【作者】 彭飞；

【导师】 宗敏华； 娄文勇；

【作者基本信息】 华南理工大学 ， 发酵工程， 2020， 博士

【摘要】 手性醇化合物在手性药物、食品香料、化学农药和液晶材料等领域发挥着重要的作用。生物法催化手性醇合成具有高对映体选择性、反应条件温和、反应溶剂绿色、可持续性等优点,受到了广泛的关注。然而,生物催化剂在催化反应过程中常面临稳定性较差和难回收等问题;另外,野生菌催化剂面临胞内酶系复杂,关键酶表达水平低和培养条件苛刻等问题,这在一定程度上限制了其应用。本课题组前期从土壤中筛选得到一株催化2-羟基苯乙酮（HAP）高对映体选择性还原合成（R）-1-苯基-1,2-乙二醇{（R）-PED}的Kurthia gibosonii SC0312菌株。在此基础上,本课题通过基因工程技术从具有自主知识产权的K.gibsonii SC0312细胞中挖掘催化HAP不对称还原的关键羰基还原酶基因,构建具有辅因子再生功能的多酶纳米反应器和重组大肠杆菌反应系统,提升催化HAP不对还原合成（R）-PED的反应效率。从Kurthia gibsonii SC0312细胞中预测8个关键羰基还原酶基因,采用基因工程技术将它们克隆在E.coli BL21（DE3）中并表达,分析重组大肠杆菌催化HAP还原的活性和对映体选择性,最终确定一个催化HAP高对映体选择性还原合成（R）-PED的关键酶Kg BDH。该酶由349个氨基酸编码,为同源二聚体,分子量为37.5 KDa,属于中链脱氢酶超家族,具有典型的辅酶结合基序和锌基序。酶学性质研究表明,重组Kg BDH在p H6.0-8.0具有较好的活性,在45℃时酶活性最高。重组酶在p H 7.5-8.0和温度20-30℃时表现出较好的稳定性。常见金属离子对重组Kg BDH无激活作用。重组Kg BDH在p H7.5和35℃条件下催化HAP还原的Km值,kcat值和酶比活力分别为5.4 mmol/L,4.9s^-1和6.7 U/mg。基于KgBDH,FDH（甲酸脱氢酶）和自组装系统Spy Catcher/Spy Tag,构建具有辅因子再生功能和催化（R）-PED不对称合成的多酶纳米反应器。通过蛋白融合技术构建含自组装元件的融合酶Kg BDH-Spy Catcher（Kg BDH-SC）和FDH-Spy Tag（FDH-ST）。在设定条件下,两个融合酶在体外自组装后,通过交联法固定在二氧化硅纳米粒子上得到共固定化酶Kg BDH-SC-ST-FDH-SiO₂,其活性是传统随机共固定化酶的2.9倍且酶活性回收率为49%。Kg BDH-SC-ST-FDH-SiO₂在p H 7.0-7.5和35℃时表现出较好的催化性能。与游离混合酶相比,Kg BDH-SC-ST-FDH-SiO₂表现出更好的p H稳定性和有机试剂稳定性。此外,Kg BDH-SC-ST-FDH-SiO₂在重复使用8批次后活性仍保留52.5%。最后,Kg BDH-SC-ST-FDH-SiO₂在磷酸缓冲液反应体系中可催化60 mmol/L HAP不对称还原合成（R）-PED,产率和光学纯度分别为83.9%和>99%。调控KgBDH-SC和FDH-ST与载体的固定化顺序,通过交联法和蛋白自组装可制备得到催化（R）-PED不对称合成的有序共固定化酶FDH-ST-SC-Kg BDH@SiO₂。在最佳制备条件下,FDH-ST-SC-Kg BDH@SiO₂的酶活回收率为50%。有序共固定化酶在p H7.0-7.5和35-40℃时具有较好的催化性能。与游离混合酶相比,有序共固定化酶具有更好的p H稳定性和有机试剂稳定性。此外,有序共固定化酶在催化HAP还原8批次后活性保留51.7%。最后,FDH-ST-SC-Kg BDH@SiO₂在磷酸缓冲液体系中可催化80 mmol/L HAP不对称还原合成（R）-PED,产率和光学纯度分别为87.4%和>99%。通过将KgBDH和GDH（葡萄糖脱氢酶）在大肠杆菌工程菌中共表达,构建得到一株重组E.coli BL21（DE3）-p ETduet1-Kg BDH-GDH工程菌。该重组工程菌的最佳诱导表达条件为IPTG浓度0.1 mmol/L,温度20℃和时间18 h。为提升游离细胞的稳定性,用活性炭-海藻酸钙复合材料包埋该重组大肠杆菌工程菌。研究发现,活性炭-海藻酸钙包埋的细胞催化HAP还原的活性显著高于海藻酸钙固定化细胞的,且与游离细胞的活性相当。活性炭-海藻酸钙固定化细胞催化HAP还原的最适p H为7.5,在p H 6.0-9.0和温度25-30℃时具有较好的稳定性。另外,该固定化细胞催化HAP还原的操作稳定性优于游离细胞,其在重复使用4批次后活性保留55%。最后,通过底物分批流加,活性炭-海藻酸钙固定化细胞催化240 mmol/L HAP不对称还原反应12 h可合成195 mmol/L（R）-PED,产率为81%,光学纯度大于99%。更多还原

【Abstract】 Chiral alcohols play crucial roles in the fields of chiral drugs,flavors,agrochemicals and liquid crystals.Biocatalytic reaction for the fabrication of chiral alcohols has attracted considerable attentions because of its several merits including high enantioselectivity,mild reaction conditions,green solvents and sustainability,etc.However,biocatalysts are also generally subjected to some demerits such as poor stabilities and difficult reuse;moreover,wild-type strains have a complex intracellular enzyme system,low expression levels of key enzymes,and rigorous culture conditions,all of which partly restrict their applications in biocatalytic reactions.Our research group previously isolated a bacterial strain（Kurthia gibosonii SC0312）from soil,which is capable of asymmetrically reducing 2-hydroxyacetophenone（HAP）to（R）-1-phenyl-1,2-ethanediol{（R）-PED}with high enantioselectivity.Based on the results,this study aimed to discover the key carbonyl reductases responsible for asymmetric reduction of HAP from Kurthia gibsonii SC0312 cells with own intellectual property rights by genetic engineering technique,and then construct the multi-enzyme nanodevice and a recombinant E.coli strain with cofactor regeneration to improve the reaction efficiency of（R）-PED biosynthesis.Eight key enzyme genes from Kurthia gibsonii SC0312 cells were predicted,then cloned into E.coli BL21（DE3）by genetic engineering technology.After inducing expression,the catalytic activity and enantioselectivity of the reduction of HAP catalyzed by the engineered E.coli strains were determined.A key enzyme Kg BDH catalyzing HAP to（R）-PED with high enantioselectivity was mined,which is a homodimer with 37.5 KDa,encoding 349 amino acid residues.Besides,it is a member of medium chain dehydrogenase superfamily and has typical coenzyme binding and zinc motifs.Studying on enzymatic characteristics showed that the recombinant Kg BDH performed good activity at p H 6.0-8.0 and the highest activity at 45℃,and preferable stabilities at p H 7.5-8.0 and 20-30℃.And common metal irons have no activation effect on the recombinant Kg BDH.Furthermore,the Km,kcat and specific activity of the recombinant enzyme for the reduction of HAP were 5.4 mmol/L,4.9 s^-1,and 6.7 U/mg,respectively,at p H 7.5 and 35℃.A multi-enzyme nanodevice with cofactor regeneration was established to prepare（R）-PED based on Kg BDH,FDH（formate dehydrogenase）and self-assembly system Spy Catcher/Spy Tag.Firstly,the fusion enzymes Kg BDH-Spy Catcher（Kg BDH-SC）and FDH-Spy Tag（FDH-ST）with self-assembling pair were constructed on the basis of protein fusion technology.After in vitro self-assembly between the two fusion proteins,the co-immobilized enzymes Kg BDH-SC-ST-FDH-SiO₂ were obtained by chemical crosslinking under the design conditions,which was 2.9 times higher than that of the conventional random co-immobilized enzymes in catalytic activity and remained 49% of initial activity.Kg BDH-SC-ST-FDH-SiO₂ also presented excellent catalytic activity at p H 7.0-7.5 and 35℃.Compared with the mixture of free enzymes,it has better p H and organic solvents stability.Moreover,the co-immobilized enzymes remained 52.5% of initial activity after eight cycles.Finally,the co-immobilized enzymes Kg BDH-SC-ST-FDH-SiO₂ could asymmetrically reduce HAP of 60 mmol/L to chiral（R）-PED in phosphate buffer,affording 83.9% yield and>99% ee.A sequential co-immobilized enzymes FDH-ST-SC-Kg BDH@SiO₂ were fabricated to prepare（R）-PED using crosslinking and protein self-assembly by adjusting the immobilized sequence of Kg BDH-SC and FDH-ST.Under the optimum immobilization conditions,FDH-ST-SC-Kg BDH@SiO₂ had the activity recovery of 50% and exhibited excellent catalytic performance under p H 7.0-7.5 and 35-40℃.Compared to the mixture of free enzymes,the sequential co-immobilized enzymes showed better p H stability and organic solvents tolerance.Additionally,the sequential co-immobilized enzymes for the reduction of HAP maintained 51.7% of initial activity after eight cycles.Ultimately,FDH-ST-SC-Kg BDH@SiO₂ could asymmetrically reduce HAP of 80 mmol/L to chiral（R）-PED in phosphate buffer,affording87.4% yield and>99% ee.A recombinant E.coli BL21（DE3）-p ETduet1-Kg BDH-GDH was constructed by co-expressing Kg BDH and GDH（glucose dehydrogenase）.The optimum expression conditions of the recombinant strain were 0.1 mmol/L IPTG,20℃,and 18 h.To improve the stability of the free cells,we used the mixture of activated carbon and calcium alginate to embed the recombinant E.coli engineered strains.The results showed that the reduction activity of HAP catalyzed by the cells immobilized by the mixture of activated carbon and calcium alginate was significantly higher than that of the cells immobilized by calcium alginate and was comparable to that of the free cells.The cells immobilized by the mixture of activated carbon and calcium alginate showed the highest activity for HAP reduction at p H 7.5 and performed good stabilities at p H 6.0-9.0 and 25-30℃.Furthermore,with the superiority of operational stability to free cells,the immobilized cells for the HAP reduction remained 55% of initial activity after four batches.Finally,the immobilized cells can asymmetrically catalyze HAP of 240 mmol/L to（R）-PED of 195 mmol/L in 12 h by a fed-batch strategy,with 81% yield and>99% ee.更多还原