新型羰基還原酶的挖掘及其應(yīng)用于手性苯乙二醇合成的研究
發(fā)布時(shí)間：2019-05-17 22:11
【摘要】：手性二醇是一類十分重要的化合物,在藥物合成、農(nóng)業(yè)化學(xué)品合成、功能性材料制備、香料和精油添加劑等方面都具有十分重要的用途。化學(xué)法制備手性二醇反應(yīng)步驟復(fù)雜繁瑣,催化劑昂貴且有毒、對(duì)環(huán)境不友好。生物法催化潛手性羥酮不對(duì)稱還原合成手性二醇因其良好的催化活性、立體選擇性和環(huán)境友好等優(yōu)點(diǎn)受到國(guó)內(nèi)外研究者們的重視。本課題通過對(duì)實(shí)驗(yàn)室已構(gòu)建的20種羰基還原酶進(jìn)行初步篩選,得到對(duì)底物2-羥基苯乙酮催化活性高、立體選擇性相反的兩種羰基還原酶BDHA和Go SCR,并對(duì)其進(jìn)行表達(dá)純化及酶學(xué)性質(zhì)表征;通過分別構(gòu)建體外和體內(nèi)羰基還原酶與葡萄糖脫氫酶雙酶偶聯(lián)體系,實(shí)現(xiàn)了輔酶高效循環(huán)再生,進(jìn)一步提高了對(duì)底物2-羥基苯乙酮的催化效率。首先,以2-羥基苯乙酮為底物,對(duì)實(shí)驗(yàn)室前期通過基因挖掘法從Bacillus subtilis 168和Gluconobac oxydans 621H中克隆得到20種羰基還原酶進(jìn)行初步篩選,得到催化活力較高的R選擇性羰基還原酶BDHA和S選擇性羰基還原酶Go SCR,將其重新構(gòu)建至表達(dá)載體p ET28a中,并在大腸桿菌中實(shí)現(xiàn)高效可溶表達(dá)。其次,對(duì)篩選獲得的羰基還原酶通過鎳柱進(jìn)行了純化,并獲得電泳純的酶,以2-羥基苯乙酮為底物對(duì)其進(jìn)行了酶學(xué)性質(zhì)表征。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)R型羰基還原酶BDHA最適p H為6.0,最適溫度為40o C;該酶在p H7.0的緩沖液中放置18 h,殘余酶活可保留80%以上;該酶對(duì)高溫敏感,大于40o C迅速失活,但在30o C放置18 h,殘余酶活可保留60%以上;Vmax為2.1 U/mg,KM值為1.0 m M,kcat值為1.3 s-1,kcat/KM值為1.3 s-1m M-1;該酶對(duì)有機(jī)溶劑二甲基亞砜(DMSO)敏感,當(dāng)DMSO濃度小于15%(v/v)時(shí),可保留70%以上的初始酶活;該酶具有良好的底物耐受性,底物濃度高達(dá)200 m M,仍能保留初始酶活的95%以上。S型羰基還原酶Go SCR最適p H為6.0,最適反應(yīng)溫度為45o C;該酶在p H7.0條件下放置18 h,殘余酶活可保留85%以上,在堿性條件下的酶活穩(wěn)定性大于BDHA;該酶熱穩(wěn)定性相比BDHA較差,20o C放置10 h,殘余酶活可保留60%以上;Vmax為1.1U/mg,約為BDHA的50%,KM值為0.8 m M,kcat值為0.5 s-1,kcat/KM值為0.6 s-1m M-1;高濃度的有機(jī)溶劑DMSO會(huì)使其喪失大多數(shù)酶活,當(dāng)DMSO濃度小于15%(v/v)時(shí),僅可保留50%以上的初始酶活;但該酶同樣具有較好的底物耐受性,底物濃度為200 m M時(shí),仍能保留初始酶活的95%以上。再次,為解決羰基還原酶反應(yīng)過程中輔酶消耗問題,將羰基還原酶與來源于枯草芽孢桿菌(Bacillus subtilis 168)的葡萄糖脫氫酶串聯(lián)構(gòu)建雙酶偶聯(lián)輔酶再生體系。分別將表達(dá)有BDHA、Go SCR和GDH的重組大腸桿菌進(jìn)行了破碎,經(jīng)離心后獲得的粗酶液體外構(gòu)建了羰基還原酶反應(yīng)輔酶再生體系。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)雙酶偶聯(lián)輔酶再生體系BDHA/GDH和Go SCR/GDH體外催化50 m M 2-羥基苯乙酮不對(duì)稱還原,在不額外添加輔酶NADH/NAD+的條件下,兩個(gè)體系底物轉(zhuǎn)化率大大提高,(R)-和(S)-1-苯基-1,2-乙二醇得率均達(dá)到99%以上,產(chǎn)物ee值均大于99%。這說明大腸桿菌細(xì)胞中自身所含輔酶通過葡萄糖脫氫酶得到了高效再生。由于整細(xì)胞反應(yīng)具有催化劑制備快速、胞內(nèi)輔因子可再生及下游處理成本低的優(yōu)點(diǎn)。本文最后構(gòu)建了羰基還原酶與葡萄糖脫氫酶在大腸桿菌細(xì)胞內(nèi)共表達(dá)體系,并對(duì)整細(xì)胞反應(yīng)條件進(jìn)行優(yōu)化。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)E.coil(BDHA-GDH)催化2-羥基苯乙酮不對(duì)稱還原的適宜反應(yīng)溫度范圍為20-35o C,適宜反應(yīng)p H范圍為6.0-8.0;E.coil(Go SCR-GDH)催化2-羥基苯乙酮不對(duì)稱還原的適宜反應(yīng)溫度范圍為25-30o C,適宜反應(yīng)p H范圍為7.0-8.0。E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)均具有較高的底物耐受性。當(dāng)?shù)孜餄舛葹?00 m M時(shí),E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)最佳細(xì)胞用量均為10 g cdw(cell dry weight)/L。繼續(xù)考察全細(xì)胞催化底物2-羥基苯乙酮反應(yīng)進(jìn)程發(fā)現(xiàn),不額外添加輔酶時(shí)E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)在最優(yōu)反應(yīng)條件下,分別催化400m M 2-羥基苯乙酮不對(duì)稱還原,產(chǎn)物(R)-和(S)-1-苯基-1,2-乙二醇得率均高達(dá)99%,ee值大于99%,時(shí)空得率為18 g·L-1·h-1。通過利用本實(shí)驗(yàn)所構(gòu)建的E.coil(BDHA-GDH)和E.coil(Go SCR-GDH)整細(xì)胞催化劑,可以高效催化底物濃度高達(dá)54 g/L的2-羥基苯乙酮,產(chǎn)物(R)-和(S)-1-苯基-1,2-乙二醇得率高達(dá)99%,ee值大于99%。本實(shí)驗(yàn)為手性苯乙二醇的工業(yè)化制備提供了一種新的生物催化方法,具有潛在的工業(yè)應(yīng)用價(jià)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.
【學(xué)位授予單位】：太原理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O643.36;TQ243.4
本文編號(hào)：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.
【學(xué)位授予單位】：太原理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O643.36;TQ243.4
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