生物催化合成（R）-β-羟基异丁酸的工艺研究
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摘要
光学纯(R)-β-羟基异丁酸(R-HIBA)是医药、食品和精细化工产品的重要手性中间体。因其具有重要的应用价值,已引起人们的广泛兴趣。利用化学法对甲基丙烯酸进行选择性催化氧化是制备(R)-β-羟基异丁酸的一个重要方法,但是存在收率低、副产品多、环境污染严重、工业化生产成本昂贵等缺点。本文通过对皱落假丝酵母不对称催化氧化异丁酸(IBA)生产(R)-β-羟基异丁酸进行研究,从优化生物发酵条件以及工艺方面,对生物法催化氧化反应条件进行了较系统的研究并建立了以气相色谱(GC),氢焰检测器(FID)为主的检测方法。
本文利用酵母全细胞催化转化(R)-β-羟基异丁酸,所以菌体生物量对其催化氧化的影响很大。通过对皱落假丝酵母自然选育以及紫外诱变对菌体进行改良,得到了高产菌株比原始菌株细胞量提高14.3%,产物(R)-β-羟基异丁酸产量提高18%。
通过对培养基条件和反应条件进行优化得出:底物IBA浓度为20g/L,葡萄糖浓度为30g/L,pH值为7.3,装液量在20%(v/v)条件下更有利于菌体的生长和转化。通过补料半批式发酵,按照优化条件对反应体系进行调控,经过112h培养,产物R-HIBA浓度达到60.9g/L,其平均生产率为0.356g/(L·h),IBA摩尔转化率达到41.2%。
本文所研究底物和产物均为有机酸,极性都比较高,还具有易吸附、高沸点、难汽化等特点,所以在对发酵液进行处理后,对样品进行酯化处理,使它们极性变小,沸点变低、有利于处理和检测采用标准图谱,添加底物和产物标准品比较对照的方法进行定性分析,并确立了以三氯甲烷为内标物的内标法进行定量测定。并对产物进行比旋光度和红外光谱测定。
Optical-pure(R)-β-hydroxyisobutyric acid(R-HIBA) is an important chiral building block in medicine,food and fine chemical products.It has attracted interest widely because of its important commercial interest.It is an important method to catalytic oxidate selectively methylacrylic acid to product R-HIBA by chemical synthesis.Howere,there are some shortage,such as low-yield,high costs,lot of by-products and serious pollution.In this study, production of R-HIBA from isobutyric acid was investigated using Candida rugosa.It studies more systematically the condition of Biological oxidation in term of optimize Bio-fermentation conditions and technology,and The gas chromatogram(GC) with FID(Fire iron deteteetor) was applied to detect and analyze their appearance.
In this study,R-HIBA is synthesized through the method of Yeast whole-cell catalytic convertion,so biomass has a considerable effect on bio-catalytic oxidation.The Candida rugosa was improved by UV-induction and isolating and screening method.High-yield mutant was obtained,which increases the biomass by 14.3%,with the yield of product of the R-HIBA increased by 18%compared with the parent strain.After optimizing the conditions and system of reaction,a conclusion was drawn:It was advantageous to cell growth and coversion of R-HIBA production,when the concentration of substrate(IBA),Glu density was 20g/L and 30g/L,respectively,pH was 7.3,the volume of liquild was maintained 20%(v/v).
Through the fed-batch fermentation,the reaction system was controlled according to the optimization conditions.when the culture time reached 112h,the concentration of production of R-HIBA was up to 60.9g/L,with the average productivity 0.356 g/L and the molar conversion yield of 41.2%.
Since substrate and product are organic acids and both of them have a higher polarity, easy adsorption,high boiling point,hardness to vaporize et.the samples were first derived for the esters,which is beneficial to treat and test.The sample of substrate and product were analyzed qualitatively comparing with gas cheromatographic chart of standard,and the internal standard method was used to determine their quantity of sample,with chloroform as internal standard sample.Specific rotation and IR was measured for the product.
本文利用酵母全细胞催化转化(R)-β-羟基异丁酸,所以菌体生物量对其催化氧化的影响很大。通过对皱落假丝酵母自然选育以及紫外诱变对菌体进行改良,得到了高产菌株比原始菌株细胞量提高14.3%,产物(R)-β-羟基异丁酸产量提高18%。
通过对培养基条件和反应条件进行优化得出:底物IBA浓度为20g/L,葡萄糖浓度为30g/L,pH值为7.3,装液量在20%(v/v)条件下更有利于菌体的生长和转化。通过补料半批式发酵,按照优化条件对反应体系进行调控,经过112h培养,产物R-HIBA浓度达到60.9g/L,其平均生产率为0.356g/(L·h),IBA摩尔转化率达到41.2%。
本文所研究底物和产物均为有机酸,极性都比较高,还具有易吸附、高沸点、难汽化等特点,所以在对发酵液进行处理后,对样品进行酯化处理,使它们极性变小,沸点变低、有利于处理和检测采用标准图谱,添加底物和产物标准品比较对照的方法进行定性分析,并确立了以三氯甲烷为内标物的内标法进行定量测定。并对产物进行比旋光度和红外光谱测定。
Optical-pure(R)-β-hydroxyisobutyric acid(R-HIBA) is an important chiral building block in medicine,food and fine chemical products.It has attracted interest widely because of its important commercial interest.It is an important method to catalytic oxidate selectively methylacrylic acid to product R-HIBA by chemical synthesis.Howere,there are some shortage,such as low-yield,high costs,lot of by-products and serious pollution.In this study, production of R-HIBA from isobutyric acid was investigated using Candida rugosa.It studies more systematically the condition of Biological oxidation in term of optimize Bio-fermentation conditions and technology,and The gas chromatogram(GC) with FID(Fire iron deteteetor) was applied to detect and analyze their appearance.
In this study,R-HIBA is synthesized through the method of Yeast whole-cell catalytic convertion,so biomass has a considerable effect on bio-catalytic oxidation.The Candida rugosa was improved by UV-induction and isolating and screening method.High-yield mutant was obtained,which increases the biomass by 14.3%,with the yield of product of the R-HIBA increased by 18%compared with the parent strain.After optimizing the conditions and system of reaction,a conclusion was drawn:It was advantageous to cell growth and coversion of R-HIBA production,when the concentration of substrate(IBA),Glu density was 20g/L and 30g/L,respectively,pH was 7.3,the volume of liquild was maintained 20%(v/v).
Through the fed-batch fermentation,the reaction system was controlled according to the optimization conditions.when the culture time reached 112h,the concentration of production of R-HIBA was up to 60.9g/L,with the average productivity 0.356 g/L and the molar conversion yield of 41.2%.
Since substrate and product are organic acids and both of them have a higher polarity, easy adsorption,high boiling point,hardness to vaporize et.the samples were first derived for the esters,which is beneficial to treat and test.The sample of substrate and product were analyzed qualitatively comparing with gas cheromatographic chart of standard,and the internal standard method was used to determine their quantity of sample,with chloroform as internal standard sample.Specific rotation and IR was measured for the product.
引文
[1]Andreas Liese,Murillo Villela Filho.Production of fine chemicals using biocatalysis Current Opinion in Biotechnology[J].Chemistry Today,1999,10:595-603
[2]Thomas C.Rosen,Thomas Daumann.Biocatalyst vs.Chemical Catalyst for asymmetric reduction[J].Chemistry Today,1999.12:43-45
[3]A.Schmid,J.S.Dordick,B.Hauer,et al.Industrial biocatalysis today and tomorrow[J].Nature,2001,409(1):258-268
[4]R.E.Parales,N.C.Bruce,A.Schmid,L.P.Wackett.Biodegradation,Biotransformation,and Biocatalysis(B3).Applied and Environmental Microbiology,2002,68(10):4699-4709
[5]M.Eckstein et al.Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One-and Two-Phase Systems[J].Biocatalysis and Biotransformation,2004,22:89-96
[6]Kataoka M,Kita K,Wada M,Yasohara Y,et al.Novel bioreduction system for the production ofchiral alcohols[J].Applied Microbiology and Biotechnology,2003,62(5):437-445
[7]Lee I Y,Hong W K,Hwang Y B.Production of D-β-Hydroxyisobutyric Acid from Isobutyric Acid by Candida rugosa[J].J Ferment Bioeng,1996,81:255-258
[8]孙万儒.手性化合物的生物合成与转化[J].药物生物技术,1996,3(4):239-345
[9]张玉彬等编,生物催化的手性合成[M].北京:化学工业出版社,2002:45-210
[10]李红利 手性化合物与2001诺贝尔奖[J].河南师范大学学报,2002,30(2):59-63
[11]黄辛、张建成.手性技术开掘分子合成新路[N].科学时报.2001,10:16-17
[12]沈同,王镜岩.生物化学[M].北京:高等教育出版社.1997:160-250
[13]贾春荣,乐嘉.萘普生拆分法概述[J].中国医药工业杂志,1990,21(3):137-140
[14]韦革宏,杨祥.发酵工程[M].北京:科学出版社,2008:150-182
[15]熊振平等编,酶工程[M],北京:化学工业出版社,1989,15-89
[16]Andreas S Bommarius,Bettina Riebel.Biocatalysis:fundamentals and Applications[M].Beijing:Chemical industral press,2005:1-43
[17]黄铃.生物催化技术在手性化合物合成中的应用[J],贵州化工,2007,32(2):23-26
[18]童海宝.催化的新领域一酶催化[J].化工催化剂及甲醇技术.2001,(5):71-72
[19]H.L.Holland.Oranic Synthesis with Oxidative Enzyme,VCH publishers,Inc,1992,p41
[20]Kinast G.,Schedel,Vierstufige 1-Desoxynojirimycin synthese miteiner Biotrabsformation alazentralem Reaktionschritt[J].Angem.Chem.1981,93:799-800
[21]Schroder T,Stubbe.M.Patent UP 4806650,1989
[22]Paulsen,H.Kohlendydrate mit stichstoff oder Schwefel in Halbacetal- Ring[J].Angen Chem.1966,78:495-510
[23]Paulsen H,Sangster L,Heyns K.Mono-saccharide mit stickstoffhaltigem Ring ⅩⅢ.Synthese and Reaktionen von keto- piperridinosen[J].Chem.Ber.1967,100..802-815
[24]Kbemick,W.Verfahren zur Herstllung von 1,5-Didesoxy -1,5-imino- D-glucitol and dessen N-Derivaten[P],European Patent:55431,1982
[25]Kinast G;Schedel M.Nerfahrenzur.Her-stellung von 6-Amino-6- desoxy-1- sorbose[P].European Patent:8031,1980a
[26]陈国强,陈耀全,陈新滋等.手性合成[M].北京:化学出版社,1997:485
[27]孙志浩.催化剂制备手性化合物技术进展[J].精细与专用化学品,2006,14(24):5-9
[28]孙志浩.生物催化工艺学[M].北京:化学工业出版社,2004:109-125
[29]孙志浩.手性技术与生物催化[J].生物加工过程,2004,2(4):6-10
[30]孙志浩.手性化合物的生物催化技术进展及工业化应用[J].医药化工,2007,12:1-6
[31]欧阳立明,许建和.生物催化与生物转化研究进展[J].生物加工过程,2008,6(3):1-8
[32]张树政,王修桓.工业微生物成就[M],北京:科学出版社,1988:78-190
[33]A.N.Collion,G.N.Sheldrake,J.Crosby.Chirality,Wiley,1992
[34]D.J.Aberhart.Bioerg.Chem,1977,6:191
[35]Hasegawa J,Hamaguchi S,Ogura M.Production of β-hydroxyisobutyric acids from aliphatic carboxylic acids by microorganisms[J].J Ferment Technol,1981,59(4):257-262
[36]费海明,叶勤.Production of β-Hydroxy-β-methylbutyric acid from β-Methylbutyric acid with galactomyces reessii[J].华东理工大学学报,2002,06:601-605
[37]Chul Ho Kim,Won Kyoung Hong,In Young Lee etc.Enhanced production of D-β-hydroxyisobutyric acid through strain improvement[J].Biotechnology.1999,69:75-79
[38]姜林海,王在利.GrapHical synthetic routes of captopeil[J].中国医药工业杂志.1993,24(6):274-276
[39]孙万儒.Biosynthesis and transformation about chiral compounds[J].化工文摘.2004,4:39-40
[40]许建和,杨立荣,孙志浩,徐岩.Rapid advancement of asymmetric biocaalysis technology[J].生物加工过程.2005,3(3):1-6
[41]余龙江 发酵工程原理与技术应用[M].北京:化学工业出版社,2002:118-140
[42]陈建华,盛勤,冯瑞山等.氧化葡萄糖酸杆菌原生质体的形成和再生研究[J].中国药科大学学报,1996,27(12):758-760
[43]杜连祥.工业微生物学实验技术[M].天津:天津科学技术出版社,1992:180
[44]叶勤 发酵过程原理[M].北京:化学工业出版社,2005:87-132
[45]王立,汪正范.色谱分析样品处理[M].北京:化学工业出版社,2006:42-90
[46]苏克曼,潘铁英,张玉兰.波谱解析法[M].上海:华东理工大学出版社,2002:80-132
[2]Thomas C.Rosen,Thomas Daumann.Biocatalyst vs.Chemical Catalyst for asymmetric reduction[J].Chemistry Today,1999.12:43-45
[3]A.Schmid,J.S.Dordick,B.Hauer,et al.Industrial biocatalysis today and tomorrow[J].Nature,2001,409(1):258-268
[4]R.E.Parales,N.C.Bruce,A.Schmid,L.P.Wackett.Biodegradation,Biotransformation,and Biocatalysis(B3).Applied and Environmental Microbiology,2002,68(10):4699-4709
[5]M.Eckstein et al.Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One-and Two-Phase Systems[J].Biocatalysis and Biotransformation,2004,22:89-96
[6]Kataoka M,Kita K,Wada M,Yasohara Y,et al.Novel bioreduction system for the production ofchiral alcohols[J].Applied Microbiology and Biotechnology,2003,62(5):437-445
[7]Lee I Y,Hong W K,Hwang Y B.Production of D-β-Hydroxyisobutyric Acid from Isobutyric Acid by Candida rugosa[J].J Ferment Bioeng,1996,81:255-258
[8]孙万儒.手性化合物的生物合成与转化[J].药物生物技术,1996,3(4):239-345
[9]张玉彬等编,生物催化的手性合成[M].北京:化学工业出版社,2002:45-210
[10]李红利 手性化合物与2001诺贝尔奖[J].河南师范大学学报,2002,30(2):59-63
[11]黄辛、张建成.手性技术开掘分子合成新路[N].科学时报.2001,10:16-17
[12]沈同,王镜岩.生物化学[M].北京:高等教育出版社.1997:160-250
[13]贾春荣,乐嘉.萘普生拆分法概述[J].中国医药工业杂志,1990,21(3):137-140
[14]韦革宏,杨祥.发酵工程[M].北京:科学出版社,2008:150-182
[15]熊振平等编,酶工程[M],北京:化学工业出版社,1989,15-89
[16]Andreas S Bommarius,Bettina Riebel.Biocatalysis:fundamentals and Applications[M].Beijing:Chemical industral press,2005:1-43
[17]黄铃.生物催化技术在手性化合物合成中的应用[J],贵州化工,2007,32(2):23-26
[18]童海宝.催化的新领域一酶催化[J].化工催化剂及甲醇技术.2001,(5):71-72
[19]H.L.Holland.Oranic Synthesis with Oxidative Enzyme,VCH publishers,Inc,1992,p41
[20]Kinast G.,Schedel,Vierstufige 1-Desoxynojirimycin synthese miteiner Biotrabsformation alazentralem Reaktionschritt[J].Angem.Chem.1981,93:799-800
[21]Schroder T,Stubbe.M.Patent UP 4806650,1989
[22]Paulsen,H.Kohlendydrate mit stichstoff oder Schwefel in Halbacetal- Ring[J].Angen Chem.1966,78:495-510
[23]Paulsen H,Sangster L,Heyns K.Mono-saccharide mit stickstoffhaltigem Ring ⅩⅢ.Synthese and Reaktionen von keto- piperridinosen[J].Chem.Ber.1967,100..802-815
[24]Kbemick,W.Verfahren zur Herstllung von 1,5-Didesoxy -1,5-imino- D-glucitol and dessen N-Derivaten[P],European Patent:55431,1982
[25]Kinast G;Schedel M.Nerfahrenzur.Her-stellung von 6-Amino-6- desoxy-1- sorbose[P].European Patent:8031,1980a
[26]陈国强,陈耀全,陈新滋等.手性合成[M].北京:化学出版社,1997:485
[27]孙志浩.催化剂制备手性化合物技术进展[J].精细与专用化学品,2006,14(24):5-9
[28]孙志浩.生物催化工艺学[M].北京:化学工业出版社,2004:109-125
[29]孙志浩.手性技术与生物催化[J].生物加工过程,2004,2(4):6-10
[30]孙志浩.手性化合物的生物催化技术进展及工业化应用[J].医药化工,2007,12:1-6
[31]欧阳立明,许建和.生物催化与生物转化研究进展[J].生物加工过程,2008,6(3):1-8
[32]张树政,王修桓.工业微生物成就[M],北京:科学出版社,1988:78-190
[33]A.N.Collion,G.N.Sheldrake,J.Crosby.Chirality,Wiley,1992
[34]D.J.Aberhart.Bioerg.Chem,1977,6:191
[35]Hasegawa J,Hamaguchi S,Ogura M.Production of β-hydroxyisobutyric acids from aliphatic carboxylic acids by microorganisms[J].J Ferment Technol,1981,59(4):257-262
[36]费海明,叶勤.Production of β-Hydroxy-β-methylbutyric acid from β-Methylbutyric acid with galactomyces reessii[J].华东理工大学学报,2002,06:601-605
[37]Chul Ho Kim,Won Kyoung Hong,In Young Lee etc.Enhanced production of D-β-hydroxyisobutyric acid through strain improvement[J].Biotechnology.1999,69:75-79
[38]姜林海,王在利.GrapHical synthetic routes of captopeil[J].中国医药工业杂志.1993,24(6):274-276
[39]孙万儒.Biosynthesis and transformation about chiral compounds[J].化工文摘.2004,4:39-40
[40]许建和,杨立荣,孙志浩,徐岩.Rapid advancement of asymmetric biocaalysis technology[J].生物加工过程.2005,3(3):1-6
[41]余龙江 发酵工程原理与技术应用[M].北京:化学工业出版社,2002:118-140
[42]陈建华,盛勤,冯瑞山等.氧化葡萄糖酸杆菌原生质体的形成和再生研究[J].中国药科大学学报,1996,27(12):758-760
[43]杜连祥.工业微生物学实验技术[M].天津:天津科学技术出版社,1992:180
[44]叶勤 发酵过程原理[M].北京:化学工业出版社,2005:87-132
[45]王立,汪正范.色谱分析样品处理[M].北京:化学工业出版社,2006:42-90
[46]苏克曼,潘铁英,张玉兰.波谱解析法[M].上海:华东理工大学出版社,2002:80-132