利用酵母细胞生物转化法合成天然2-苯乙醇的研究
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摘要
2-苯乙醇是一种具玫瑰气味的芳香醇,作为香料广泛用于食品、日化和轻工等领域。人们对天然香料消费需求的日益增长,推动了利用生物技术方法生产天然2-苯乙醇的研究。以发酵法或酶法生产的L-苯丙氨酸为前体,利用酵母细胞将其转化为2-苯乙醇,产品具有天然属性,可以取代从玫瑰或其它植物精油中提取的天然2-苯乙醇,具有广阔的开发前景。本文以提高2-苯乙醇转化浓度和生产率为目标,从上、中、下游系统地研究了2-苯乙醇转化合成工艺,大幅度提高了2-苯乙醇的转化浓度和生产率,为生物转化法合成天然2-苯乙醇的工业化应用奠定了良好的基础。
从15个酵母菌株中,筛选出1株对2-苯乙醇耐受性强、转化合成2-苯乙醇浓度高的酿酒酵母(Saccharomyces cerevisiae)BD菌株,对其进行纯化并结合紫外诱变,得到酿酒酵母BD18菌株,在未经优化的培养基中,该菌株转化合成2-苯乙醇浓度达到2.04g/L,是1株适用于2-苯乙醇的生物转化合成的理想菌种。
采用单因素试验和正交试验,优化了转化合成2-苯乙醇的培养基组成、种子培养基组成和转化培养条件,使得2-苯乙醇浓度有大幅度提高。摇瓶转化合成2-苯乙醇的最佳工艺是:种子培养基组成为葡萄糖40g/L、蛋白胨20g/L、酵母浸出粉10g/L,装量为40mL/250mL三角瓶;转化培养基组成为蔗糖120g/L、酵母浸出粉5g/L、KH_2PO_4 7.5g/L、K_2HPO_4 9.6g/L、MgSO_4·7H_2O 0.5g/L,装量为30mL/250mL三角瓶;种子培养基接种后于30℃培养24h,按10%的接种量移种至转化培养基,再加入10g/L的L-苯丙氨酸;转化体系于30℃、200r/min条件下培养18h,2-苯乙醇的浓度可达到4.64g/L,摩尔产率为62.7%,生产率为0.26g/(L·h)。
采用Box-Behnken中心组合设计和响应面分析,建立了2-苯乙醇浓度与蔗糖、酵母浸出粉和L-苯丙氨酸之间的二次多项式回归模型,模型具有较高的准确性和实用性,可为生物转化法合成2-苯乙醇的最优化生产提供理论基础。
采用油酸萃取,聚丙二醇2000萃取和D101大孔树脂吸附3种产物原位分离法转化合成2-苯乙醇,2-苯乙醇的摩尔产率均有所提高。在油酸与培养基体积比为1:3,振荡转速为250r/min,转化温度为30℃,底物浓度为14g/L的条件下,转化培养18h,油酸和水相中2-苯乙醇的浓度分别达到14.9g/L和1.74g/L,2-苯乙醇的摩尔产率达到64.7%,生产率达到0.37g/(L·h),较单一水相体系生物转化合成2-苯乙醇的生产率0.26g/(L·h)提高了44.3%。以聚丙二醇2000为萃取溶剂,加入体积与培养基体积比为1:2,底物浓度为12g/L时,转化18h,聚丙二醇中的2-苯乙醇浓度可达11.1g/L,摩尔产率为68.1%,生产率为0.31g/(L·h)。在30mL培养基中加入2g湿大孔树脂D101,底物浓度为12g/L,转化24h,2-苯乙醇总浓度可达6.17g/L,其中3.15g/L保留在培养基中,3.02g/L吸附到D101中,摩尔产率达到69.5%,生产率为0.26g/(L·h)。
在5L发酵罐中,进行常规水相体系、有机溶剂萃取法和大孔树脂吸附法转化合成2-苯乙醇的放大实验,取得的结果与摇瓶工艺相近,表明摇瓶转化工艺放大较为容易。基于5L发酵罐生物转化合成2-苯乙醇得到的菌体得率、2-苯乙醇浓度、蔗糖消耗实验数据,建立了转化过程中菌体生长的Logistic模型、2-苯乙醇生成的Luedeking-Piret模型和蔗糖消耗的Luedeking-Piret相似模型。3个动力学模型具有较高的拟合精度,能准确反映2-苯乙醇生物转化过程及其动力学特征,可用于酿酒酵母转化生产2-苯乙醇过程的预测。
通过对多种有机溶剂的筛选,得出乙酸乙酯是萃取分离2-苯乙醇的最佳溶剂,其萃取的最佳相比0.5,萃取液经减压蒸馏除去乙酸乙酯,便得到提纯的2-苯乙醇,收率为93.6%,纯度可以达到90.7%。静态吸附实验结果表明大孔树脂D101也是很好的2-苯乙醇分离吸附剂。采用大孔树脂D101柱分离2-苯乙醇中,当转化液中2-苯乙醇浓度为4.5g/L左右时,最佳上样量为5.5柱体积(BV),上样速度10BV/h。样品上柱后,用上样量同等体积的蒸馏水洗涤树脂,再用3BV的95%乙醇进行洗脱,收集洗脱液,减压蒸馏去除乙醇,此工艺条件下分离得到的2-苯乙醇样品收率为84.4%,纯度为85.2%。
2-phenylethanol is a higher aromatic alcohol with a rose-like odor,and takes on extensive applications in food,daily chemical and light industries as flavors or fragrances. The worldwide demand for natural products impels the research and development on production of 2-phenylethanol by biotechnology.The most efficient biotechnological approach used to obtain natural 2-phenylethanol is the bioconversion of L-phenylalanine to 2-phenylethanol by yeast cells.Since the precursor L-phenylalanine is now produced microbiologically,2-phenylethanol produced in this way can undoubtedly be labeled "natural",and it is considered to be the best substitute for the product extracted from essential oils of roses or other plants.The future for development and utilization of this technology is therefore bright.Aimed at improving the concentration and productivity of 2-phenylethanol,the thesis comprehensively studied the bioconversion process of 2-phenylethanol from L-phenylalanine by yeast cells.Some remarkable results were obtained finally,in which the 2-phenylethanol concentration and productivity were improved significantly,and provided an available foundation for the industrial application.
A 2-phenylethanol-tolerant strain,Saccharomyces cerevisiae BD,capable of producing high concentration of 2-phenylethanol from L-phenylalanine,was screened out from fifteen yeast strains collected.After separation and ultraviolet radiation of the strain BD,BD18 was selected as a desirable strain for 2-phenylethanol production with the 2-phenylethanol concentration of 2.04 g/L in initial medium.
The experiments of single factor and orthogonal design were carried out to optimize the compositions of bioconversion and seed medium,as well as cultivation conditions for 2-phenylethanol production.The best operations process was as following:The strain maintained in slant medium was inoculated into 40 mL of preculturing medium containing 40 g/L glucose,20 g/L peptone and 10 g/L yeast extract,and incubated at 30℃and 200 rpm for 24 h.Then a 3 mL cell suspension was transferred to 250 mL Erlenmeyer flasks containing 30 mL of biotransformation medium consisting of 120 g/L sucrose,5 g/L yeast extract,7.5 g/L KH_2PO_4,9.6 g/L K_2HPO_4 and 0.5 g/L MgSO_4·7H_2O. After inoculation,10 g/L L-phenylalanine,without prior sterilization,were added to the bioconversion medium and incubated at 30℃and 200 rpm.This process resulted in a improvement of 2-phenylethanol concentration,up to 4.64 g/L with a molar yield of 62.7%and productivity of 0.26 g/(L·h).
A model of polynomial regressive equation between the 2-phenylethanol concentration and sucrose,yeast extract and L-phenylalanine in the medium was established after the Box-Behnken central composite design and the response surface analysis were put into practice.The model exhibited fine exactitude and provided a predictive model for optimizing the medium in the production of 2-phenylethanol by bioconversion.
In situ product recovery techniques were applied to production of 2-phenylethanol by bioconversion,which was performed in aqueous/organic solvent and aqueous/solid absorbent two-phase system respectively.The selection of organic solvents and absorbents favorable for establishing two-phase system was conducted,and the bioconversion conditions in two-phase system for production of 2-phenylethanol were investigated too.When oleic acid with 1/3 volume of medium was added into medium, and shaking speed,temperature and concentration of substrate were set at 250 r/min, 30℃,14 g/L respectively,the 2-phenylethanol concentration could reach 14.9 g/L and 1.74 g/L in oleic acid phase and aqueous phase respectively after 18 h cultivation,with a molar yield of 64.7%.The productivity of 2-phenylethanol achieved at 0.37 g/(L·h). When polypropylene glycol 2000 with 1/2 volume of medium was added into medium, the concentration of substrate were set at 12 g/L,the 2-phenylethanol concentration could reach 11.1 g/L in polypropylene glycol 2000 phase after 18 h cultivation,with a molar yield of 68.1%.The productivity of 2-phenylethanol achieved at 0.31 g/(L·h).When 2g macroporous resin D101 used as adsorbent and 12 g/L L-phenylalanine were added into 30 mL medium,the total 2-phenylethanol concentration achieved was 6.17 g/L,of which 3.15 g/L remained in the aqueous phase and 3.02 g/L was adsorbed onto the resin.After 24 h cultivation,the molar yield and productivity of 2-phenylethanol reached 69.5%and 0.26 g/(L·h)respectively.
Production of 2-phenylethanol by conventional bioconversion process and with organic solvent extraction or macroporous resin adsorption were scaled up in 5 L bioreactor,and the results were almost same as in flask scale.It means that scale-up of the bioconversion from flask scale achieved success easily for production of 2-phenylethanol.
The kinetic models for cell growth,2-phenylethanol production and sucrose consumption of Saccharomyces cerevisiae were established based on the data of bioconversion process in 5 L bioreactor.Three kinetic models were proposed by the Logistic equation for the cell growth,the Luedeking-Piret equation for 2-phenylethanol production and the Luedeking-Piret-like equation for sucrose consumption respectively. The models exhibiting high precision for simulation could exactly describe and predict 2-phenylethanol production by biotransformation in practice.
The selection of organic solvent favorable for extraction separation of 2-phenylethanol was performed,and it was found that acetic ether is the best extract for 2-phenylethanol separation from bioconversion liquid.In extracting separation,if phase ratio of acetic ether to bioconversion liquid was set at 0.5,the recovery and purity of 2-phenylethanol could reach 93.6%and 90.7%respectively.Through static adsorption experiments,the macroporous resin D101 was also found to be the best absorbent for 2-phenylethanol separation.If separation was conducted on column packed with D101 the optimal loading volume was 5.5 BV(bed volume),and the flow rate was 10 BV/h when the concentration of 2-phenylethanol in bioconversion liquid was about 4.5 g/L. After sample loading,D 101 was firstly leached by distilled water with the same volume of loaded sample,and then eluted by 95%ethanol with 3 BV.By this process,the product with recovery of 84.4%and purity of 85.2%was obtained after the ethanol was evaporated.
从15个酵母菌株中,筛选出1株对2-苯乙醇耐受性强、转化合成2-苯乙醇浓度高的酿酒酵母(Saccharomyces cerevisiae)BD菌株,对其进行纯化并结合紫外诱变,得到酿酒酵母BD18菌株,在未经优化的培养基中,该菌株转化合成2-苯乙醇浓度达到2.04g/L,是1株适用于2-苯乙醇的生物转化合成的理想菌种。
采用单因素试验和正交试验,优化了转化合成2-苯乙醇的培养基组成、种子培养基组成和转化培养条件,使得2-苯乙醇浓度有大幅度提高。摇瓶转化合成2-苯乙醇的最佳工艺是:种子培养基组成为葡萄糖40g/L、蛋白胨20g/L、酵母浸出粉10g/L,装量为40mL/250mL三角瓶;转化培养基组成为蔗糖120g/L、酵母浸出粉5g/L、KH_2PO_4 7.5g/L、K_2HPO_4 9.6g/L、MgSO_4·7H_2O 0.5g/L,装量为30mL/250mL三角瓶;种子培养基接种后于30℃培养24h,按10%的接种量移种至转化培养基,再加入10g/L的L-苯丙氨酸;转化体系于30℃、200r/min条件下培养18h,2-苯乙醇的浓度可达到4.64g/L,摩尔产率为62.7%,生产率为0.26g/(L·h)。
采用Box-Behnken中心组合设计和响应面分析,建立了2-苯乙醇浓度与蔗糖、酵母浸出粉和L-苯丙氨酸之间的二次多项式回归模型,模型具有较高的准确性和实用性,可为生物转化法合成2-苯乙醇的最优化生产提供理论基础。
采用油酸萃取,聚丙二醇2000萃取和D101大孔树脂吸附3种产物原位分离法转化合成2-苯乙醇,2-苯乙醇的摩尔产率均有所提高。在油酸与培养基体积比为1:3,振荡转速为250r/min,转化温度为30℃,底物浓度为14g/L的条件下,转化培养18h,油酸和水相中2-苯乙醇的浓度分别达到14.9g/L和1.74g/L,2-苯乙醇的摩尔产率达到64.7%,生产率达到0.37g/(L·h),较单一水相体系生物转化合成2-苯乙醇的生产率0.26g/(L·h)提高了44.3%。以聚丙二醇2000为萃取溶剂,加入体积与培养基体积比为1:2,底物浓度为12g/L时,转化18h,聚丙二醇中的2-苯乙醇浓度可达11.1g/L,摩尔产率为68.1%,生产率为0.31g/(L·h)。在30mL培养基中加入2g湿大孔树脂D101,底物浓度为12g/L,转化24h,2-苯乙醇总浓度可达6.17g/L,其中3.15g/L保留在培养基中,3.02g/L吸附到D101中,摩尔产率达到69.5%,生产率为0.26g/(L·h)。
在5L发酵罐中,进行常规水相体系、有机溶剂萃取法和大孔树脂吸附法转化合成2-苯乙醇的放大实验,取得的结果与摇瓶工艺相近,表明摇瓶转化工艺放大较为容易。基于5L发酵罐生物转化合成2-苯乙醇得到的菌体得率、2-苯乙醇浓度、蔗糖消耗实验数据,建立了转化过程中菌体生长的Logistic模型、2-苯乙醇生成的Luedeking-Piret模型和蔗糖消耗的Luedeking-Piret相似模型。3个动力学模型具有较高的拟合精度,能准确反映2-苯乙醇生物转化过程及其动力学特征,可用于酿酒酵母转化生产2-苯乙醇过程的预测。
通过对多种有机溶剂的筛选,得出乙酸乙酯是萃取分离2-苯乙醇的最佳溶剂,其萃取的最佳相比0.5,萃取液经减压蒸馏除去乙酸乙酯,便得到提纯的2-苯乙醇,收率为93.6%,纯度可以达到90.7%。静态吸附实验结果表明大孔树脂D101也是很好的2-苯乙醇分离吸附剂。采用大孔树脂D101柱分离2-苯乙醇中,当转化液中2-苯乙醇浓度为4.5g/L左右时,最佳上样量为5.5柱体积(BV),上样速度10BV/h。样品上柱后,用上样量同等体积的蒸馏水洗涤树脂,再用3BV的95%乙醇进行洗脱,收集洗脱液,减压蒸馏去除乙醇,此工艺条件下分离得到的2-苯乙醇样品收率为84.4%,纯度为85.2%。
2-phenylethanol is a higher aromatic alcohol with a rose-like odor,and takes on extensive applications in food,daily chemical and light industries as flavors or fragrances. The worldwide demand for natural products impels the research and development on production of 2-phenylethanol by biotechnology.The most efficient biotechnological approach used to obtain natural 2-phenylethanol is the bioconversion of L-phenylalanine to 2-phenylethanol by yeast cells.Since the precursor L-phenylalanine is now produced microbiologically,2-phenylethanol produced in this way can undoubtedly be labeled "natural",and it is considered to be the best substitute for the product extracted from essential oils of roses or other plants.The future for development and utilization of this technology is therefore bright.Aimed at improving the concentration and productivity of 2-phenylethanol,the thesis comprehensively studied the bioconversion process of 2-phenylethanol from L-phenylalanine by yeast cells.Some remarkable results were obtained finally,in which the 2-phenylethanol concentration and productivity were improved significantly,and provided an available foundation for the industrial application.
A 2-phenylethanol-tolerant strain,Saccharomyces cerevisiae BD,capable of producing high concentration of 2-phenylethanol from L-phenylalanine,was screened out from fifteen yeast strains collected.After separation and ultraviolet radiation of the strain BD,BD18 was selected as a desirable strain for 2-phenylethanol production with the 2-phenylethanol concentration of 2.04 g/L in initial medium.
The experiments of single factor and orthogonal design were carried out to optimize the compositions of bioconversion and seed medium,as well as cultivation conditions for 2-phenylethanol production.The best operations process was as following:The strain maintained in slant medium was inoculated into 40 mL of preculturing medium containing 40 g/L glucose,20 g/L peptone and 10 g/L yeast extract,and incubated at 30℃and 200 rpm for 24 h.Then a 3 mL cell suspension was transferred to 250 mL Erlenmeyer flasks containing 30 mL of biotransformation medium consisting of 120 g/L sucrose,5 g/L yeast extract,7.5 g/L KH_2PO_4,9.6 g/L K_2HPO_4 and 0.5 g/L MgSO_4·7H_2O. After inoculation,10 g/L L-phenylalanine,without prior sterilization,were added to the bioconversion medium and incubated at 30℃and 200 rpm.This process resulted in a improvement of 2-phenylethanol concentration,up to 4.64 g/L with a molar yield of 62.7%and productivity of 0.26 g/(L·h).
A model of polynomial regressive equation between the 2-phenylethanol concentration and sucrose,yeast extract and L-phenylalanine in the medium was established after the Box-Behnken central composite design and the response surface analysis were put into practice.The model exhibited fine exactitude and provided a predictive model for optimizing the medium in the production of 2-phenylethanol by bioconversion.
In situ product recovery techniques were applied to production of 2-phenylethanol by bioconversion,which was performed in aqueous/organic solvent and aqueous/solid absorbent two-phase system respectively.The selection of organic solvents and absorbents favorable for establishing two-phase system was conducted,and the bioconversion conditions in two-phase system for production of 2-phenylethanol were investigated too.When oleic acid with 1/3 volume of medium was added into medium, and shaking speed,temperature and concentration of substrate were set at 250 r/min, 30℃,14 g/L respectively,the 2-phenylethanol concentration could reach 14.9 g/L and 1.74 g/L in oleic acid phase and aqueous phase respectively after 18 h cultivation,with a molar yield of 64.7%.The productivity of 2-phenylethanol achieved at 0.37 g/(L·h). When polypropylene glycol 2000 with 1/2 volume of medium was added into medium, the concentration of substrate were set at 12 g/L,the 2-phenylethanol concentration could reach 11.1 g/L in polypropylene glycol 2000 phase after 18 h cultivation,with a molar yield of 68.1%.The productivity of 2-phenylethanol achieved at 0.31 g/(L·h).When 2g macroporous resin D101 used as adsorbent and 12 g/L L-phenylalanine were added into 30 mL medium,the total 2-phenylethanol concentration achieved was 6.17 g/L,of which 3.15 g/L remained in the aqueous phase and 3.02 g/L was adsorbed onto the resin.After 24 h cultivation,the molar yield and productivity of 2-phenylethanol reached 69.5%and 0.26 g/(L·h)respectively.
Production of 2-phenylethanol by conventional bioconversion process and with organic solvent extraction or macroporous resin adsorption were scaled up in 5 L bioreactor,and the results were almost same as in flask scale.It means that scale-up of the bioconversion from flask scale achieved success easily for production of 2-phenylethanol.
The kinetic models for cell growth,2-phenylethanol production and sucrose consumption of Saccharomyces cerevisiae were established based on the data of bioconversion process in 5 L bioreactor.Three kinetic models were proposed by the Logistic equation for the cell growth,the Luedeking-Piret equation for 2-phenylethanol production and the Luedeking-Piret-like equation for sucrose consumption respectively. The models exhibiting high precision for simulation could exactly describe and predict 2-phenylethanol production by biotransformation in practice.
The selection of organic solvent favorable for extraction separation of 2-phenylethanol was performed,and it was found that acetic ether is the best extract for 2-phenylethanol separation from bioconversion liquid.In extracting separation,if phase ratio of acetic ether to bioconversion liquid was set at 0.5,the recovery and purity of 2-phenylethanol could reach 93.6%and 90.7%respectively.Through static adsorption experiments,the macroporous resin D101 was also found to be the best absorbent for 2-phenylethanol separation.If separation was conducted on column packed with D101 the optimal loading volume was 5.5 BV(bed volume),and the flow rate was 10 BV/h when the concentration of 2-phenylethanol in bioconversion liquid was about 4.5 g/L. After sample loading,D 101 was firstly leached by distilled water with the same volume of loaded sample,and then eluted by 95%ethanol with 3 BV.By this process,the product with recovery of 84.4%and purity of 85.2%was obtained after the ethanol was evaporated.
引文
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