醋酸杆菌催化手性醇的不对称合成及其反-Prelog羰基还原酶的酶学特性研究
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摘要
光学纯的手性醇及其衍生物是合成手性药物、农药及功能材料的重要中间体，其中对映体纯的芳香醇类是合成多种治疗神经系统和心血管疾病药物以及抗炎药物、抗过敏药物的手性砌块；光学纯脂肪醇类如R型或S型的2-辛醇是合成手性液晶材料、类固醇、昆虫性外激素等的重要手性前体物质。目前，采用酶或微生物细胞催化手性醇合成的生物催化法受到广泛的关注。
本课题组从“中华开菲尔”菌粒中成功筛选得到一株新型醋酸杆菌Acetobacter sp.CCTCC M209061，该菌能遵循反-Prelog规则催化4-三甲基硅基-3-丁炔-2-酮（TMSBO）高效、高选择性的不对称还原合成对映体纯的(R)-4-三甲基硅基-3-丁炔-2-醇[(R)-TMSBL]。前述研究采用番茄汁培养基培养该新菌株，生物量较低，细胞的还原活性及稳定性也不甚理想，这限制了该新型菌株的工业化应用。因此，本文首先考察了培养基组成及培养条件对醋酸杆菌Acetobacter sp. CCTCC M209061的生长及催化活性的影响，提高了该菌株的生物量及还原活性；随后将该活性细胞固定化，获得了稳定性好、重复性佳且催化活性高的固定化Acetobacter sp. CCTCC M209061细胞；同时探讨了不同反应介质对固定化细胞催化(R)-2-辛醇不对称合成的影响规律，建立可广泛用于高效合成对映体纯手性醇的生物催化体系；并分离纯化得到在不对称还原反应中起关键作用的反-Prelog羰基还原酶，系统研究了其酶学特性，为该酶的进一步研究及实际应用奠定基础。
结合单因素实验方法和响应面分析方法（中心组合旋转设计）考察了培养基组成和培养条件对醋酸杆菌Acetobacter sp. CCTCC M209061的生长及还原活性的影响，优化后的培养基组成和培养条件分别为：葡萄糖8.3g/L，果糖2.5g/L，大豆蛋白胨83.9g/L，MnSO4·H2O0.088g/L，初始pH值为5.7，培养温度30℃，摇床转速为80r/min，接种量为10%（v/v）。在此条件下培养30h后，所得生物量达1.10g/L，为初始条件下的9.5倍，细胞催化4'-氯苯乙酮不对称还原为(R)-1-(4-氯苯基)乙醇的比活由29.34μmol/min/g提高到39.49μmol/min/g，产物的e.e.值为99%以上。此外，优化培养所得的Acetobactersp. CCTCC M209061细胞催化TMSBO不对称还原为药物中间体(R)-TMSBL的催化效率也得到了提高。
尽管优化培养所得的Acetobacter sp. CCTCC M209061细胞具有较好的催化活性，但其稳定性和重复利用性相对其他生物催化剂而言仍然较差。因此，考察了不同固定化方法对醋酸杆菌细胞操作稳定性的影响。研究发现，海藻酸钙包埋的Acetobacter sp.CCTCC M209061细胞具有较好的催化活性，利用壳聚糖覆膜可进一步提高其机械强度和抗溶胀性能。以催化4'-氯苯乙酮不对称还原的活性及重复批次为标准，确定最优的固定化条件为：海藻酸钠2.5%（w/v），CaCl20.2mol/L，壳聚糖0.9%（w/v），壳聚糖溶液的pH值5.0，覆膜时间20min，细胞载量为每克湿固定化颗粒中含细胞干重17.2mg。在此条件下制备的固定化Acetobacter sp. CCTCC M209061细胞的活性为等量游离细胞活性的85%，其热稳定性、pH稳定性、溶剂耐受性以及贮藏稳定性相对游离细胞均有所提高，连续反应25批后，其催化活性保持50%以上，是游离细胞重复批次的3.5倍（7批次）。反应25批次后的固定化细胞在新鲜培养基中复活培养24h后，可再重复反应25批次。在反应体系中，达姆科勒系数Da﹤﹤1，说明外部传质阻力可忽略；效率因子i﹤1和蒂勒模数0.3﹤﹤1，表明内部传质阻力对催化反应有一定影响但并非主要限制性因素。海藻酸钙包埋壳聚糖覆膜有效地固定Acetobacter sp. CCTCC M209061细胞，提高其重复利用性。
随后考察了不同反应介质中固定化Acetobacter sp. CCTCC M209061细胞催化2-辛酮的不对称还原反应，进一步扩展其在手性醇合成中的应用。在水相反应体系中，该反应的最适pH值、反应温度、辅底物、辅底物浓度、底物浓度和振荡速度分别为5.5、35℃、葡萄糖、100mmol/L、6mmol/L、200r/min。在上述反应条件下，固定化醋酸杆菌细胞（0.3g/mL）催化2-辛酮不对称还原反应的初速度、最大产率及产物的e.e.值分别为0.393mmol/L/min，99.0%和97.3%。在水相反应体系中，因底物溶解度低，底物及产物抑制严重，故反应效率较低。为此，探讨了向水相缓冲液体系中添加亲水性离子液体对该反应的影响。研究发现，添加[C4MIM]·Ac有利于增加细胞膜的通透性，降低胞内产物浓度，提高反应效率。最适[C4MIM]·Ac浓度为3%（m/v），最适底物浓度、反应初速度和产物的e.e.值均高于水相体系的对应值，分别为10mmol/L、0.558mmol/L/min和99.3%。为进一步提高反应效率，在含[C4MIM]·Ac缓冲液体系中引入正十四烷第二相，以有效萃取2-辛酮和2-辛醇，控制水相中的底物和产物浓度处于较低水平，减轻底物和产物抑制。最适两相体积比（Vaq/Vor）和底物浓度分别为3:1和500mmol/L，产率为53.4%，产物浓度可达267mmol/L，产物的e.e.值﹥99%。可见含[C4MIM]·Ac缓冲液/正十四烷双相反应体系显著提高了该反应的效率。
在Acetobacter sp. CCTCC M209061全细胞催化羰基化合物高选择性不对称还原的过程中，起关键催化作用的是细胞内的反-Prelog羰基还原酶（AcCR），本研究通过4步将之分离纯化，总酶活得率为0.4%，纯化倍数为27.5倍，比活力为3.85U/mg。该酶的表观分子量为104kDa，亚基分子量为27kDa，是同源四聚体结构，含有四个相同亚基。
AcCR能催化羰基的还原和对应醇的氧化，NAD(H)和NADP(H)均可作为其辅酶。该酶以NADH为辅酶催化4'-氯苯乙酮不对称还原时，最适pH值为5.0，最适温度为25℃，与全细胞催化反应的最适条件相似；而以NADPH为辅酶时，最适pH值为7.5，最适温度为25℃。在催化异丙醇的氧化时，以NAD+或NADP+为辅酶时AcCR表现出相似的催化特性，最适pH值和最适温度相同，分别为8.0和35℃。Mn2+，Ni2+和Fe2+对AcCR具有显著的激活作用，Zn2+和Ca2+次之，Co2+和Mg2+也有一定的激活作用。Hg2+和Ag+对该酶的活性有显著的抑制作用，Cu2+次之。碘乙酰胺和β-巯基乙醇对AcCR的活性影响较小，5mmol/L的EDTA完全抑制了该酶的活力。此外，该酶可催化多种羰基化合物如芳香酮类、脂肪酮类、α-酮酯类的高效、高选择性不对称还原。
对AcCR催化4'-氯苯乙酮不对称还原反应的动力学研究发现，AcCR对NADH的Km值是对NADPH的Km值的25倍（0.66mmol/L vs0.026mmol/L），说明该酶对NADPH的亲和力远远高于对NADH的亲和力。当NADH作为辅酶时，反应的初速度随底物4'-氯苯乙酮浓度的变化曲线呈S型，符合Hill模型，所得Hill系数为3.1，表明四个亚基的底物结合位点之间存在相互协同作用，反应的Vmax值稍高于NADPH为辅酶时的Vmax值（0.21mmol/L/min vs0.17mmol/L/min）。同样研究了AcCR催化异丙醇氧化反应的动力学性质，以NAD+为辅酶时，Km和Vmax值分别为1.33mmol/L（NAD+），48.32mmol/L（异丙醇），0.069mmol/L/min；以NADP+为辅酶时，Km和Vmax值分别为1.12mmol/L（NADP+），67.82mmol/L（异丙醇），0.074mmol/L/min。
本研究不仅有助于丰富对生物催化和生物转化的认识，还提供了高效、高选择性制备对映体纯手性醇的新途径。
Optically pure chiral alcohols and their derivatives are important intermediates for thesynthesis of chiral pharmaceuticals, pesticides and functional materials. For examples,enantiopure aromatic alcohols are chiral building blocks for the synthesis of pharmaceuticalswhich could be used for the treatment of diseases of the cardiovascular and nervous systems,as well as allergic response and inflammation. Optically pure aliphatic alcohols such as(S)-2-octanol and (R)-2-octanol are key chiral synthons for the synthesis of chiral liquidmaterials, steroid, insect sex pheromones. Currently, among the studies on the production ofchiral alcohols, biocatalysis using enzymes or microbial cells as catalysts has attracted muchattention.
Our research group has successfully isolated a novel strain Acetobacter sp. CCTCCM209061from Chinese kefir grains, which showed exclusive anti-Prelog stereoselectivity forthe reduction of4-(trimethylsilyl)-3-butyn-2-one (TMSBO) to(R)-4-(trimethylsilyl)-3-butyn-2-ol [(R)-TMSBL]. In the previous works, it was found thatthis new strain exhibited low biomass when cultivated in tomato juice medium withdisappointingly low activity and stability, which limited its industrial application. Therefore,in this dissertation, the effects of medium composition and culture conditions on growth andcatalytic activity of Acetobacter sp. CCTCC M209061were explored to improve the biomassand the reduction activity of this strain; and immobilized Acetobacter sp. CCTCC M209061cells with good stability, reusability and activity were prepared by immobilizing the activecells. The effects of different reaction media on the asymmetric synthesis of (R)-2-octanolwith immobilized Acetobacter sp. CCTCC M209061cells were also examined and thebiocatalytic reaction systems used for highly efficient production of enantiopure alcohols havebeen well established. Moreover, purification and characterization of the carbonyl reductasecatalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelogstereoselectivity from Acetobacter sp. CCTCC M209061were carried out. The enzymologicalproperties of AcCR were systematically investigated for further studies and practicalapplications.
The effects of medium components and culture conditions on the strain’s growth andreduction activity were explored using a one-at-a-time method and a central compositerotatable design (CCRD). The optimal medium and culture conditions were found to be asfollows: glucose8.3g/L, fructose2.5g/L, soy peptone83.9g/L, MnSO4·H2O0.088g/L, pH5.7,30°C,80r/min and10%(v/v) inoculum. Under the above-mentioned conditions, the biomass after30h cultivation reached1.10g/L, which is9.5-fold higher than that obtainedwith basic medium. Also, the reduction activity towards4'-chloroacetophenone was markedlyenhanced to39.49μmol/min/g from29.34μmol/min/g, with the product e.e. being above99%.Comparable improvements were also seen with the enantioselective bioreduction of TMSBOto the key pharmaceutical precursor (R)-TMSBL.
Although Acetobacter sp. CCTCC M209061cells cultivated in optimized medium hadpromising catalytic properties, its stability and reusability were relatively poor compared toother biocatalysts. Hence, the effects of various immobilizing methods on the operationalstability of the cells were examined here. It was found that Ca-alginate gave the bestimmobilized biocatalyst, which was then coated with chitosan to further improve itsmechanical strength and swelling-resistance properties. Conditions were optimized forformation of reusable immobilized beads which can keep high activity and be used forrepeated batch asymmetric reduction of4'-chloroacetophenone. The optimal immobilizationconditions were found to be as follows: sodium alginate2.5%(w/v), CaCl20.2mol/L,chitosan0.9%(w/v), pH of chitosan solution5.0, coating time20min, cell loading17.2mg-dw cell/g-ww catalyst. The resulting immobilized biocatalyst was very promising, with aspecific activity of85%that of the free-cell biocatalyst. The immobilized cells showed betterthermal stability, pH stability, solvent tolerance and storability compared with free cells. After25cycles reaction, the immobilized beads still retained>50%catalytic activity, which was3.5times higher than that of free cells (7cycles). The immobilized cells could be recultured infresh medium to regain full activity and perform a further25cycles of the reduction reaction.The external mass transfer resistances were negligible as deduced from Damkohler modulusDa <<1, and internal mass transfer restriction affected the reaction but was not therate-controlling step according to effectiveness factors i<1and Thiele modulus0.3<<1. Ca-alginate coated with chitosan is a highly effective material for immobilization ofAcetobacter sp. CCTCC M209061cells for repeated use.
The asymmetric reduction of2-octanone catalyzed by immobilized Acetobacter sp.CCTCC M209061cells in various media was explored to broaden its application inproduction of chiral alcohols. In the aqueous monophasic system, the optimal pH value,reaction temperature, co-substrate and its concentration, substrate concentration and shakingrate were5.5,35°C, glucose,100mmol/L,6mmol/L,200r/min, respectively, under which,the initial reaction rate, the yield and the product e.e. with0.3g/mL immobilized cells were0.393mmol/L/min,99.0%and97.3%, respectively. However, the poor solubility of substrates and the pronounced inhibition of the reactants and the products were observed in aqueousmonophasic system, resulting in dissatisfactory reaction efficiency. Therefore, water-miscibleionic liquids were firstly added into aqueous buffer system for evaluating the effects ofvarious ionic liquids on asymmetric reduction of2-octanone to (R)-2-octanol catalyzed byimmobilized Acetobacter sp. CCTCC M209061cells. It was found that the addition of[C4MIM]·Ac could effectively make the cell membrane more permeable, and lower theproduct concentration in the cells, thus enhancing the efficiency of the reaction. The optimal[C4MIM]·Ac content was3%(w/v), and the concentration of2-octanone, the initial reactionrate and the product e.e. were10mmol/L,0.558mmol/L/min and99.3%, respectively, whichwere higher than the corresponding values in the aqueous buffer system. To further improvethe reaction efficiency, n-tetradecane as a second phase was introduced into the[C4MIM]·Ac-containing buffer system, which could effectively extract2-octanone and2-octanol to keep substrate and product concentrations at low level in aqueous phase, thusalleviating the inhibition of the substrate and the product. The optimal volume ratio ofaqueous phase to organic phase and2-octanone concentration were3:1and500mmol/L,respectively, under which the obtained yield, product concentration and product e.e. for thebioreduction were53.4%,267mmol/L and>99%, respectively. Obviously, the[C4MIM]·Ac-containing buffer/n-tetradecane biphasic system significantly enhanced thereaction efficiency.
The anti-Prelog carbonyl reductase，the key enzyme in cytoplasm of Acetobacter sp.CCTCC M209061(AcCR) for the asymmetric reduction of carbonyl compounds with highstereoselectivity, was isolated and purified via four steps. The enzyme was enriched27.5-foldafter purification with an overall yield of0.4%and specific activity of3.85U/mg. AcCR hada homotetrameric structure with an apparent molecular mass of104kDa and each subunit of27kDa.
AcCR can catalyze the reduction of carbonyl groups and the oxidation of correspondingalcohols utilizing either NAD(H) or NADP(H) as coenzyme. For the reduction of4'-chloroacetophenone using NADH as coenzyme, its optimum activity was at pH5.0and25°C, which were similar with the optimum conditions of whole cell-catalyzed reduction. WhenNADPH acted as the cofactor, the optimum pH and the reaction temperature were7.5and25°C. For isopropanol oxidation, similar catalytic properties of AcCR were found using eitherNAD+or NADP+as cofactor. The optimum pH and reaction temperature were8.0and35°C.Metal ions such as Mn2+, Ni2+and Fe2+remarkably activated the enzyme, followed by Zn2+and Ca2+. Co2+and Mg2+showed weak activation on the enzyme. Hg2+and Ag+completely inhibited the activity of the enzyme, while Cu2+showed relatively weaker inhibition on theenzyme. Iodoacetamide and β-mercaptoethanol showed little effect on enzymatic activity.5mmol/L EDTA completely inhibited the activity of AcCR. Furthermore, a broad range ofcarbonyl compounds such as aryl ketones, α-ketoesters and aliphatic ketones could beenantioselectively reduced by this enzyme with high activity.
The reaction kinetics of4'-chloroacetophenone reduction catalyzed by AcCR was explored.It was found that the Kmvalue of AcCR for NADH was over25-fold greater than that forNADPH (0.66mmol/L vs0.026mmol/L), showing that the enzyme had a preference forNADPH over NADH. When NADH was used as cofactor, the response of carbonyl reductaseactivity to increasing concentration of4'-chloroacetophenone was clearly sigmoidal with aHill coefficient of3.1, suggesting that the enzyme might possess four substrate-binding sitescooperated with each other when NADH was present. The Vmaxvalue for NADH-basedreduction of4'-chloroacetophenone was slightly higher than that for NADPH-based reduction(0.21mmol/L/min vs0.17mmol/L/min). The reaction kenitics of isopropanol oxidationcatalyzed by AcCR was also explored. When NAD+acted as cofactor, the Kmand Vmaxvalueswere1.33mmol/L (NAD+),48.32mmol/L (isopropanol) and0.069mmol/L/min. WhenNADP+acted as cofactor, the Kmvalues for NADP+and isopropanol were1.12mmol/L and67.82mmol/L, respectively, and the Vmaxvalue was0.074mmol/L/min.
This study provides not only a better understanding of biocatalysis and biotransformations,but also a novel route to the preparation of enantiopure chiral alcohols.
本课题组从“中华开菲尔”菌粒中成功筛选得到一株新型醋酸杆菌Acetobacter sp.CCTCC M209061，该菌能遵循反-Prelog规则催化4-三甲基硅基-3-丁炔-2-酮（TMSBO）高效、高选择性的不对称还原合成对映体纯的(R)-4-三甲基硅基-3-丁炔-2-醇[(R)-TMSBL]。前述研究采用番茄汁培养基培养该新菌株，生物量较低，细胞的还原活性及稳定性也不甚理想，这限制了该新型菌株的工业化应用。因此，本文首先考察了培养基组成及培养条件对醋酸杆菌Acetobacter sp. CCTCC M209061的生长及催化活性的影响，提高了该菌株的生物量及还原活性；随后将该活性细胞固定化，获得了稳定性好、重复性佳且催化活性高的固定化Acetobacter sp. CCTCC M209061细胞；同时探讨了不同反应介质对固定化细胞催化(R)-2-辛醇不对称合成的影响规律，建立可广泛用于高效合成对映体纯手性醇的生物催化体系；并分离纯化得到在不对称还原反应中起关键作用的反-Prelog羰基还原酶，系统研究了其酶学特性，为该酶的进一步研究及实际应用奠定基础。
结合单因素实验方法和响应面分析方法（中心组合旋转设计）考察了培养基组成和培养条件对醋酸杆菌Acetobacter sp. CCTCC M209061的生长及还原活性的影响，优化后的培养基组成和培养条件分别为：葡萄糖8.3g/L，果糖2.5g/L，大豆蛋白胨83.9g/L，MnSO4·H2O0.088g/L，初始pH值为5.7，培养温度30℃，摇床转速为80r/min，接种量为10%（v/v）。在此条件下培养30h后，所得生物量达1.10g/L，为初始条件下的9.5倍，细胞催化4'-氯苯乙酮不对称还原为(R)-1-(4-氯苯基)乙醇的比活由29.34μmol/min/g提高到39.49μmol/min/g，产物的e.e.值为99%以上。此外，优化培养所得的Acetobactersp. CCTCC M209061细胞催化TMSBO不对称还原为药物中间体(R)-TMSBL的催化效率也得到了提高。
尽管优化培养所得的Acetobacter sp. CCTCC M209061细胞具有较好的催化活性，但其稳定性和重复利用性相对其他生物催化剂而言仍然较差。因此，考察了不同固定化方法对醋酸杆菌细胞操作稳定性的影响。研究发现，海藻酸钙包埋的Acetobacter sp.CCTCC M209061细胞具有较好的催化活性，利用壳聚糖覆膜可进一步提高其机械强度和抗溶胀性能。以催化4'-氯苯乙酮不对称还原的活性及重复批次为标准，确定最优的固定化条件为：海藻酸钠2.5%（w/v），CaCl20.2mol/L，壳聚糖0.9%（w/v），壳聚糖溶液的pH值5.0，覆膜时间20min，细胞载量为每克湿固定化颗粒中含细胞干重17.2mg。在此条件下制备的固定化Acetobacter sp. CCTCC M209061细胞的活性为等量游离细胞活性的85%，其热稳定性、pH稳定性、溶剂耐受性以及贮藏稳定性相对游离细胞均有所提高，连续反应25批后，其催化活性保持50%以上，是游离细胞重复批次的3.5倍（7批次）。反应25批次后的固定化细胞在新鲜培养基中复活培养24h后，可再重复反应25批次。在反应体系中，达姆科勒系数Da﹤﹤1，说明外部传质阻力可忽略；效率因子i﹤1和蒂勒模数0.3﹤﹤1，表明内部传质阻力对催化反应有一定影响但并非主要限制性因素。海藻酸钙包埋壳聚糖覆膜有效地固定Acetobacter sp. CCTCC M209061细胞，提高其重复利用性。
随后考察了不同反应介质中固定化Acetobacter sp. CCTCC M209061细胞催化2-辛酮的不对称还原反应，进一步扩展其在手性醇合成中的应用。在水相反应体系中，该反应的最适pH值、反应温度、辅底物、辅底物浓度、底物浓度和振荡速度分别为5.5、35℃、葡萄糖、100mmol/L、6mmol/L、200r/min。在上述反应条件下，固定化醋酸杆菌细胞（0.3g/mL）催化2-辛酮不对称还原反应的初速度、最大产率及产物的e.e.值分别为0.393mmol/L/min，99.0%和97.3%。在水相反应体系中，因底物溶解度低，底物及产物抑制严重，故反应效率较低。为此，探讨了向水相缓冲液体系中添加亲水性离子液体对该反应的影响。研究发现，添加[C4MIM]·Ac有利于增加细胞膜的通透性，降低胞内产物浓度，提高反应效率。最适[C4MIM]·Ac浓度为3%（m/v），最适底物浓度、反应初速度和产物的e.e.值均高于水相体系的对应值，分别为10mmol/L、0.558mmol/L/min和99.3%。为进一步提高反应效率，在含[C4MIM]·Ac缓冲液体系中引入正十四烷第二相，以有效萃取2-辛酮和2-辛醇，控制水相中的底物和产物浓度处于较低水平，减轻底物和产物抑制。最适两相体积比（Vaq/Vor）和底物浓度分别为3:1和500mmol/L，产率为53.4%，产物浓度可达267mmol/L，产物的e.e.值﹥99%。可见含[C4MIM]·Ac缓冲液/正十四烷双相反应体系显著提高了该反应的效率。
在Acetobacter sp. CCTCC M209061全细胞催化羰基化合物高选择性不对称还原的过程中，起关键催化作用的是细胞内的反-Prelog羰基还原酶（AcCR），本研究通过4步将之分离纯化，总酶活得率为0.4%，纯化倍数为27.5倍，比活力为3.85U/mg。该酶的表观分子量为104kDa，亚基分子量为27kDa，是同源四聚体结构，含有四个相同亚基。
AcCR能催化羰基的还原和对应醇的氧化，NAD(H)和NADP(H)均可作为其辅酶。该酶以NADH为辅酶催化4'-氯苯乙酮不对称还原时，最适pH值为5.0，最适温度为25℃，与全细胞催化反应的最适条件相似；而以NADPH为辅酶时，最适pH值为7.5，最适温度为25℃。在催化异丙醇的氧化时，以NAD+或NADP+为辅酶时AcCR表现出相似的催化特性，最适pH值和最适温度相同，分别为8.0和35℃。Mn2+，Ni2+和Fe2+对AcCR具有显著的激活作用，Zn2+和Ca2+次之，Co2+和Mg2+也有一定的激活作用。Hg2+和Ag+对该酶的活性有显著的抑制作用，Cu2+次之。碘乙酰胺和β-巯基乙醇对AcCR的活性影响较小，5mmol/L的EDTA完全抑制了该酶的活力。此外，该酶可催化多种羰基化合物如芳香酮类、脂肪酮类、α-酮酯类的高效、高选择性不对称还原。
对AcCR催化4'-氯苯乙酮不对称还原反应的动力学研究发现，AcCR对NADH的Km值是对NADPH的Km值的25倍（0.66mmol/L vs0.026mmol/L），说明该酶对NADPH的亲和力远远高于对NADH的亲和力。当NADH作为辅酶时，反应的初速度随底物4'-氯苯乙酮浓度的变化曲线呈S型，符合Hill模型，所得Hill系数为3.1，表明四个亚基的底物结合位点之间存在相互协同作用，反应的Vmax值稍高于NADPH为辅酶时的Vmax值（0.21mmol/L/min vs0.17mmol/L/min）。同样研究了AcCR催化异丙醇氧化反应的动力学性质，以NAD+为辅酶时，Km和Vmax值分别为1.33mmol/L（NAD+），48.32mmol/L（异丙醇），0.069mmol/L/min；以NADP+为辅酶时，Km和Vmax值分别为1.12mmol/L（NADP+），67.82mmol/L（异丙醇），0.074mmol/L/min。
本研究不仅有助于丰富对生物催化和生物转化的认识，还提供了高效、高选择性制备对映体纯手性醇的新途径。
Optically pure chiral alcohols and their derivatives are important intermediates for thesynthesis of chiral pharmaceuticals, pesticides and functional materials. For examples,enantiopure aromatic alcohols are chiral building blocks for the synthesis of pharmaceuticalswhich could be used for the treatment of diseases of the cardiovascular and nervous systems,as well as allergic response and inflammation. Optically pure aliphatic alcohols such as(S)-2-octanol and (R)-2-octanol are key chiral synthons for the synthesis of chiral liquidmaterials, steroid, insect sex pheromones. Currently, among the studies on the production ofchiral alcohols, biocatalysis using enzymes or microbial cells as catalysts has attracted muchattention.
Our research group has successfully isolated a novel strain Acetobacter sp. CCTCCM209061from Chinese kefir grains, which showed exclusive anti-Prelog stereoselectivity forthe reduction of4-(trimethylsilyl)-3-butyn-2-one (TMSBO) to(R)-4-(trimethylsilyl)-3-butyn-2-ol [(R)-TMSBL]. In the previous works, it was found thatthis new strain exhibited low biomass when cultivated in tomato juice medium withdisappointingly low activity and stability, which limited its industrial application. Therefore,in this dissertation, the effects of medium composition and culture conditions on growth andcatalytic activity of Acetobacter sp. CCTCC M209061were explored to improve the biomassand the reduction activity of this strain; and immobilized Acetobacter sp. CCTCC M209061cells with good stability, reusability and activity were prepared by immobilizing the activecells. The effects of different reaction media on the asymmetric synthesis of (R)-2-octanolwith immobilized Acetobacter sp. CCTCC M209061cells were also examined and thebiocatalytic reaction systems used for highly efficient production of enantiopure alcohols havebeen well established. Moreover, purification and characterization of the carbonyl reductasecatalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelogstereoselectivity from Acetobacter sp. CCTCC M209061were carried out. The enzymologicalproperties of AcCR were systematically investigated for further studies and practicalapplications.
The effects of medium components and culture conditions on the strain’s growth andreduction activity were explored using a one-at-a-time method and a central compositerotatable design (CCRD). The optimal medium and culture conditions were found to be asfollows: glucose8.3g/L, fructose2.5g/L, soy peptone83.9g/L, MnSO4·H2O0.088g/L, pH5.7,30°C,80r/min and10%(v/v) inoculum. Under the above-mentioned conditions, the biomass after30h cultivation reached1.10g/L, which is9.5-fold higher than that obtainedwith basic medium. Also, the reduction activity towards4'-chloroacetophenone was markedlyenhanced to39.49μmol/min/g from29.34μmol/min/g, with the product e.e. being above99%.Comparable improvements were also seen with the enantioselective bioreduction of TMSBOto the key pharmaceutical precursor (R)-TMSBL.
Although Acetobacter sp. CCTCC M209061cells cultivated in optimized medium hadpromising catalytic properties, its stability and reusability were relatively poor compared toother biocatalysts. Hence, the effects of various immobilizing methods on the operationalstability of the cells were examined here. It was found that Ca-alginate gave the bestimmobilized biocatalyst, which was then coated with chitosan to further improve itsmechanical strength and swelling-resistance properties. Conditions were optimized forformation of reusable immobilized beads which can keep high activity and be used forrepeated batch asymmetric reduction of4'-chloroacetophenone. The optimal immobilizationconditions were found to be as follows: sodium alginate2.5%(w/v), CaCl20.2mol/L,chitosan0.9%(w/v), pH of chitosan solution5.0, coating time20min, cell loading17.2mg-dw cell/g-ww catalyst. The resulting immobilized biocatalyst was very promising, with aspecific activity of85%that of the free-cell biocatalyst. The immobilized cells showed betterthermal stability, pH stability, solvent tolerance and storability compared with free cells. After25cycles reaction, the immobilized beads still retained>50%catalytic activity, which was3.5times higher than that of free cells (7cycles). The immobilized cells could be recultured infresh medium to regain full activity and perform a further25cycles of the reduction reaction.The external mass transfer resistances were negligible as deduced from Damkohler modulusDa <<1, and internal mass transfer restriction affected the reaction but was not therate-controlling step according to effectiveness factors i<1and Thiele modulus0.3<<1. Ca-alginate coated with chitosan is a highly effective material for immobilization ofAcetobacter sp. CCTCC M209061cells for repeated use.
The asymmetric reduction of2-octanone catalyzed by immobilized Acetobacter sp.CCTCC M209061cells in various media was explored to broaden its application inproduction of chiral alcohols. In the aqueous monophasic system, the optimal pH value,reaction temperature, co-substrate and its concentration, substrate concentration and shakingrate were5.5,35°C, glucose,100mmol/L,6mmol/L,200r/min, respectively, under which,the initial reaction rate, the yield and the product e.e. with0.3g/mL immobilized cells were0.393mmol/L/min,99.0%and97.3%, respectively. However, the poor solubility of substrates and the pronounced inhibition of the reactants and the products were observed in aqueousmonophasic system, resulting in dissatisfactory reaction efficiency. Therefore, water-miscibleionic liquids were firstly added into aqueous buffer system for evaluating the effects ofvarious ionic liquids on asymmetric reduction of2-octanone to (R)-2-octanol catalyzed byimmobilized Acetobacter sp. CCTCC M209061cells. It was found that the addition of[C4MIM]·Ac could effectively make the cell membrane more permeable, and lower theproduct concentration in the cells, thus enhancing the efficiency of the reaction. The optimal[C4MIM]·Ac content was3%(w/v), and the concentration of2-octanone, the initial reactionrate and the product e.e. were10mmol/L,0.558mmol/L/min and99.3%, respectively, whichwere higher than the corresponding values in the aqueous buffer system. To further improvethe reaction efficiency, n-tetradecane as a second phase was introduced into the[C4MIM]·Ac-containing buffer system, which could effectively extract2-octanone and2-octanol to keep substrate and product concentrations at low level in aqueous phase, thusalleviating the inhibition of the substrate and the product. The optimal volume ratio ofaqueous phase to organic phase and2-octanone concentration were3:1and500mmol/L,respectively, under which the obtained yield, product concentration and product e.e. for thebioreduction were53.4%,267mmol/L and>99%, respectively. Obviously, the[C4MIM]·Ac-containing buffer/n-tetradecane biphasic system significantly enhanced thereaction efficiency.
The anti-Prelog carbonyl reductase，the key enzyme in cytoplasm of Acetobacter sp.CCTCC M209061(AcCR) for the asymmetric reduction of carbonyl compounds with highstereoselectivity, was isolated and purified via four steps. The enzyme was enriched27.5-foldafter purification with an overall yield of0.4%and specific activity of3.85U/mg. AcCR hada homotetrameric structure with an apparent molecular mass of104kDa and each subunit of27kDa.
AcCR can catalyze the reduction of carbonyl groups and the oxidation of correspondingalcohols utilizing either NAD(H) or NADP(H) as coenzyme. For the reduction of4'-chloroacetophenone using NADH as coenzyme, its optimum activity was at pH5.0and25°C, which were similar with the optimum conditions of whole cell-catalyzed reduction. WhenNADPH acted as the cofactor, the optimum pH and the reaction temperature were7.5and25°C. For isopropanol oxidation, similar catalytic properties of AcCR were found using eitherNAD+or NADP+as cofactor. The optimum pH and reaction temperature were8.0and35°C.Metal ions such as Mn2+, Ni2+and Fe2+remarkably activated the enzyme, followed by Zn2+and Ca2+. Co2+and Mg2+showed weak activation on the enzyme. Hg2+and Ag+completely inhibited the activity of the enzyme, while Cu2+showed relatively weaker inhibition on theenzyme. Iodoacetamide and β-mercaptoethanol showed little effect on enzymatic activity.5mmol/L EDTA completely inhibited the activity of AcCR. Furthermore, a broad range ofcarbonyl compounds such as aryl ketones, α-ketoesters and aliphatic ketones could beenantioselectively reduced by this enzyme with high activity.
The reaction kinetics of4'-chloroacetophenone reduction catalyzed by AcCR was explored.It was found that the Kmvalue of AcCR for NADH was over25-fold greater than that forNADPH (0.66mmol/L vs0.026mmol/L), showing that the enzyme had a preference forNADPH over NADH. When NADH was used as cofactor, the response of carbonyl reductaseactivity to increasing concentration of4'-chloroacetophenone was clearly sigmoidal with aHill coefficient of3.1, suggesting that the enzyme might possess four substrate-binding sitescooperated with each other when NADH was present. The Vmaxvalue for NADH-basedreduction of4'-chloroacetophenone was slightly higher than that for NADPH-based reduction(0.21mmol/L/min vs0.17mmol/L/min). The reaction kenitics of isopropanol oxidationcatalyzed by AcCR was also explored. When NAD+acted as cofactor, the Kmand Vmaxvalueswere1.33mmol/L (NAD+),48.32mmol/L (isopropanol) and0.069mmol/L/min. WhenNADP+acted as cofactor, the Kmvalues for NADP+and isopropanol were1.12mmol/L and67.82mmol/L, respectively, and the Vmaxvalue was0.074mmol/L/min.
This study provides not only a better understanding of biocatalysis and biotransformations,but also a novel route to the preparation of enantiopure chiral alcohols.
引文
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