新型羰基还原酶的挖掘及其应用于手性苯乙二醇合成的研究
发布时间：2019-05-17 22:11
【摘要】：手性二醇是一类十分重要的化合物,在药物合成、农业化学品合成、功能性材料制备、香料和精油添加剂等方面都具有十分重要的用途。化学法制备手性二醇反应步骤复杂繁琐,催化剂昂贵且有毒、对环境不友好。生物法催化潜手性羟酮不对称还原合成手性二醇因其良好的催化活性、立体选择性和环境友好等优点受到国内外研究者们的重视。本课题通过对实验室已构建的20种羰基还原酶进行初步筛选,得到对底物2-羟基苯乙酮催化活性高、立体选择性相反的两种羰基还原酶BDHA和Go SCR,并对其进行表达纯化及酶学性质表征;通过分别构建体外和体内羰基还原酶与葡萄糖脱氢酶双酶偶联体系,实现了辅酶高效循环再生,进一步提高了对底物2-羟基苯乙酮的催化效率。首先,以2-羟基苯乙酮为底物,对实验室前期通过基因挖掘法从Bacillus subtilis 168和Gluconobac oxydans 621H中克隆得到20种羰基还原酶进行初步筛选,得到催化活力较高的R选择性羰基还原酶BDHA和S选择性羰基还原酶Go SCR,将其重新构建至表达载体p ET28a中,并在大肠杆菌中实现高效可溶表达。其次,对筛选获得的羰基还原酶通过镍柱进行了纯化,并获得电泳纯的酶,以2-羟基苯乙酮为底物对其进行了酶学性质表征。实验结果发现R型羰基还原酶BDHA最适p H为6.0,最适温度为40o C;该酶在p H7.0的缓冲液中放置18 h,残余酶活可保留80%以上;该酶对高温敏感,大于40o C迅速失活,但在30o C放置18 h,残余酶活可保留60%以上;Vmax为2.1 U/mg,KM值为1.0 m M,kcat值为1.3 s-1,kcat/KM值为1.3 s-1m M-1;该酶对有机溶剂二甲基亚砜(DMSO)敏感,当DMSO浓度小于15%(v/v)时,可保留70%以上的初始酶活;该酶具有良好的底物耐受性,底物浓度高达200 m M,仍能保留初始酶活的95%以上。S型羰基还原酶Go SCR最适p H为6.0,最适反应温度为45o C;该酶在p H7.0条件下放置18 h,残余酶活可保留85%以上,在碱性条件下的酶活稳定性大于BDHA;该酶热稳定性相比BDHA较差,20o C放置10 h,残余酶活可保留60%以上;Vmax为1.1U/mg,约为BDHA的50%,KM值为0.8 m M,kcat值为0.5 s-1,kcat/KM值为0.6 s-1m M-1;高浓度的有机溶剂DMSO会使其丧失大多数酶活,当DMSO浓度小于15%(v/v)时,仅可保留50%以上的初始酶活;但该酶同样具有较好的底物耐受性,底物浓度为200 m M时,仍能保留初始酶活的95%以上。再次,为解决羰基还原酶反应过程中辅酶消耗问题,将羰基还原酶与来源于枯草芽孢杆菌(Bacillus subtilis 168)的葡萄糖脱氢酶串联构建双酶偶联辅酶再生体系。分别将表达有BDHA、Go SCR和GDH的重组大肠杆菌进行了破碎,经离心后获得的粗酶液体外构建了羰基还原酶反应辅酶再生体系。实验结果发现双酶偶联辅酶再生体系BDHA/GDH和Go SCR/GDH体外催化50 m M 2-羟基苯乙酮不对称还原,在不额外添加辅酶NADH/NAD+的条件下,两个体系底物转化率大大提高,(R)-和(S)-1-苯基-1,2-乙二醇得率均达到99%以上,产物ee值均大于99%。这说明大肠杆菌细胞中自身所含辅酶通过葡萄糖脱氢酶得到了高效再生。由于整细胞反应具有催化剂制备快速、胞内辅因子可再生及下游处理成本低的优点。本文最后构建了羰基还原酶与葡萄糖脱氢酶在大肠杆菌细胞内共表达体系,并对整细胞反应条件进行优化。实验结果发现E.coil(BDHA-GDH)催化2-羟基苯乙酮不对称还原的适宜反应温度范围为20-35o C,适宜反应p H范围为6.0-8.0;E.coil(Go SCR-GDH)催化2-羟基苯乙酮不对称还原的适宜反应温度范围为25-30o C,适宜反应p H范围为7.0-8.0。E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)均具有较高的底物耐受性。当底物浓度为100 m M时,E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)最佳细胞用量均为10 g cdw(cell dry weight)/L。继续考察全细胞催化底物2-羟基苯乙酮反应进程发现,不额外添加辅酶时E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)在最优反应条件下,分别催化400m M 2-羟基苯乙酮不对称还原,产物(R)-和(S)-1-苯基-1,2-乙二醇得率均高达99%,ee值大于99%,时空得率为18 g·L-1·h-1。通过利用本实验所构建的E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)整细胞催化剂,可以高效催化底物浓度高达54 g/L的2-羟基苯乙酮,产物(R)-和(S)-1-苯基-1,2-乙二醇得率高达99%,ee值大于99%。本实验为手性苯乙二醇的工业化制备提供了一种新的生物催化方法,具有潜在的工业应用价值。
[Abstract]:The chiral diol is a very important compound, and has very important application in the aspects of drug synthesis, agricultural chemical synthesis, functional material preparation, perfume and essential oil additive. The method for preparing the chiral diol by the chemical method is complex and complex, the catalyst is expensive and toxic, and the environment is not friendly. The synthesis of the chiral diol by the biocatalysis of the asymmetric reduction of the prochiral hydroxy-ketone has been paid more and more attention by the researchers at home and abroad because of its good catalytic activity, stereoselectivity and environmental protection. In this paper,20 kinds of base-base reductases, which have been constructed in the laboratory, were selected for preliminary screening, and the two kinds of base-base reductase BDHA and Go SCR with high catalytic activity and stereoselectivity to the substrate 2-hydroxyacetophenone were obtained, and the expression and purification of 2-hydroxyacetophenone and the characterization of the enzymatic properties were obtained. By respectively constructing a double-enzyme coupling system of an in vitro and an in vivo yeast-based reductase and a glucose dehydrogenase, the high-efficiency cyclic regeneration of the coenzyme is realized, and the catalytic efficiency of the substrate 2-hydroxyacetophenone is further improved. in that first step, the 2-hydroxyacetophenone is used as a substrate, and 20 kinds of base-based reductases are first screened from the Bacillus subtilis 168 and the Gluconidium oxydans 621H by a gene digging method in the early stage of the laboratory to obtain the R-selective calcium-base reductase BDHA and the S-selective salt-base reductase Go SCR with higher catalytic activity, It was re-constructed into expression vector pET28a and high-efficiency soluble expression was achieved in E. coli. Secondly, the base-base reductase obtained by screening was purified by a nickel column, and an electrophoresis-pure enzyme was obtained, and the enzymatic property was characterized by using 2-hydroxyacetophenone as a substrate. The results showed that the optimum pH of the R-type base-base reductase (BDHA) was 6.0, the optimum temperature was 40 o C, the enzyme was placed in the buffer of p-H7.0 for 18 h, and the residual enzyme activity could be retained by more than 80%; the enzyme was sensitive to the high temperature, which was more than 40 o C, but it was placed at 30 o C for 18 h. The residual enzyme activity can be retained by more than 60%; Vmax is 2.1 U/ mg, the KM value is 1.0m M, the kcat value is 1.3 s-1, the kcat/ KM value is 1.3 s-1 m M-1, the enzyme is sensitive to the organic solvent dimethylara (DMSO), and when the concentration of DMSO is less than 15% (v/ v), more than 70% of the initial enzyme activity can be retained; and the enzyme has good substrate tolerance, The substrate has a concentration of up to 200 m and still retains more than 95% of the initial enzyme activity. The optimum pH of the S-type yeast-based reductase-Go SCR is 6.0, the optimum reaction temperature is 45o C, the enzyme is placed for 18 h under the condition of p H7.0, the activity of the residual enzyme can be kept above 85%, the activity stability of the enzyme under the alkaline condition is greater than that of the BDHA, the thermal stability of the enzyme is poor compared with the BDHA, and the 20 o C is placed for 10 h, The residual enzyme activity can be retained by more than 60%; Vmax is 1.1 U/ mg, about 50% of BDHA, the KM value is 0.8m M, the kcat value is 0.5s-1, the kcat/ KM value is 0.6 s-1 m M-1, the high-concentration organic solvent DMSO will lose most of the enzyme activity, and when the concentration of DMSO is less than 15% (v/ v), only 50% of the initial enzyme activity can be retained; However, the enzyme also has good substrate tolerance, and when the substrate concentration is 200 m M, more than 95% of the initial enzyme activity can be retained. In ord to solve that problem of the consumption of the coenzyme during the reaction of the base-base reductase, a double-enzyme-coupled coenzyme regeneration system is construct in series with the glucose dehydrogenase derived from the bacillus subtilis 168. The recombinant E. coli, which is expressed as BDHA, Go SCR and GDH, is broken, and the enzyme-based reductase-reactive coenzyme regeneration system is constructed out of the crude enzyme liquid obtained after centrifugation. The results show that the double-enzyme-coupled coenzyme regeneration system BDHA/ GDH and Go SCR/ GDH catalyze the asymmetric reduction of 50 m M 2-hydroxyacetophenone in vitro, and under the condition that the coenzyme NADH/ NAD + is not added, the conversion rate of the two system substrates is greatly improved, (R)-and (S) -1-phenyl-1,2-ethanediol yield is above 99%, and the product ee value is more than 99%. This indicates that the coenzyme itself contained in the E. coli cell is efficiently regenerated by the glucose dehydrogenase. Because the whole cell reaction has the advantages of rapid preparation of the catalyst, the regeneration of the intracellular cofactor and the low downstream treatment cost. In this paper, the co-expression system of yeast-base reductase and glucose dehydrogenase in E. coli cells was constructed, and the reaction conditions of the whole cell were optimized. The experimental results show that the suitable reaction temperature range of E. coil (BDHA-GDH) for asymmetric reduction of 2-hydroxyacetophenone is 20-35o C, the appropriate reaction p H range is 6.0-8.0, and the suitable reaction temperature range of E. coil (Go SCR-GDH) to catalyze the asymmetric reduction of 2-hydroxyacetophenone is 25-30o C, The suitable reaction p H ranges from 7.0-8.0.E.coil (BDHA-GDH) and E. coil (Go SCR-GDH) to have higher substrate tolerance. When the substrate concentration was 100 m M, the optimal cell dosage of E. coil (BDHA-GDH) and E. coil (Go SCR-GDH) was 10 g cdw (cell dry weight)/ L. It was found that E. coil (BDHA-GDH) and E. coil (Go SCR-GDH) were not added with coenzymes under the optimal reaction conditions. The yield of the product (R)-and (S) -1-phenyl-1,2-ethanediol is up to 99%, the ee value is more than 99%, and the space-time yield is 18 g 路 L-1 路 h-1, respectively. By using E. coil (BDHA-GDH) and E. coil (Go SCR-GDH) whole cell catalyst constructed in this experiment,2-hydroxyacetophenone with a substrate concentration of up to 54 g/ L can be efficiently catalyzed, and the yield of the product (R)-and (S)-1-phenyl-1,2-ethanediol is as high as 99%, and the ee value is more than 99%. The experiment provides a new biocatalysis method for the industrial preparation of the chiral benzene glycol, and has the potential industrial application value.
【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O643.36;TQ243.4
本文编号：2479441
[Abstract]:The chiral diol is a very important compound, and has very important application in the aspects of drug synthesis, agricultural chemical synthesis, functional material preparation, perfume and essential oil additive. The method for preparing the chiral diol by the chemical method is complex and complex, the catalyst is expensive and toxic, and the environment is not friendly. The synthesis of the chiral diol by the biocatalysis of the asymmetric reduction of the prochiral hydroxy-ketone has been paid more and more attention by the researchers at home and abroad because of its good catalytic activity, stereoselectivity and environmental protection. In this paper,20 kinds of base-base reductases, which have been constructed in the laboratory, were selected for preliminary screening, and the two kinds of base-base reductase BDHA and Go SCR with high catalytic activity and stereoselectivity to the substrate 2-hydroxyacetophenone were obtained, and the expression and purification of 2-hydroxyacetophenone and the characterization of the enzymatic properties were obtained. By respectively constructing a double-enzyme coupling system of an in vitro and an in vivo yeast-based reductase and a glucose dehydrogenase, the high-efficiency cyclic regeneration of the coenzyme is realized, and the catalytic efficiency of the substrate 2-hydroxyacetophenone is further improved. in that first step, the 2-hydroxyacetophenone is used as a substrate, and 20 kinds of base-based reductases are first screened from the Bacillus subtilis 168 and the Gluconidium oxydans 621H by a gene digging method in the early stage of the laboratory to obtain the R-selective calcium-base reductase BDHA and the S-selective salt-base reductase Go SCR with higher catalytic activity, It was re-constructed into expression vector pET28a and high-efficiency soluble expression was achieved in E. coli. Secondly, the base-base reductase obtained by screening was purified by a nickel column, and an electrophoresis-pure enzyme was obtained, and the enzymatic property was characterized by using 2-hydroxyacetophenone as a substrate. The results showed that the optimum pH of the R-type base-base reductase (BDHA) was 6.0, the optimum temperature was 40 o C, the enzyme was placed in the buffer of p-H7.0 for 18 h, and the residual enzyme activity could be retained by more than 80%; the enzyme was sensitive to the high temperature, which was more than 40 o C, but it was placed at 30 o C for 18 h. The residual enzyme activity can be retained by more than 60%; Vmax is 2.1 U/ mg, the KM value is 1.0m M, the kcat value is 1.3 s-1, the kcat/ KM value is 1.3 s-1 m M-1, the enzyme is sensitive to the organic solvent dimethylara (DMSO), and when the concentration of DMSO is less than 15% (v/ v), more than 70% of the initial enzyme activity can be retained; and the enzyme has good substrate tolerance, The substrate has a concentration of up to 200 m and still retains more than 95% of the initial enzyme activity. The optimum pH of the S-type yeast-based reductase-Go SCR is 6.0, the optimum reaction temperature is 45o C, the enzyme is placed for 18 h under the condition of p H7.0, the activity of the residual enzyme can be kept above 85%, the activity stability of the enzyme under the alkaline condition is greater than that of the BDHA, the thermal stability of the enzyme is poor compared with the BDHA, and the 20 o C is placed for 10 h, The residual enzyme activity can be retained by more than 60%; Vmax is 1.1 U/ mg, about 50% of BDHA, the KM value is 0.8m M, the kcat value is 0.5s-1, the kcat/ KM value is 0.6 s-1 m M-1, the high-concentration organic solvent DMSO will lose most of the enzyme activity, and when the concentration of DMSO is less than 15% (v/ v), only 50% of the initial enzyme activity can be retained; However, the enzyme also has good substrate tolerance, and when the substrate concentration is 200 m M, more than 95% of the initial enzyme activity can be retained. In ord to solve that problem of the consumption of the coenzyme during the reaction of the base-base reductase, a double-enzyme-coupled coenzyme regeneration system is construct in series with the glucose dehydrogenase derived from the bacillus subtilis 168. The recombinant E. coli, which is expressed as BDHA, Go SCR and GDH, is broken, and the enzyme-based reductase-reactive coenzyme regeneration system is constructed out of the crude enzyme liquid obtained after centrifugation. The results show that the double-enzyme-coupled coenzyme regeneration system BDHA/ GDH and Go SCR/ GDH catalyze the asymmetric reduction of 50 m M 2-hydroxyacetophenone in vitro, and under the condition that the coenzyme NADH/ NAD + is not added, the conversion rate of the two system substrates is greatly improved, (R)-and (S) -1-phenyl-1,2-ethanediol yield is above 99%, and the product ee value is more than 99%. This indicates that the coenzyme itself contained in the E. coli cell is efficiently regenerated by the glucose dehydrogenase. Because the whole cell reaction has the advantages of rapid preparation of the catalyst, the regeneration of the intracellular cofactor and the low downstream treatment cost. In this paper, the co-expression system of yeast-base reductase and glucose dehydrogenase in E. coli cells was constructed, and the reaction conditions of the whole cell were optimized. The experimental results show that the suitable reaction temperature range of E. coil (BDHA-GDH) for asymmetric reduction of 2-hydroxyacetophenone is 20-35o C, the appropriate reaction p H range is 6.0-8.0, and the suitable reaction temperature range of E. coil (Go SCR-GDH) to catalyze the asymmetric reduction of 2-hydroxyacetophenone is 25-30o C, The suitable reaction p H ranges from 7.0-8.0.E.coil (BDHA-GDH) and E. coil (Go SCR-GDH) to have higher substrate tolerance. When the substrate concentration was 100 m M, the optimal cell dosage of E. coil (BDHA-GDH) and E. coil (Go SCR-GDH) was 10 g cdw (cell dry weight)/ L. It was found that E. coil (BDHA-GDH) and E. coil (Go SCR-GDH) were not added with coenzymes under the optimal reaction conditions. The yield of the product (R)-and (S) -1-phenyl-1,2-ethanediol is up to 99%, the ee value is more than 99%, and the space-time yield is 18 g 路 L-1 路 h-1, respectively. By using E. coil (BDHA-GDH) and E. coil (Go SCR-GDH) whole cell catalyst constructed in this experiment,2-hydroxyacetophenone with a substrate concentration of up to 54 g/ L can be efficiently catalyzed, and the yield of the product (R)-and (S)-1-phenyl-1,2-ethanediol is as high as 99%, and the ee value is more than 99%. The experiment provides a new biocatalysis method for the industrial preparation of the chiral benzene glycol, and has the potential industrial application value.
【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O643.36;TQ243.4
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