Acetobacter sp. CCTCC M209061细胞催化潜手性酮不对称还原反应的研究
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摘要
手性醇是重要的的手性中间体,可广泛应用于手性药物和功能材料的合成。如对映体纯的(R)-4-三甲基硅基-3-丁炔-2-醇{(R)-TMSBL}可用于合成治疗老年痴呆症药物的中间体(R)-苯甲基-4-羟基-2-戊炔酸。
与化学合成法相比,生物法反应条件温和、立体选择性高、环境友好,故日益受到人们的青睐。全细胞催化无需添加昂贵的辅酶,同时酶和辅酶都被保护在天然的细胞环境中,有利于保持其活性。因此,利用微生物细胞催化潜手性4-三甲基硅基-3-丁炔-2-酮(TMSBO)不对称还原合成对映体纯的(R)-TMSBL具有重要的意义。近年来的研究发现,许多酶和微生物细胞在含离子液体介质中具有较高的活性、选择性和稳定性。基于以上情况,本论文从可能遵循反Prelog规则催化TMSBO不对称还原合成(R)-TMSBL的微生物样品中筛选高效菌株,通过对比研究不同介质中该微生物细胞催化TMSBO不对称还原反应特性,阐明不同离子液体对该反应的影响规律及机理,揭示在含离子液体介质中该微生物细胞的催化特性,并建立可用于高效、高选择性合成对映体纯(R)-TMSBL的生物催化反应体系。
从可能遵循反Prelog规则催化TMSBO不对称还原反应的微生物样品中,筛选到多株能催化TMSBO不对称还原的微生物菌株。经复筛发现,从“中华开菲尔”菌粒中筛选得到的微生物菌株XZY003催化TMSBO不对称还原生成(R)-TMSBL的效果最好。通过微生物形态、生理生化特性和16S rDNA基因序列逐级鉴定出菌株XZY003属于Acetobacter ghanaensis或其亚种,命名为Acetobacter sp. CCTCC M209061。与已报道的遵循反Prelog规则的微生物相比,在TEA-HCl缓冲液反应体系中Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原生成(R)-TMSBL的效率最高; Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的最适条件为:pH值5.0,辅底物为异丙醇,其浓度130.6 mmol/L,反应温度30℃,底物浓度6.0 mmol/L以及振荡速度180 r/min;在此条件下,反应的初速度、最大产率及产物的e.e.值分别可达0.50μmol/min、71%和99%以上。此外,Acetobacter sp. CCTCC M209061细胞能催化其它一系列潜手性酮不对称还原生成对映体纯手性醇。试验表明,水相中存在较为严重的底物和产物抑制,导致最大产率和最适底物浓度较低。
为解决这一问题,通过代谢调控途径,利用咪唑类离子液体能改善细胞膜通透性的特性,在反应体系中添加与水互溶的咪唑类离子液体,以改变底物和产物在细胞内的浓度,进而提高底物浓度和产率;同时探讨不同亲水性离子液体对Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的影响规律。研究表明,组成离子液体的阴、阳离子类型对Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应有较大影响,特定阴阳离子的匹配对其发挥最佳催化效果至关重要。当组成离子液体的阴离子为BF_4~-和TfO-时,Acetobacter sp. CCTCC M209061细胞的催化活性较低;当阳离子为C_nMIM~+时,随着n值的增加,Acetobacter sp. CCTCC M209061细胞的催化活性呈下降趋势;只有当C_2OHMIM+和NO_3-匹配时,Acetobacter sp. CCTCC M209061细胞的催化活性最高。在含离子液体C_2OHMIM·NO_3的反应介质中,Acetobacter sp. CCTCC M209061细胞催化TMSBO的不对称还原反应的最适底物浓度可达9.0 mmol/L,反应初速度达1.7μmol/min,最大产率为91%,均高于水相体系的对应值,并且产物的e.e.也保持99%以上。此外,在含C2OHMIM·NO_3介质中,Acetobacter sp. CCTCC M209061能高效地催化一系列潜手性酮不对称还原反应。在所考察的15种亲水性离子液体中,Acetobacter sp. CCTCC M209061细胞在含离子液体C2OHMIM·NO_3介质中具有较高的糖代谢活性和适宜的细胞膜完整性,表明C_2OHMIM·NO_3对Acetobacter sp. CCTCC M209061细胞具有较好的生物相容性,同时该离子液体可能适度地改善细胞膜的通透性,有助于生成的产物迅速运输到胞外,部分解除产物的抑制和降低其对细胞的毒性作用。这很好地解释了在含C2OHMIM·NO_3介质中细胞催化效率较高。但是,该反应的最适底物浓度仍较低(9.0 mmol/L),最大产率有待于进一步提高。
为此,尝试采用相转移的方法,利用第二相对底物和产物的萃取作用,解决上述问题。有机溶液/缓冲液是常见的双相反应体系,故首先在不同有机溶剂与TEA-HCl缓冲液组成的双相体系中进行Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应。在所研究的有机溶剂范围内,当其Log P低于3.5时,该不对称还原反应的初速度和最大产率随着有机溶剂Log P值的升高而升高;Log P高于3.5时,该不对称还原反应的初速度和最大产率随着有机溶剂Log P值的升高而降低。与其他有机溶剂相比,正己烷为最适有机介质。在正己烷/TEA-HCl缓冲液双相反应体系中,该反应的初速度最高为1.8μmol/h,产率最大为90%。显然,正己烷/TEA-HCl缓冲液不能有效地提高反应效率。实验表明,有机溶剂对Acetobacter sp. CCTCC M209061细胞的毒性较大,导致细胞的催化活性及稳定性大大下降。
用具有良好生物相容性的疏水性离子液体替代有机溶剂作为萃取相可提高细胞活性。为此,研究了不同疏水性离子液体对Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的影响。在所研究的疏水性离子液体中,C_4MIM·PF_6最适充当该反应的第二相。在TEA-HCl缓冲液/C_4MIM·PF_6双相反应体系中,Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的最适条件为:缓冲液与C_4MIM·PF_6两相体积比4:1,底物浓度60.0 mmol/L,缓冲液pH值5.0,辅底物浓度555.7 mmol/L,反应温度30℃,振荡速度200 r/min。在此条件下,反应初速度为9.4μmol /h,最大产率和产物e.e.值分别达到93%和99%以上。与正己烷/缓冲液双相体系相比,最适底物浓度(60.0 mmol/L vs. 9.0 mmol/L)和最大产率(93% vs. 90%)均有所提高。在所研究的范围内,Acetobacter sp. CCTCC M209061细胞在TEA-HCl缓冲液/ C_4MIM·PF_6双相反应体系中的糖代谢活性,且细胞膜较为完整,表明C_4MIM·PF_6对Acetobacter sp. CCTCC M209061细胞具有较好的生物相容性;另外,离子液体C_4MIM·PF_6对底物和产物具有较高的萃取效率,这很好地解释了Acetobacter sp. CCTCC M209061细胞在TEA-HCl缓冲液/C_4MIM·PF_6双相体系中催化TMSBO不对称还原反应的效率较高这一现象。另一方面,红外光谱法分析表明,离子液体C_4MIM·PF_6能进入Acetobacter sp. CCTCC M209061细胞内,且在细胞膜中富集,提示该离子液体可能与胞内的醇脱氢酶系存在相互作用。此外,在缓冲液/C_4MIM·PF_6双相体系中,Acetobacter sp. CCTCC M209061细胞能高效催化其它一系列潜手性酮不对称还原反应。
在以上所考察的四种反应体系中, Acetobacter sp. CCTCC M209061细胞在缓冲液/C_4MIM·PF_6双相反应体系中的操作稳定性最好,而在正己烷/缓冲液双相反应体系中的操作稳定性最差。
本研究不仅奠定离子液体用于微生物细胞催化的理论基础,还提供一条高效制备对映体纯手性醇的新途径。
Chiral alcohols are important building blocks for the synthesis of chiral pharmaceuticals and functional materials. For example, enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol {(R)-TMSBL} is a key chiral block for the synthesis of (R)-benzyl-4-hydroxyl-2-pentynoate with the potential therapeutical function for Alzheimer’s disease.
Compared with the chemical ways, the biocatalytic routes have gained more and more attention owing to its mild reaction conditions, high enantioselectivity, high efficiency, and low environmental concerns. Using whole cells can avoid the need for coenzyme addition, protect the related enzymes and coenzyme from inactivation by keeping them in natural environment of cells. As a result, it is of great significance to prepare enantiopure (R)-TMSBL throught microbial cell-mediated asymmetric reduction of prochiral 4-(trimethylsilyl)-3-butyn-2-one (TMSBO). In recent years, a number of investigations have showed that many enzymes and microbial cells have higher activity, selectivity and stability in IL-containing systems. In this dissertation, the microbial strains, which are highly effective and enantioselective in catalyzing asymmetric reduction of TMSBO to enantiopure (R)-TMSBL, were screened out from various possible microbial samples following anti-Prelog rule for stereoselective reduction reactions, and a comparative study was made of the biocatalytic asymmetric reduction of TMSBO catalyzed by the novel microbial cells in various reaction media. The effects of various ionic liquids (ILs) on the bioreduction of TMSBO were examined and the catalytic performances exhibited by the cells in IL-containing systems were characterized. Additionally, the novel biocatalytic reaction system used for highly efficient and enantioselective synthesis of enantiopure (R)-TMSBL was established.
Several microbial strains, capable of catalyzing the asymmetric reduction of TMSBO to enantiopure (R)-TMSBL have been screened out from the microbial samples. Among them, the strain XZY003 isolated from China kefir grains has proven to be the best one for the asymmetric reduction of TMSBO. It has been identified to be a member of Acetobacter species by morphological, physiological and biochemical tests and further 16S rDNA gene sequence analysis, and named as Acetobacter sp. CCTCC M209061. In comparison with the reported microbial strains following anti-Prelog rule, Acetobacter sp. CCTCC M209061 cells exhibited higher efficiency and enantioselectivity in catalyzing the anti-Prelog reduction of TMSBO to enantiopure (R)-TMSBL in TEA-HCl buffer. The optimal reaction conditions for the bioreduction of TMSBO with Acetobacter sp. CCTCC M209061 cells were as follows: buffer pH 5.0, cosubstrate 2-propanol, 2-propanol concentration 130.6 mmol/L, reaction temperature 30 oC, substrate concentration 6.0 mmol/L and shaking rate 180 r/min. Under these conditions, the initial reaction rate, the maximum yield and the product e.e. were 0.50μmol/min, 71% and >99%, respectively. Additionally, the novel strain Acetobacter sp. CCTCC M209061 was capable of catalyzing the anti-Prelog asymmetric reduction of a series of other prochiral ketones to the corresponding enantiopure chiral alcohols in aqueous monophasic system. However, the maximum yield and the optimal substrate concentration for the bioreduction carried out in aqueous monophasic system were relatively low, possibly due to the pronounced inhibition of the reaction by the substrate and/or the product in this system.
In order to solve this problem, a metabolic regulation way, namely adding water-miscible imidazolium ILs to the aqueous buffer to make the cell membrane more permeable and thus lower the product concentration in the cells and enhance the reaction efficiency, was attempted. Also, the effects of various hydrophilic ILs on the bioreduction of TMSBO were examined and the catalytic performances of the cells in the IL-containing systems were characterized. It was found that the catalytic performance of the biocatalyst depended not only on the types of anion and cation of the IL, but also on their combination. Acetobacter sp. CCTCC M209061 cells displayed drastically low catalytic activity in the co-solvent systems involving BF4-- or TfO--based ILs. In the case of C_nMIM~+-based ILs, the catalytic activity of the cells decreased with the increasing n value. With the pairing of C2OHMIM+ with NO3-, the cells manifested the highest catalytic activity and therefore the IL 1-(2'-hydroxyl) ethyl-3-methylimidazolium nitrate (C_2OHMIM·NO_3) was considered as the optimal IL for the bioreduction reaction. In the C_2OHMIM·NO_3-containing system, several crucial influential variables were examined. Under the optimized reaction conditions for the bioreduction in the C_2OHMIM·NO_3-containing system, the substrate concentration was 9.0 mmol/L, and the initial reaction rate and the maximum yield weres 1.7μmol/min and 91%, resepectively, which were higher than the corresponding values with the aqueous monophasic system. Also, the product e.e. was not affected and remained above 99%. Furthermore, the biocatalytic anti-Prelog asymmetric reduction of a series of other prochiral ketones catalyzed by Acetobacter sp. CCTCC M209061 cells were successfully performed in the C_2OHMIM·NO_3-containing system. Of all the tested 15 hydrophilic ILs, Acetobacter sp. CCTCC M209061 cells had the highest sugar metabolic activity and suitable cell membrane integrity in the reaction system involving the IL C_2OHMIM·NO_3, showing the better biocompatibility of the IL with Acetobacter sp. CCTCC M209061 cells. Besides, the IL C_2OHMIM·NO_3 can properly increase the cell membrane permeability, which is helpful for the rapid transport of the formed product out of cells and thus partly relieves inhibitory and toxic effects by the product. All these can well account for the highest catalytic efficiency of the cells in the C_2OHMIM·NO_3-containing system. However, it should be noted that the optimal substrate concentration is rather low (9 mmol/L), and the maximum yield needs further improvement.
To tackle this problem, phase transfer method was used in this work with the expectation that the second phase can effectively extract the substrate and the product. Generally, organic solvent/buffer biphasic system is adopted for this purpose. So the asymmetric reduction of TMSBO with Acetobacter sp. CCTCC M209061 cells was initially carried out in various organic solvent/buffer biphasic systems. The effects of organic solvents on the bioreduction of TMSBO varied widely. The initial reaction rate and the maximum yield both increased with increasing Log P values of organic solvents when Log P values was lower than 3.5 and the initial reaction rate and the maximum yield both reduced with increasing Log P values of organic solvents when Log P values was higher than 3.5. n-Hexane was found to be the most suitable organic solvent for the bioreduction among all the tested organic solvents. For the bioreduction performed in the n-hexane/buffer biphasic system, the initial reaction rate, the maximum yield and the product e.e. were1.8μmol/h, 90% and >99%, respectively, under the optimal reaction conditions. Obviously, using n-hexane/buffer biphasic system failed to enhance the efficiency of the reaction efficiently. As shown by our experiments, the organic solvent n-hexane was quite toxic to the cells, leading to a substantial drop in the catalytic activity of the cells.
Using ILs, instead of traditional organic solvents, could improve the cell activity. So the effects of various hydrophobic ILs on the bioreduction of TMSBO were examined. Of all the tested hydrophobic ILs, 1-butyl-3-methylimidzolum hexafluorophosphate (C_4MIM·PF_6) showed to be the most suitable one for the bioreduction. In the buffer/C_4MIM·PF_6 biphasic system, the optimal volume ratio of buffer to C_4MIM·PF_6, substrate concentration, buffer pH, co-substrate concentration, reaction temperature and shaking rate were 4/1, 60.0 mmol/L, 5.0, 555.7 mmol/L, 30 oC and 200 r/min, respectively, under which the initial reaction rate, the maximum yield and the product e.e. were 9.4μmol/h, 93% and >99%, respectively. The optimal substrate concentration (60.0 mmol/L vs. 9.0 mmol/L) and the maximum yield (93% vs. 91%) in the C_4MIM·PF_6-based biphasic systems were much higher than those achieved in n-hexane/buffer biphasic system. Among the examined 7 hydrophobic ILs, Acetobacter sp. CCTCC M209061 cells showed the highest sugar metabolic activity and cell membrane integrity in buffer/C_4MIM·PF_6 biphasic system, indicating the IL’s excellent biocompatibility with the cell. Additionally, the IL C_4MIM·PF_6 can extrac the substrate and the product efficiently. The above-described results contributes to the observation that the efficiency of the asymmetric bioreduction catalyzed by Acetobacter sp. CCTCC M209061 cells was relatively higher in the C_4MIM·PF_6-based biphasic system.On the other hand, the FT-IR analysis showed that the IL C_4MIM·PF_6 was able to enter Acetobacter sp. CCTCC M209061 cells and accumulate within cell membrane, suggesting that the IL was likely to interact with the enzymes associated with the bioreduction within the cells. Furthermore, the biocatalytic anti-Prelog asymmetric reduction of a series of other prochiral ketones using Acetobacter sp. CCTCC M209061 cells were successfully carried out in the C_4MIM·PF_6-based biphasic system.
Among the above-mentioned four reaction systems examined, the best operational stability of Acetobacter sp. CCTCC M209061 cells was observed in the buffer/C_4MIM·PF_6 biphasic system, and the poorest one was recorded in the n-hexane/buffer biphasic system.
The study will not only establish the underlying theories for applications of ILs in microbial whole-cell biocatalysis, but also provide a novel and efficient route to enantiopure chiral alcohols.
与化学合成法相比,生物法反应条件温和、立体选择性高、环境友好,故日益受到人们的青睐。全细胞催化无需添加昂贵的辅酶,同时酶和辅酶都被保护在天然的细胞环境中,有利于保持其活性。因此,利用微生物细胞催化潜手性4-三甲基硅基-3-丁炔-2-酮(TMSBO)不对称还原合成对映体纯的(R)-TMSBL具有重要的意义。近年来的研究发现,许多酶和微生物细胞在含离子液体介质中具有较高的活性、选择性和稳定性。基于以上情况,本论文从可能遵循反Prelog规则催化TMSBO不对称还原合成(R)-TMSBL的微生物样品中筛选高效菌株,通过对比研究不同介质中该微生物细胞催化TMSBO不对称还原反应特性,阐明不同离子液体对该反应的影响规律及机理,揭示在含离子液体介质中该微生物细胞的催化特性,并建立可用于高效、高选择性合成对映体纯(R)-TMSBL的生物催化反应体系。
从可能遵循反Prelog规则催化TMSBO不对称还原反应的微生物样品中,筛选到多株能催化TMSBO不对称还原的微生物菌株。经复筛发现,从“中华开菲尔”菌粒中筛选得到的微生物菌株XZY003催化TMSBO不对称还原生成(R)-TMSBL的效果最好。通过微生物形态、生理生化特性和16S rDNA基因序列逐级鉴定出菌株XZY003属于Acetobacter ghanaensis或其亚种,命名为Acetobacter sp. CCTCC M209061。与已报道的遵循反Prelog规则的微生物相比,在TEA-HCl缓冲液反应体系中Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原生成(R)-TMSBL的效率最高; Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的最适条件为:pH值5.0,辅底物为异丙醇,其浓度130.6 mmol/L,反应温度30℃,底物浓度6.0 mmol/L以及振荡速度180 r/min;在此条件下,反应的初速度、最大产率及产物的e.e.值分别可达0.50μmol/min、71%和99%以上。此外,Acetobacter sp. CCTCC M209061细胞能催化其它一系列潜手性酮不对称还原生成对映体纯手性醇。试验表明,水相中存在较为严重的底物和产物抑制,导致最大产率和最适底物浓度较低。
为解决这一问题,通过代谢调控途径,利用咪唑类离子液体能改善细胞膜通透性的特性,在反应体系中添加与水互溶的咪唑类离子液体,以改变底物和产物在细胞内的浓度,进而提高底物浓度和产率;同时探讨不同亲水性离子液体对Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的影响规律。研究表明,组成离子液体的阴、阳离子类型对Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应有较大影响,特定阴阳离子的匹配对其发挥最佳催化效果至关重要。当组成离子液体的阴离子为BF_4~-和TfO-时,Acetobacter sp. CCTCC M209061细胞的催化活性较低;当阳离子为C_nMIM~+时,随着n值的增加,Acetobacter sp. CCTCC M209061细胞的催化活性呈下降趋势;只有当C_2OHMIM+和NO_3-匹配时,Acetobacter sp. CCTCC M209061细胞的催化活性最高。在含离子液体C_2OHMIM·NO_3的反应介质中,Acetobacter sp. CCTCC M209061细胞催化TMSBO的不对称还原反应的最适底物浓度可达9.0 mmol/L,反应初速度达1.7μmol/min,最大产率为91%,均高于水相体系的对应值,并且产物的e.e.也保持99%以上。此外,在含C2OHMIM·NO_3介质中,Acetobacter sp. CCTCC M209061能高效地催化一系列潜手性酮不对称还原反应。在所考察的15种亲水性离子液体中,Acetobacter sp. CCTCC M209061细胞在含离子液体C2OHMIM·NO_3介质中具有较高的糖代谢活性和适宜的细胞膜完整性,表明C_2OHMIM·NO_3对Acetobacter sp. CCTCC M209061细胞具有较好的生物相容性,同时该离子液体可能适度地改善细胞膜的通透性,有助于生成的产物迅速运输到胞外,部分解除产物的抑制和降低其对细胞的毒性作用。这很好地解释了在含C2OHMIM·NO_3介质中细胞催化效率较高。但是,该反应的最适底物浓度仍较低(9.0 mmol/L),最大产率有待于进一步提高。
为此,尝试采用相转移的方法,利用第二相对底物和产物的萃取作用,解决上述问题。有机溶液/缓冲液是常见的双相反应体系,故首先在不同有机溶剂与TEA-HCl缓冲液组成的双相体系中进行Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应。在所研究的有机溶剂范围内,当其Log P低于3.5时,该不对称还原反应的初速度和最大产率随着有机溶剂Log P值的升高而升高;Log P高于3.5时,该不对称还原反应的初速度和最大产率随着有机溶剂Log P值的升高而降低。与其他有机溶剂相比,正己烷为最适有机介质。在正己烷/TEA-HCl缓冲液双相反应体系中,该反应的初速度最高为1.8μmol/h,产率最大为90%。显然,正己烷/TEA-HCl缓冲液不能有效地提高反应效率。实验表明,有机溶剂对Acetobacter sp. CCTCC M209061细胞的毒性较大,导致细胞的催化活性及稳定性大大下降。
用具有良好生物相容性的疏水性离子液体替代有机溶剂作为萃取相可提高细胞活性。为此,研究了不同疏水性离子液体对Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的影响。在所研究的疏水性离子液体中,C_4MIM·PF_6最适充当该反应的第二相。在TEA-HCl缓冲液/C_4MIM·PF_6双相反应体系中,Acetobacter sp. CCTCC M209061细胞催化TMSBO不对称还原反应的最适条件为:缓冲液与C_4MIM·PF_6两相体积比4:1,底物浓度60.0 mmol/L,缓冲液pH值5.0,辅底物浓度555.7 mmol/L,反应温度30℃,振荡速度200 r/min。在此条件下,反应初速度为9.4μmol /h,最大产率和产物e.e.值分别达到93%和99%以上。与正己烷/缓冲液双相体系相比,最适底物浓度(60.0 mmol/L vs. 9.0 mmol/L)和最大产率(93% vs. 90%)均有所提高。在所研究的范围内,Acetobacter sp. CCTCC M209061细胞在TEA-HCl缓冲液/ C_4MIM·PF_6双相反应体系中的糖代谢活性,且细胞膜较为完整,表明C_4MIM·PF_6对Acetobacter sp. CCTCC M209061细胞具有较好的生物相容性;另外,离子液体C_4MIM·PF_6对底物和产物具有较高的萃取效率,这很好地解释了Acetobacter sp. CCTCC M209061细胞在TEA-HCl缓冲液/C_4MIM·PF_6双相体系中催化TMSBO不对称还原反应的效率较高这一现象。另一方面,红外光谱法分析表明,离子液体C_4MIM·PF_6能进入Acetobacter sp. CCTCC M209061细胞内,且在细胞膜中富集,提示该离子液体可能与胞内的醇脱氢酶系存在相互作用。此外,在缓冲液/C_4MIM·PF_6双相体系中,Acetobacter sp. CCTCC M209061细胞能高效催化其它一系列潜手性酮不对称还原反应。
在以上所考察的四种反应体系中, Acetobacter sp. CCTCC M209061细胞在缓冲液/C_4MIM·PF_6双相反应体系中的操作稳定性最好,而在正己烷/缓冲液双相反应体系中的操作稳定性最差。
本研究不仅奠定离子液体用于微生物细胞催化的理论基础,还提供一条高效制备对映体纯手性醇的新途径。
Chiral alcohols are important building blocks for the synthesis of chiral pharmaceuticals and functional materials. For example, enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol {(R)-TMSBL} is a key chiral block for the synthesis of (R)-benzyl-4-hydroxyl-2-pentynoate with the potential therapeutical function for Alzheimer’s disease.
Compared with the chemical ways, the biocatalytic routes have gained more and more attention owing to its mild reaction conditions, high enantioselectivity, high efficiency, and low environmental concerns. Using whole cells can avoid the need for coenzyme addition, protect the related enzymes and coenzyme from inactivation by keeping them in natural environment of cells. As a result, it is of great significance to prepare enantiopure (R)-TMSBL throught microbial cell-mediated asymmetric reduction of prochiral 4-(trimethylsilyl)-3-butyn-2-one (TMSBO). In recent years, a number of investigations have showed that many enzymes and microbial cells have higher activity, selectivity and stability in IL-containing systems. In this dissertation, the microbial strains, which are highly effective and enantioselective in catalyzing asymmetric reduction of TMSBO to enantiopure (R)-TMSBL, were screened out from various possible microbial samples following anti-Prelog rule for stereoselective reduction reactions, and a comparative study was made of the biocatalytic asymmetric reduction of TMSBO catalyzed by the novel microbial cells in various reaction media. The effects of various ionic liquids (ILs) on the bioreduction of TMSBO were examined and the catalytic performances exhibited by the cells in IL-containing systems were characterized. Additionally, the novel biocatalytic reaction system used for highly efficient and enantioselective synthesis of enantiopure (R)-TMSBL was established.
Several microbial strains, capable of catalyzing the asymmetric reduction of TMSBO to enantiopure (R)-TMSBL have been screened out from the microbial samples. Among them, the strain XZY003 isolated from China kefir grains has proven to be the best one for the asymmetric reduction of TMSBO. It has been identified to be a member of Acetobacter species by morphological, physiological and biochemical tests and further 16S rDNA gene sequence analysis, and named as Acetobacter sp. CCTCC M209061. In comparison with the reported microbial strains following anti-Prelog rule, Acetobacter sp. CCTCC M209061 cells exhibited higher efficiency and enantioselectivity in catalyzing the anti-Prelog reduction of TMSBO to enantiopure (R)-TMSBL in TEA-HCl buffer. The optimal reaction conditions for the bioreduction of TMSBO with Acetobacter sp. CCTCC M209061 cells were as follows: buffer pH 5.0, cosubstrate 2-propanol, 2-propanol concentration 130.6 mmol/L, reaction temperature 30 oC, substrate concentration 6.0 mmol/L and shaking rate 180 r/min. Under these conditions, the initial reaction rate, the maximum yield and the product e.e. were 0.50μmol/min, 71% and >99%, respectively. Additionally, the novel strain Acetobacter sp. CCTCC M209061 was capable of catalyzing the anti-Prelog asymmetric reduction of a series of other prochiral ketones to the corresponding enantiopure chiral alcohols in aqueous monophasic system. However, the maximum yield and the optimal substrate concentration for the bioreduction carried out in aqueous monophasic system were relatively low, possibly due to the pronounced inhibition of the reaction by the substrate and/or the product in this system.
In order to solve this problem, a metabolic regulation way, namely adding water-miscible imidazolium ILs to the aqueous buffer to make the cell membrane more permeable and thus lower the product concentration in the cells and enhance the reaction efficiency, was attempted. Also, the effects of various hydrophilic ILs on the bioreduction of TMSBO were examined and the catalytic performances of the cells in the IL-containing systems were characterized. It was found that the catalytic performance of the biocatalyst depended not only on the types of anion and cation of the IL, but also on their combination. Acetobacter sp. CCTCC M209061 cells displayed drastically low catalytic activity in the co-solvent systems involving BF4-- or TfO--based ILs. In the case of C_nMIM~+-based ILs, the catalytic activity of the cells decreased with the increasing n value. With the pairing of C2OHMIM+ with NO3-, the cells manifested the highest catalytic activity and therefore the IL 1-(2'-hydroxyl) ethyl-3-methylimidazolium nitrate (C_2OHMIM·NO_3) was considered as the optimal IL for the bioreduction reaction. In the C_2OHMIM·NO_3-containing system, several crucial influential variables were examined. Under the optimized reaction conditions for the bioreduction in the C_2OHMIM·NO_3-containing system, the substrate concentration was 9.0 mmol/L, and the initial reaction rate and the maximum yield weres 1.7μmol/min and 91%, resepectively, which were higher than the corresponding values with the aqueous monophasic system. Also, the product e.e. was not affected and remained above 99%. Furthermore, the biocatalytic anti-Prelog asymmetric reduction of a series of other prochiral ketones catalyzed by Acetobacter sp. CCTCC M209061 cells were successfully performed in the C_2OHMIM·NO_3-containing system. Of all the tested 15 hydrophilic ILs, Acetobacter sp. CCTCC M209061 cells had the highest sugar metabolic activity and suitable cell membrane integrity in the reaction system involving the IL C_2OHMIM·NO_3, showing the better biocompatibility of the IL with Acetobacter sp. CCTCC M209061 cells. Besides, the IL C_2OHMIM·NO_3 can properly increase the cell membrane permeability, which is helpful for the rapid transport of the formed product out of cells and thus partly relieves inhibitory and toxic effects by the product. All these can well account for the highest catalytic efficiency of the cells in the C_2OHMIM·NO_3-containing system. However, it should be noted that the optimal substrate concentration is rather low (9 mmol/L), and the maximum yield needs further improvement.
To tackle this problem, phase transfer method was used in this work with the expectation that the second phase can effectively extract the substrate and the product. Generally, organic solvent/buffer biphasic system is adopted for this purpose. So the asymmetric reduction of TMSBO with Acetobacter sp. CCTCC M209061 cells was initially carried out in various organic solvent/buffer biphasic systems. The effects of organic solvents on the bioreduction of TMSBO varied widely. The initial reaction rate and the maximum yield both increased with increasing Log P values of organic solvents when Log P values was lower than 3.5 and the initial reaction rate and the maximum yield both reduced with increasing Log P values of organic solvents when Log P values was higher than 3.5. n-Hexane was found to be the most suitable organic solvent for the bioreduction among all the tested organic solvents. For the bioreduction performed in the n-hexane/buffer biphasic system, the initial reaction rate, the maximum yield and the product e.e. were1.8μmol/h, 90% and >99%, respectively, under the optimal reaction conditions. Obviously, using n-hexane/buffer biphasic system failed to enhance the efficiency of the reaction efficiently. As shown by our experiments, the organic solvent n-hexane was quite toxic to the cells, leading to a substantial drop in the catalytic activity of the cells.
Using ILs, instead of traditional organic solvents, could improve the cell activity. So the effects of various hydrophobic ILs on the bioreduction of TMSBO were examined. Of all the tested hydrophobic ILs, 1-butyl-3-methylimidzolum hexafluorophosphate (C_4MIM·PF_6) showed to be the most suitable one for the bioreduction. In the buffer/C_4MIM·PF_6 biphasic system, the optimal volume ratio of buffer to C_4MIM·PF_6, substrate concentration, buffer pH, co-substrate concentration, reaction temperature and shaking rate were 4/1, 60.0 mmol/L, 5.0, 555.7 mmol/L, 30 oC and 200 r/min, respectively, under which the initial reaction rate, the maximum yield and the product e.e. were 9.4μmol/h, 93% and >99%, respectively. The optimal substrate concentration (60.0 mmol/L vs. 9.0 mmol/L) and the maximum yield (93% vs. 91%) in the C_4MIM·PF_6-based biphasic systems were much higher than those achieved in n-hexane/buffer biphasic system. Among the examined 7 hydrophobic ILs, Acetobacter sp. CCTCC M209061 cells showed the highest sugar metabolic activity and cell membrane integrity in buffer/C_4MIM·PF_6 biphasic system, indicating the IL’s excellent biocompatibility with the cell. Additionally, the IL C_4MIM·PF_6 can extrac the substrate and the product efficiently. The above-described results contributes to the observation that the efficiency of the asymmetric bioreduction catalyzed by Acetobacter sp. CCTCC M209061 cells was relatively higher in the C_4MIM·PF_6-based biphasic system.On the other hand, the FT-IR analysis showed that the IL C_4MIM·PF_6 was able to enter Acetobacter sp. CCTCC M209061 cells and accumulate within cell membrane, suggesting that the IL was likely to interact with the enzymes associated with the bioreduction within the cells. Furthermore, the biocatalytic anti-Prelog asymmetric reduction of a series of other prochiral ketones using Acetobacter sp. CCTCC M209061 cells were successfully carried out in the C_4MIM·PF_6-based biphasic system.
Among the above-mentioned four reaction systems examined, the best operational stability of Acetobacter sp. CCTCC M209061 cells was observed in the buffer/C_4MIM·PF_6 biphasic system, and the poorest one was recorded in the n-hexane/buffer biphasic system.
The study will not only establish the underlying theories for applications of ILs in microbial whole-cell biocatalysis, but also provide a novel and efficient route to enantiopure chiral alcohols.
引文
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