微生物完整细胞在有机介质中进行还原生物转化的基础研究
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摘要
有机相生物转化是当前生物技术中一个具有理论研究意义和应用价值的领域，目前该领域的研究大多集中在利用分离的酶进行生物催化，利用微生物完整细胞进行的研究比较少。本论文以Baker's酵母细胞作为生物催化剂，采用香叶醇还原转化为香茅醇的反应为模型反应，探讨了微生物在非水介质中催化还原反应的影响因素，得出如下结论：
1．根据该模型反应中底物和产物都是微量的(μmol/ml)这一特点，建立了以气相色谱(GC)、火焰离子检测器(FID)作为手段的检测方法。采用标准图谱比较和添加产物标准品对照的方法进行定性分析；确立了苯乙酮为内标物和用内标法进行定量分析的方法。
2．比较了八种酵母的生物转化能力，发现在有机合成生物转化中应用最广泛的生物催化剂Baker's酵母最适合催化该模型反应；
3．从生物相容性和有利于转化的角度对两种溶剂，正己烷和乙酸丁酯进行了比较，确定了正己烷—忆二醇为该模型反应的溶剂体系。
4．借助SAS软件，利用响应面分析实验设计(包括部分因子重复实验设计、最陡爬坡实验、中心组合实验设计)对生物催化剂的活化培养基进行了优化，详细讨论了培养基成分及各组分含量对转化的影响。其培养基优化的结果表明：葡萄糖、硫酸镁、氯化钠三个因子对转化有显著的影响，并得到以转化率为响应的拟合方程：
根据模型寻优得出了最佳培养基配方，即：酵母膏8g/L，蛋白胨15g/L，葡萄糖26g/L，氯化钠2.8g/L，硫酸镁0.3g/L，磷酸二氢钾0.3g/L。
5．探讨了不同活化时间对模型反应的影响，结果表明，将生物催化剂培养30小时，进行有机相中转化可以得到最高的转化率。
6．在后处理阶段，比较了几种固定化方式对模型反应的影响，确立了以海藻酸钠作为包埋剂、Ca~(2+)为交联剂进行生物催化剂固定化的方法。进一步的研究表明，海藻酸钠为3％(质量浓度)、氯化钙为0.6mol/L的情况下最有利于模型反应的进行。
7．pH记忆、水活度及通透性处理的实验结果表明，pH记忆对转化有明显的影响，水活度对模型反应的影响不大；作为完整细胞生物催化，细胞的通透性可能是传质阻力的重要组成部分，利用不同浓度的乙醇和超声波对细胞进行处理，结果表明，在有机相完整细胞生物催化中，通透性处理对转化产生的是负面影响。
8．对生物催化剂用量、底物浓度以及反应进程三个主要的动力学因素进行了探讨，结果表明三个因素都有不同程度的影响，实验结果表明，在10ml溶剂中，生物
浙江大学硕上论文
催化剂用量为 15g（固定化方式人底物浓度为 90…、转化时间为 32小时的条件下，
最有利于模型反应的转化，最终使转化从初期的产物浓度2．29apOUIlll、转化率5．92％
（10m反应体系中，香叶醇7（）p）提高到产物浓度7．77删Ufnl、转化率15石2％（10m
反应体系中，香叶醇go…人
Biotransformation in organic solvent is an attractived field in nowadays. Compared with isolated enzyme whole cell is not used broadly as biocatalyst. In this research the cells of Baker's yeast is adopted to mediated a model reaction in organic solvent, in which geraniol is converted to citronellol reductively. Several factors which maybe have effect on the reaction are discussed with transformation rate as target.
In this model reaction the concentration of product and substrate in solvent system is at a level of umol/ml. as the same time Gas Chromatogram (GC) permits rapid analysis of microliter quantities of sample with high resolution. According to these the quantitative analysis for product citronellol is carried out by GC with FID (fire iron detector) as detector using hypnone as an internal standard.
The result of using eight kinds of different yeast strain as biocatalyst reveals that baker's yeast is the best biocatalyst in this transformation. As to the solvent, hexane and butvul acetate is discussed and former is more suitable for biotransformation. Among
seven kinds of energy source: menthoK alcohoK glycok glyceroU 1-hexanoK butyl alcohoL octyl alcohol, glycol is selected as energy source, so hexane-glycol is used as reaction system.
In the preparation stage of experiment the influence of yeast activity condition on transformation rate is observed and composition of culture media is discussed in detail. Using SAS software with Response Surface Methode(RSM)(including Fraction Factor design ^ the steepest ascent design and Central Composite Design) design the analysis result indicates that there component: Glucose N Magnesium vitrioK Sodium chloride in media have remarkable effect on transformation, model equation with transformation rate as responding value is gotten:
Tr=l.404500A1-0.219500A4+0.818000A5-0.789333Ai2+0.022000AiA4-0. 63900 OAiAs-0. 575333x42+0.125000x4x5-0. 552333x52
The most optimized media is decided, that is Glucose 26g/L, MgSOa 0.3g/L, NaCl 2.8g/L, yeast extraction 8g/L,peptide 15g/L, KJHbPO4 0.3g/L.Study of correlation between culturing time and transformation rate reflects that 30 hours is needed.
During down processing biocatalyst several immobilization method are discussed and the result reveals that the method with Alginate Sodium as entrapment and CaCh as cross-linking reagent is best for the conversion, and the concentrate of those are 3% and 0.6mol/L. Study of "pH memory" and Activity water(Aw) in processing of immobilization indicates that former factor has much effect on transformation and the latter just has little effect. Permeabilization treating of Baker's cell with alcohol and ultrasonic leads to lower transformation rate, which illustrates that permeabilization procedure has negative role on biotransformation with whole cell as biocatalyst in organic solvent.
In simple dynamics research amount of biocatalysu concentration of substrate and
reaction course are considered and all of them have remarkable effect on transformation. At last transformation rate from initiate 5.92% rise to 15.62% and concentration of product from 2.29nmol/ml to 7.77mol/ml.
1．根据该模型反应中底物和产物都是微量的(μmol/ml)这一特点，建立了以气相色谱(GC)、火焰离子检测器(FID)作为手段的检测方法。采用标准图谱比较和添加产物标准品对照的方法进行定性分析；确立了苯乙酮为内标物和用内标法进行定量分析的方法。
2．比较了八种酵母的生物转化能力，发现在有机合成生物转化中应用最广泛的生物催化剂Baker's酵母最适合催化该模型反应；
3．从生物相容性和有利于转化的角度对两种溶剂，正己烷和乙酸丁酯进行了比较，确定了正己烷—忆二醇为该模型反应的溶剂体系。
4．借助SAS软件，利用响应面分析实验设计(包括部分因子重复实验设计、最陡爬坡实验、中心组合实验设计)对生物催化剂的活化培养基进行了优化，详细讨论了培养基成分及各组分含量对转化的影响。其培养基优化的结果表明：葡萄糖、硫酸镁、氯化钠三个因子对转化有显著的影响，并得到以转化率为响应的拟合方程：
根据模型寻优得出了最佳培养基配方，即：酵母膏8g/L，蛋白胨15g/L，葡萄糖26g/L，氯化钠2.8g/L，硫酸镁0.3g/L，磷酸二氢钾0.3g/L。
5．探讨了不同活化时间对模型反应的影响，结果表明，将生物催化剂培养30小时，进行有机相中转化可以得到最高的转化率。
6．在后处理阶段，比较了几种固定化方式对模型反应的影响，确立了以海藻酸钠作为包埋剂、Ca~(2+)为交联剂进行生物催化剂固定化的方法。进一步的研究表明，海藻酸钠为3％(质量浓度)、氯化钙为0.6mol/L的情况下最有利于模型反应的进行。
7．pH记忆、水活度及通透性处理的实验结果表明，pH记忆对转化有明显的影响，水活度对模型反应的影响不大；作为完整细胞生物催化，细胞的通透性可能是传质阻力的重要组成部分，利用不同浓度的乙醇和超声波对细胞进行处理，结果表明，在有机相完整细胞生物催化中，通透性处理对转化产生的是负面影响。
8．对生物催化剂用量、底物浓度以及反应进程三个主要的动力学因素进行了探讨，结果表明三个因素都有不同程度的影响，实验结果表明，在10ml溶剂中，生物
浙江大学硕上论文
催化剂用量为 15g（固定化方式人底物浓度为 90…、转化时间为 32小时的条件下，
最有利于模型反应的转化，最终使转化从初期的产物浓度2．29apOUIlll、转化率5．92％
（10m反应体系中，香叶醇7（）p）提高到产物浓度7．77删Ufnl、转化率15石2％（10m
反应体系中，香叶醇go…人
Biotransformation in organic solvent is an attractived field in nowadays. Compared with isolated enzyme whole cell is not used broadly as biocatalyst. In this research the cells of Baker's yeast is adopted to mediated a model reaction in organic solvent, in which geraniol is converted to citronellol reductively. Several factors which maybe have effect on the reaction are discussed with transformation rate as target.
In this model reaction the concentration of product and substrate in solvent system is at a level of umol/ml. as the same time Gas Chromatogram (GC) permits rapid analysis of microliter quantities of sample with high resolution. According to these the quantitative analysis for product citronellol is carried out by GC with FID (fire iron detector) as detector using hypnone as an internal standard.
The result of using eight kinds of different yeast strain as biocatalyst reveals that baker's yeast is the best biocatalyst in this transformation. As to the solvent, hexane and butvul acetate is discussed and former is more suitable for biotransformation. Among
seven kinds of energy source: menthoK alcohoK glycok glyceroU 1-hexanoK butyl alcohoL octyl alcohol, glycol is selected as energy source, so hexane-glycol is used as reaction system.
In the preparation stage of experiment the influence of yeast activity condition on transformation rate is observed and composition of culture media is discussed in detail. Using SAS software with Response Surface Methode(RSM)(including Fraction Factor design ^ the steepest ascent design and Central Composite Design) design the analysis result indicates that there component: Glucose N Magnesium vitrioK Sodium chloride in media have remarkable effect on transformation, model equation with transformation rate as responding value is gotten:
Tr=l.404500A1-0.219500A4+0.818000A5-0.789333Ai2+0.022000AiA4-0. 63900 OAiAs-0. 575333x42+0.125000x4x5-0. 552333x52
The most optimized media is decided, that is Glucose 26g/L, MgSOa 0.3g/L, NaCl 2.8g/L, yeast extraction 8g/L,peptide 15g/L, KJHbPO4 0.3g/L.Study of correlation between culturing time and transformation rate reflects that 30 hours is needed.
During down processing biocatalyst several immobilization method are discussed and the result reveals that the method with Alginate Sodium as entrapment and CaCh as cross-linking reagent is best for the conversion, and the concentrate of those are 3% and 0.6mol/L. Study of "pH memory" and Activity water(Aw) in processing of immobilization indicates that former factor has much effect on transformation and the latter just has little effect. Permeabilization treating of Baker's cell with alcohol and ultrasonic leads to lower transformation rate, which illustrates that permeabilization procedure has negative role on biotransformation with whole cell as biocatalyst in organic solvent.
In simple dynamics research amount of biocatalysu concentration of substrate and
reaction course are considered and all of them have remarkable effect on transformation. At last transformation rate from initiate 5.92% rise to 15.62% and concentration of product from 2.29nmol/ml to 7.77mol/ml.
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73． Sergey A.Piletsky.Susan Alcock and Anthony P.F.Turner. Molecular imprintingrat the edge of the third millemmium. Trends in Biotechnology,2001,Vo119,No.1: 9-12． ,
74． Peter A.G. Cormack,Klaus Mosbach. Molecular imprinting:recent developments and the road ahead. Reactive and Functional Polymers, 1999,41:115-124．
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81． Ewa Zymanczyk-Duda,Pawel Kafarski.Barbara Lejczak. Reduction biotransformation of diethyl β-, γ-and δ-oxoalkylphosphonates by cells of Baker' s yeast, Enzyme and Microbial Technology, 2000,26:265-270．
82． 林耀红、谈锋 林产化学与工业，1999，19(1) ：47-54。
83． Maki-Arvela Paivi, Tiainen Lasse-Pekka. Catalysis Today, 1999 Vol. 18 January Issue 1-4: 57-63 .
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86．李浩春，卢佩章，气相色谱法，分析化学丛书，第三卷，第二册，科学出版社，1993年
87．北京大学化学系仪器分析教学组，仪器分析教程，北京大学出版社，1997年
88．范秀容，李广武，沈萍，微生物学实验，高等教育出版社，1993年
89. Griffin D.R, Yang F, Gainer J.L.. Asmmetric reduction of acetophenone with calcium-alginate entrapped baker's yeast in organic solvents. Biotechnol.Prog, 1998,14: 588-593。
90．李孱，白景华，蔡昭铃等，细菌素发酵培养基的优化及动力学初步分析，生物工程学报，2001，Vol 17，No．2：187-192。
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92．汪仁官，陈荣昭，实验设计与分析，中国统计出版社，1998年。
93. I.M. Lima, H.S. Guraya and E.T. Champagne. Improved Peanut Flour for a reduced-fat peanut butter product, Journal of food science, 2000, No. 5, Vol. 65:854-861。
94．赠胤新，产菊糖酶固定化细胞包埋剂的筛选，食品科学，1998，19：20-22。
95．田小光，彭万霖，于得水等，海藻酸铝固定化酵母生产高浓度酒精的研究，微生物学通报，1995，22：282-284。
96．微生物学教程，周德庆，高等教育出版社，1995年
97．杨启瑞等，固定化啤酒酵母强化技术的研究，工业微生物 1993 23(1)：24-26。
98．诸葛健，王正祥编著，工业微生物实验技术手册，中国轻工业出版社，1994年6月。
99. Akihiko Kondo, Yan Liu, Motoyuki Furuta, et al. Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol. Enzyme and Microbial Technology, 2000,27:806-811。
100．朱彤波，陈军，张益棻，提高三角酵母细胞DAO表观活力的透性化研究，生物工程学报，2001，17：73-77。
101. Yuli L Khmelnitsky and Joseph Rich.Biocatalysis in nonaqueous solvents. Current Opinion in Chemical Biology, 1999,3:47-53。