………26
3.2儀器設備……………………………………………………26
3.2.1發酵儀器………………………………………………26
3.2.2分析儀器………………………………………………27
3.2.3反應儀器………………………………………………28
3.3實驗方法……………………………………………………..28
3.3.1試驗菌株……………………………………………...28
3.3.2菌種保存………………………………………………28
3.3.2.1保存於 -70℃冷凍櫃及液態氮中…………...28
3.3.2.2平板培養基中保存…………………………..29
3.3.3培養基及誘導劑對菌體生長及酵素活性影響測試…………………………………………………….29
3.3.4 Latin Square 實驗設計法…………………………..30
3.3.5平板培養基…………………………………………32
3.3.6菌種的培養基(培養基A.)…………………………32
3.3.7發酵槽培養基(培養基B.)………………………….32
3.3.8饋料培養基(培養基C.)…………………………….33
3.3.9發酵槽批式培養……………………………………33
3.3.10 菌體誘導效應分析……………………………….33
3.3.11饋料批式發酵之高細胞密度培養………………..34
3.3.12饋料批式培養,μs對菌體比活性之影響………35
3.3.13 A. radiobacter生長曲線的測定………………….35
3.3.14 葡萄糖濃度之測定法(DNS法)………………….36
3.3.15 HPLC分析方法…………………………………..36
3.3.16菌體中酵素活性的分析…………………………..37
3.3.17總氮( Total nitrogen, TN )…………………………37
3.3.18菌體固定化………………………………………..38
3.3.19 自由態菌體轉化pHPH生產D-pHPG穩定性測試…………………………………………………...38
3.3.20固定化菌體轉化pHPH生產D-pHPG穩定性測試…………………………………………………39
第四章 結果與討論…………………………………………………...40
4.1反應基質及產物HPLC定量分析……………………….40
4.2培養基與誘導劑對菌體生長及酵素活性的影響………45
4.3 A. radiobacter批式培養之生長曲線……………………49
4.4菌體活性之誘導表現……………………………………53
4.5饋料批式發酵方法之比較………………………………59
4.6自由態與固定化A. radiobacter菌體轉化pHPH生產D-pHPG之穩定性比較…………………………………….73
第五章 結論與建議…………………………………………………..74
參考文獻………………………………………………………………76
參 考 文 獻
吳文騰,應用生物技術人才培訓計劃-發酵工程技術,財團法人自 強工業科學基金會,2-9~2-10,1998。
林志偉,國立清華大學化學工程所研究所碩士論文,以生物轉化法進行p-hydroxyphenylhydantoin光學切割反應之研究,1996。
林豐儀, 國立台灣工業技術學院化學工程研究所碩士論文,半批式BA乳化聚合反應:具官能基單體之效應,1995。
李宗賢,國立台灣科技大學化學工程研究所碩士論文, Agrobacterium radiobacter NRRL B11291細胞膜對其轉化乙內醯生產D- 氨基酸之影響,1998。
周世明,國立台灣大學農業化學研究所碩士論文,類胰島素生長因子之發酵生產,1997。
郭錦富, 國立台灣大學農業化學研究所碩士論文,醬油在釀造過程中香味之變化及不同成品之比較,1986。
Achary, A.;Hariharan K. A.;Bandhyopadhyaya, S.;Ramachandran, R.;Jayaraman, K.;Application of numericul modeling for the development of optimized complex medium for D-hydantoinase production from Agrobacterum radiobacter NRRL B 11291, Biotechnol. Bioeng., 55, 148-154, 1997
Bauer, K.A.;Ben-Bassat, A.;Dawson, M.;de la Puente, V.T. ; Neway, J.O.;Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Eschirichia coli K-12 by a phosphotransacetylase mutant, Appl. Environ. Microbiol., 56,1296-1302,1990
Bech, J. E.;Carlsen, S.;Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts,36,1-11,1990
Bernheim, F.;Brenheim, M. L. C.; The hydrolysis of hydantoin by various tissues , J. Biol. Chem., 163, 683-685, 1946
Chevalier, P.;Roy, D.;Morin, A.;Hydantoinase activity of immobilized non-growing Pseudomonas putida Cells, Appl. Microbiol. Biotechnol., 30, 482-486, 1989
Durham, D. R.;Weber, J. E.;Properties of D-hydantoinase form Agrobacterium tumefaciens and its use for the preparation of N-carbamyl D-amino acids,22,1095-1100,1995
Eadie, G.S.;Bernheim, F.;The partial purification and properties of animal and plant hydantoinases, J. Biol. Chem., 181, 449, 1949
Fiesch, K. J.;Ritch, T.;Production of human alpha consensue interferon in recombinant Escherichia coli, Chem. Eng. Commun. 145,229-240,1986
Gleiser, I. E.; Bauer S.;Growth of E. coli W to high cell concentration by oxygen level linked control of carbon source concentration, Biotechnol. Bioeng., 23, 1015-1021, 1981
Gokhale, D. V.;Bastawde, K. B.;Patil, S. G.;Kalkote, U. R.;Joshi, R. R.;Joshi, R. A.;Ravindranathan, T.;Gaikwad, B. G.; Jogdand, V. V. ;Nene, S.;Chemoenzymatic synthesis of D(-)phenylglycine using hydantoinase of Pseudomonas desmolyticum resting cells , Enzyme Microb. Technol, 18, 353-357,1996.
Helmuth, K.;Kore, D.J.;Sanders, E.A.;Dwckwer, W-D.;Effect of growth rate on stability and gene expression of recombinant plasmids curing continuous and high cell density cultivation of Escherichia coli TGl,J.Biotechnol.,32,289-298,1994
Kim, D. M.;Kim, H. S.;Enzymatic synthesis of D-p-hydroxylphenylglycine from DL-p-hydroxyphenylhydantoin in the presence of organic cosolvent, Enzyme Microb. Technol. 15, 530-534, 1993
Kim, D.-M.;Kim, G.-J.;Kim, H.-S.;Enhancement of operational stability of immobilized whole cell D-hydantoinase, Biotechnol. Lett., 16, 11-16, 1994
Kim, G.-J.;Kim H.-S.;Optimization of the enzymatic synthesis of D-p-hydroxyphenylglycine from DL-5-substituted hydantoin using D-hydantoinase and N-carbamoylase ,Enzyme Micro.Technol., 17, 63-67,1995.
Klaus, B. A.;Kaspar, V. M.;Are growth rate of Escherichia coli in bath cultures limited by respiration, J. Bacteriol., 144, 114-123, 1980
Korz, D. J.;Rinas, U.;Hellmuth, K.;Sanders, E.A.; Deckwer, W.-D.;Simple fed-batch technique for high cell density cultivation of Escherichia coli, J. Biotechnol., 39, 59-65, 1995
Lapointe, G.;Viau, S.;Leblanc, D.; Robert, N.; Morin, A. ; Cloning sequencing expression in of the D-hydantoinase gene from Pseudomonas putida and other microorganisms, Appl. Environ. Microbiol. , 60, 888-895, 1994
Lee, D-C.;Lee, S-G.;Kim, H-S.;Production of D-phydroxyphenylgycline form D,L-5-(4-hydroxyphenyl)hydantion using immobilized thermostable D-hydantionase form Bacillus sterothermophilus SD-1,Enzyme Micro. Technol.,18,35-40,1996A
Lee, J.-H.;Choi, Y.-H.;Kang, S.-K.;Park, H. H.;Kwon, I.-B.;Production of human leukocyte interferon in Escherichia coli by control of growth rate in fed-batch fermentation, Biotechnol. Lett., 2, 695-698, 1989
Lee, S.Y.;Yim, K.S.;Chang, H.N. ;Chang, Y.K.;Construction of plasmids estimation of plasmid stability and use of stable plasmids for the production of poly (3-hydroxybutyric acid) by recombinant Escherichia coli, J. Biotechnol., 32,203-211,1994
Lee, S. Y.;Chang, H. N.;High cell density cultivation of Escherichia coli w using sucrose as a carbon source, Biotechnol. Lett., 15, 971-974, 1993
Lee, S. Y.;High cell-density culture of Escherichia coli, Trends Biotechnol., 14, 98-105, 1996B
Lee, Y.L. ;Chang, H.N.;High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor, Biotechnol. Bioeng., 41,316-324,1990
Luksa, V.;Starkuviene, V.;Starkuviene, B.;Dagys, R.;Purification and characterization of the D-hydantoinase from Bacillus circulans , Appl. Bioch. Biotech., 62, 219, 1997
Mac Donald, H.L.;Neway, J.O.;Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12,Appl.Environ. Microbiol.,56,640-645,1990
Mark1, H.;Zenneck, C.;Dubach, A. ;Ogbonna, J.C.;Cultivation of Escherichia coli to high cell densities in a dialysis reactor, Appl. Microbiol. Biotechnol.,39,48-52,1993
Meyer, P.;Runser, S.; Efficient production of the industrial biocatalysts hydantoinase and N-carbamoyl amino acid amidohydrolase: novel non-metabolozable inducers, FEMS Micro. Biol. Lett., 109, 67-74, 1993
Mizutani, S.;Mori, H.;Shimizu, S.;Sakaguchi, K. ;Kobayashi, T.;Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid, Biotechnol. Bioeng., 28,204-209,1986
Moller, A.;Syldatk, C.;Schulze, M. ;Wagner F.;Stereo-and substrate-specificity of a D-hydantoinase and a D-N-carbamoyl-amino acid amidohydrolase of Arthrobacter crystallopoietes AM2 , Enzyme Microb. Technol., 10, 618-625, 1988
Mori, H.;Yano, T.;Kobayashi, T.;Shimizu, S.;High density cultiration of biomass in fed-batch system with DO-stat, J. Chem. Eng. Jpn, 12, 313-319, 1979
Morin, A.;Hummel W.;Kula, M. R.;Production of hydantoinase from Pseudomonas fluorescens strain DSM 84, Appl. Microbiol. Biotechnol. 25, 91-96, 1986B。
Morin, A.;Hummel, W.;Schutte, H.;Kula, M. —R.; Characterization of hydantoinase from Pseudomoanas fluorescens Strain DSM 84 , Biotechnol. Appl. Biochem, 8, 564, 1986A
Ogawa, J. ;Schimizu, S.;Yamada, H.; N-carbamoyl-D-amino acid amidohydrolase from Comamonas sp. E 222C purification and characterization, Eur. J. Biochem., 212, 685-691, 1993
Ogawa, J.;Honda, M.;Soong, C-L.;Schimizu, S.;Diversity of cyclic uride compound-dihydropyrimidine and hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4 , Biosci. Biotech. Biochem., 59, 1960-1962, 1995
Ogawa, J.;Schimizu, S.;Diversity and versatility of hydantoin-transforming enzymes , Journal of Molecular Catalysis B:Enzymatic, 2, 163-176, 1997
Ogawa, J.;Chung, M. C.-M.;Hida, S.;Yamada, H.;Shimizu, S.;Thermostable N-carbamoyl-D-amino acid amidohydrolase: screening, purification and characterization, J. Biotechnol., 38, 11-19, 1994
Olivieri, R.;Fastcetti, E.;Angelini, L.;Degen, L.;Enzymatic conversion of N-carbamoyl-D-amino acid to D-amino acids, Enzyme Microb. Technol., 1, 201-204, 1979
Olivieri, R.;Fascetti, E.;Angelini, L.;Degen, L.;Microbial transformation of racemic hydantoins to D-amino acid, Biotechnol. Bioeng. 23, 2173-2183, 1981。
Paalme, T.;Tiisma, K.;Kahru, A.;Vanatalu, K.;Vilu, R.;Glucose-limited fed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate, Biotechnol. Bioeng., 35, 312-319, 1990
Pack, P.;Kujau, M.;Schroeckh, V.;Knupfer, U.;Wenderoth, R.;Riesenberg, D.;Pluckthum, A.;Improved bivalent miniantibodies with identical avidity as whole antibodies produced by high cell density fermentation of Escherichia coli, Bio/Technology,11,1271-1276,1993
Peter, L.;Tord, H.;Removal of inhibitors of bacterial growth by dialysis culture, J. Gen. Microbiol., 103, 345-352, 1977
Renata, G.;Claudio, P.;Giuliano, G.;Guido, G.;Topological mapping of the cysteine residues of N-carbamoyl-D-amino-acid amidohydrolase and their role in enzymatic activity, J. Biol. Chem., 271, 9326-9331, 1996
Riesenberg, D.;Schulz, V.;Knorre, W. A.;Pohl, H.-D.;Korz, D.; Sanders, E. A.;Ro, A.;Deckwer, W.-D.;High cell density cultivation of Escherichia coli at controlled specific growth rate, J. Biotechnol., 20, 17-28, 1991
Riesenberg, D.;Menzek, K.;Schulz, V.;Schumann, K.;Veith, G.;Zuber, G.;Knorre, W.-A.;High cell density fermentation of recombinant Escherichia coli expreeing human interferon alpha 1,Appl. Microbiol. Biotechnol.,34,77-82,1990
Runser, S.;Chinski, N.;Ohleyer, E.;D-p-hydroxyphenylglycine production from DL-5-p-hydroxyphenylhydantoin by Agrobacterium sp. , Appl. Microbiol. Biotechnol., 33, 382-388, 1990B
Runser, S.M.;Meyer, C.;Purification and biotechemical characterization of the hydrolyzing enzyme form Agrobacterium sp.,Eur. J. Biochem,213,1315-1324,1993
Runser, S.; Ohleyer, E.;Properties of the hydantoinase from Agrobacterium sp. IP-I-671, Biotechnol. Lett., 12, 259-264, 1990A
Scragg, A. H.;Bioreactors in biotechnology a practical approach, Ellis Horwood Limited, 59, 1991
Shimizu, N.;Fukuzono, S.;Harada, Y.;Fujimori, K.;Gotoh, K.;Yamazaki, Y.;Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli, Biotechnol. Bioeng., 38,37-42,1991
Shimizu, O. S.;Yamada, H.;N-carbamoyl-D-amino acid amidohydrolase from Commamonas sp. E222c purification and characterization , Eur. J. Biochem, 212, 685-691,1993.
Shimizu, S.;Shimada, H.;Takahashi, S.;Ohashi, T.;Tani, Y. ;Yamada, H.;Synthesis of N-carbamoyl-D-2-thienylglycine and D-2-thienylglycine by microbial hydantoinase, Agric. Biol. Chem., 44, 2233-2234, 1980
Strandberg, L.;Enfors, S-O.;Batch and fed-batch cultivations for the temperature induced production of a recombinant protein in Escherichia coli, Biotechnol.Lett.,13,690-614,1991
Syldatk, C.;Laufer, A.;Muller, R.;Hoke, H.;Production of optically pure D- and L-α-amino acids by bioconversin of D, L-5-monosubstituted hydantoin derivatives. , Advances in biochemical engineering/biotechnology, 41 ,29-75, 1990
Syldatk, C.;Muller, R.;Pirtsh, M.;Wagner, F.;Mircobial and enzymatic production of D-α-amino acids from D, L-5- monosubstituted hydantoins ,Biocatalytic production of amino acids & derivatives, 75-176, 1994
Syldatk, C.;Muller, R.;Sieman, M.;Krohn, K.;Wagner, F.;Mircobial and enzymatic production of D-α-amino acids from D, L-5- monosubstituted hydantoins ,Biocatalytic production of amino acids & derivatives, 75-128, 1992
Takahashi, S.;Kii, Y.;Kumagai, H.;Yamada, H.;Purification, crystallization and properties of hydantoinase from Pseudomonas striata., J. Ferment. Technol, 56, 492-498, 1978
Takahiro, S.;Tsuneo, Y.;Shoichi, S.;Phenomenological background and some preliminary trials of automated substrate supply in pH-stst modal fed-batch culture using a setpoint of high limit, J. Ferment. Bioeng., 69, 292-297, 1990
Takuo, Y.;Hironori, M.;Takeshi, K.;Control system for dissolved oxygen concentration in aerobic cultivation, J. Ferment. Technol., 57, 91-98, 1979
Takuo, Y.;Takeshi, K.;Shoichi, S.;Fed-batch culture of methanol-utilizing bacterium with DO-stat, J. Ferment. Technol., 56, 416-420, 1978
Yamada, H.;Takahashi, S.;Kii, Y.;Kumagai, H.;Distribution of hydantoin hydrolyzing activity in microorganisms , J. Ferment. Technol., 56,484, 1978
Yang, X-M;Xu, L.;Eppstein, L.;Production of recombinant human interferon-γ1 by Escherichia coli using a computer-controlled cultivation process, J. Biotechnol.,23,291-301,1992
Yee, L.;Blanch, H.W.;Recombinant Trypsin production in high cell density fed-batch cultures in Escherichia coli, Biotechnol. Bioeng., 41,781-790,1993
Yokozeki, K.;Kubota, K.;Mechanism of asymmetric production of D-amino acids from the corresponding hydantoins by Pseudomonas sp , Agric. Biol. Chem., 51, 721-728, 1987
Yoon, S.K.;Kang, W.K.;Park, T.H.;Fed-batch operation.of recombinant Escherichia coli containing trp promoter with controlled specific growth rate,Biotechnol.Bioeng.,43,995-999,1994
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