(一)技术领域(1) Technical field
本发明涉及一种6-氰基-(3R,5R)-二羟基己酸叔丁酯的制备方法，特别涉及一种基于功能化树脂固定化酶填充的循环式连续流反应器高效绿色生产6-氰基-(3R,5R)-二羟基己酸叔丁酯的方法。The present invention relates to a preparation method of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, in particular to a circular continuous flow reactor based on functionalized resin immobilized enzyme filled efficient green production6 - The method of tert-butyl cyano-(3R,5R)-dihydroxyhexanoate.
(二)背景技术(2) Background technology
阿托伐他汀和瑞舒伐他汀、匹伐他汀等“超级他汀”是治疗心脑血管疾病的重大降脂药品种，具有高效的降脂功效、长期安全性和临床益处，显著降低心脑血管疾病的发病率和死亡率。血浆中胆固醇的正常含量为0-200mg/dL，当LDL胆固醇浓度升高沉积于心脑部位血管动脉内壁，导致动脉粥样硬化性斑块，从而引发心血管疾病。而他汀类药物能够作为胆固醇合成限速酶的竞争性抑制剂，降低血液中低密度脂蛋白(Low Density Lipoprotein,LDL)胆固醇水平。在降低心脑血管疾病发生率及抗氧化、消炎、抗癌，预防中风和老年痴呆症等方面也得到了广泛研究应用。"Super statins" such as atorvastatin, rosuvastatin, and pitavastatin are important lipid-lowering drugs for the treatment of cardiovascular and cerebrovascular diseases. Disease morbidity and mortality. The normal content of cholesterol in plasma is 0-200mg/dL. When the concentration of LDL cholesterol increases, it deposits on the inner wall of blood vessels and arteries in the heart and brain, leading to atherosclerotic plaque, which leads to cardiovascular diseases. Statins can act as competitive inhibitors of the rate-limiting enzymes of cholesterol synthesis and reduce the level of low-density lipoprotein (Low Density Lipoprotein, LDL) cholesterol in the blood. It has also been widely researched and applied in reducing the incidence of cardiovascular and cerebrovascular diseases, anti-oxidation, anti-inflammation, anti-cancer, and preventing stroke and Alzheimer's disease.
6-氰基-(3R,5R)-二羟基己酸叔丁酯、6-氯-(3R,5S)-二羟基己酸叔丁酯是阿托伐他汀、瑞舒伐他汀及匹伐他汀合成的关键手性中间体。由于6-取代-(3R,5R/S)-二羟基己酸叔丁酯具有两个手性中心，因此，研究光学纯6-取代-(3R,5R/S)-二羟基己酸叔丁酯的手性合成方法学和合成技术具有重要意义。化学法合成6-氰基-(3R,5R)-二羟基己酸叔丁酯反应条件较为苛刻，其第二个手性中心的合成，需要在超低温-90℃条件下，二异丙基胺基锂(LDA)脱质子缩合，硼烷作为手性诱导剂，硼氢化钠作为还原剂进行反应。该反应存在选择性不强，产物光学纯度低，能耗大、成本高、环境不友好等问题。而生物催化合成6-氰基-(3R,5R)-二羟基己酸叔丁酯，具有原子利用率高，区域/立体选择性高，反应条件温和，有机试剂用量少，安全绿色等优势，可避免化学法的多步保护与去保护步骤。6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester, 6-chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester are atorvastatin, rosuvastatin and pitavastatin Synthetic key chiral intermediates. Since tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoate has two chiral centers, the study of optically pure tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoate The chiral synthesis methodology and synthesis technology of esters are of great significance. The chemical synthesis of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate requires harsh reaction conditions, and the synthesis of its second chiral center requires diisopropylamine at ultra-low temperature -90°C. Lithium-based lithium (LDA) deprotonation condensation, borane as a chiral inducer, sodium borohydride as a reducing agent for the reaction. This reaction has problems such as poor selectivity, low optical purity of the product, high energy consumption, high cost, and unfriendly environment. The biocatalytic synthesis of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate has the advantages of high atom utilization, high regio/stereoselectivity, mild reaction conditions, less organic reagent consumption, and safety and greenness. , can avoid the multi-step protection and deprotection steps of chemical method.
现有生物催化合成6-氰基-(3R,5R)-二羟基己酸叔丁酯反应体系主要包括醛酮还原酶偶联葡萄糖脱氢酶双酶双底物体系以及羰基还原酶偶联辅底物异丙醇的单酶双底物体系，而目前主要是通过全细胞或游离酶构筑的酶-偶联与底物-偶联体系来实现6-氰基-(3R,5R)-二羟基己酸叔丁酯的不对称还原合成。The existing biocatalytic synthesis of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate mainly includes aldehyde and ketone reductase coupled glucose dehydrogenase dual-enzyme dual-substrate system and carbonyl reductase coupled co- The single-enzyme dual-substrate system of the substrate isopropanol, and at present, the enzyme-coupling and substrate-coupling systems constructed by whole cells or free enzymes are mainly used to realize the 6-cyano-(3R,5R)-di Asymmetric reductive synthesis of tert-butyl hydroxycaproate.
而采用游离细胞或者酶作为催化剂，游离细胞或者酶的稳定性较差，易变性失活；细胞或者酶只能一次性使用，增加了酶制剂成本与废水、废渣排放；酶与底物和产物的混合加大了产物的分离纯化的难度，影响产品收率，增加生产成本。而酶固定化技术因其稳定性高、与反应混合物分离简单、可回收性强等优点，可解决上述问题。但是，将第一代热点材料(硅藻土、树脂、多孔玻璃、多糖类材料等)作为固定化酶的基质，普遍存在回收率和稳定性不高等缺点。However, when free cells or enzymes are used as catalysts, the stability of free cells or enzymes is poor, and the variability is inactivated; cells or enzymes can only be used once, which increases the cost of enzyme preparations and the discharge of waste water and waste residue; enzymes and substrates and products Mixing increases the difficulty of the separation and purification of the product, affects the product yield, and increases the production cost. The enzyme immobilization technology can solve the above problems because of its high stability, simple separation from the reaction mixture, and strong recyclability. However, using the first-generation hot-spot materials (diatomaceous earth, resin, porous glass, polysaccharide materials, etc.)
因此，设计理想的固定化方案，筛选合适的具备工业属性的固定化材料，采用静电吸附的方法，为提高醛酮还原酶固定化酶的稳定性及酶活回收率，自动化、高效连续式生产6-氰基-(3R,5R)-二羟基己酸叔丁酯，成为酶法合成他汀类药物关键手性中间体的瓶颈。Therefore, to design an ideal immobilization scheme, select suitable immobilization materials with industrial properties, and adopt electrostatic adsorption method, in order to improve the stability of immobilized enzyme of aldehyde and ketone reductase and the recovery rate of enzyme activity, automatic and efficient continuous production 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester has become a bottleneck in enzymatic synthesis of key chiral intermediates of statins.
(三)发明内容(3) Contents of the invention
本发明的目的在于提供一种醛酮还原酶-葡萄糖脱氢酶共固定化酶及其在他汀类药物手性中间体连续流合成中的应用，特别是合成6-氰基-(3R,5R)-二羟基己酸叔丁酯中的应用，本发明借助化学交联与静电吸附的方法，筛选得到能提高醛酮还原酶与葡萄糖脱氢酶共固定化酶的酶活回收率及稳定性的表面功能化树脂做共固定化的基质，极大地提高了固定化酶物理化学相关的性能，促进了固定化酶走向工业应用；同时将共固定化酶用于连续流反应器中，实现了辅因子再生以及连续、高效合成阿托伐他汀钙的关键中间体6-氰基-(3R,5R)-二羟基己酸叔丁酯的目的，为生物化工、生物催化、医药合成相关领域的新一代工业化应用技术革新提出了新的思路和理论基础。The object of the present invention is to provide a co-immobilized enzyme of aldehyde and ketone reductase-glucose dehydrogenase and its application in the continuous flow synthesis of chiral intermediates of statins, especially the synthesis of 6-cyano-(3R, 5R )-dihydroxyhexanoic acid tert-butyl ester, the present invention screens and obtains the enzyme activity recovery rate and the stability that can improve the co-immobilization enzyme of aldehyde ketone reductase and glucose dehydrogenase by means of chemical cross-linking and electrostatic adsorption The surface functionalized resin is used as the co-immobilization matrix, which greatly improves the physical and chemical properties of the immobilized enzyme and promotes the industrial application of the immobilized enzyme; at the same time, the co-immobilized enzyme is used in a continuous flow reactor to achieve The purpose of cofactor regeneration and continuous and efficient synthesis of atorvastatin calcium's key intermediate 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester is for biochemical industry, biocatalysis, and pharmaceutical synthesis related fields. The new generation of industrial application technology innovation has put forward new ideas and theoretical basis.
本发明采用的技术方案主要如下：The technical scheme that the present invention adopts is mainly as follows:
本发明提供了一种醛酮还原酶-葡萄糖脱氢酶共固定化酶，所述共固定化酶(优选KmAKR&BmGDH@aminoresin(C6)-PEI)以氨基树脂为载体，以戊二醛为交联剂，以聚乙烯亚胺(PEI)为修饰聚合物，以醛酮还原酶纯酶液和葡萄糖脱氢酶纯酶液为活性组分，于0.2M，pH7.0的磷酸钠缓冲溶液中，在28℃、200rpm条件下搅拌孵育18h，离心，收集沉淀，获得醛酮还原酶-葡萄糖脱氢酶共固定化酶；所述氨基树脂包括氨基树脂(amino resinC6)LXTE-700，氨基树脂(amino resinC2-1)LXTE-703，氨基树脂(amino resinC2-2)LXTE-704，氨基树脂(amino resinC2-3)LXTE-705。优选的氨基树脂LXTE-700。The invention provides a co-immobilized enzyme of aldehyde and ketone reductase-glucose dehydrogenase, the co-immobilized enzyme (preferably KmAKR&BmGDH@aminoresin(C6)-PEI) uses amino resin as carrier and glutaraldehyde as cross-link Agent, with polyethyleneimine (PEI) as a modified polymer, with aldehyde ketone reductase pure enzyme solution and glucose dehydrogenase pure enzyme solution as active components, in 0.2M, pH7.0 sodium phosphate buffer solution, Stir and incubate at 28°C and 200rpm for 18h, centrifuge, collect the precipitate, and obtain the co-immobilized enzyme of aldehyde and ketone reductase-glucose dehydrogenase; the amino resin includes amino resin (amino resinC6) LXTE-700, amino resin (amino resinC6) resinC2-1) LXTE-703, amino resin (amino resinC2-2) LXTE-704, amino resin (amino resinC2-3) LXTE-705. Preferred amino resin LXTE-700.
优选的，所述戊二醛以体积浓度0.5-2％(优选0.5％)戊二醛的磷酸钠缓冲液(0.2M，pH 7.0)的形式加入，所述戊二醛的磷酸钠缓冲液体积加入量以载体质量计为5-15mL/g，优选10mL/g；所述聚乙烯亚胺以体积浓度2-6％聚乙烯亚胺水溶液的形式加入，所述聚乙烯亚胺水溶液体积加入量以载体质量计为10-30mL/g，优选20mL/g；所述醛酮还原酶纯酶液以蛋白含量计与载体质量比为0.0025-0.05:1，优选0.005:1；所述葡萄糖脱氢酶纯酶液以蛋白含量计与载体质量比为0.001-0.02:1，优选0.005:1。Preferably, the glutaraldehyde is added in the form of a sodium phosphate buffer (0.2M, pH 7.0) with a volume concentration of 0.5-2% (preferably 0.5%) glutaraldehyde, and the volume of the glutaraldehyde sodium phosphate buffer is The amount added is 5-15mL/g in terms of carrier mass, preferably 10mL/g; the polyethyleneimine is added in the form of a polyethyleneimine aqueous solution with a volume concentration of 2-6%, and the volumetric addition amount of the polyethyleneimine aqueous solution is The carrier mass is 10-30mL/g, preferably 20mL/g; the mass ratio of the aldehyde and ketone reductase pure enzyme liquid to the carrier is 0.0025-0.05:1, preferably 0.005:1 in terms of protein content; the glucose dehydrogenation The mass ratio of the pure enzyme liquid to the carrier in terms of protein content is 0.001-0.02:1, preferably 0.005:1.
优选的，所述醛酮还原酶纯酶液按如下方法制备：Preferably, the aldehyde and ketone reductase pure enzyme solution is prepared as follows:
(1)工程菌：将源自马克斯克鲁维酵母的醛酮还原酶KmAKR基因(核苷酸序列如SEQID NO.1所示)人工合成后，连接至pET28a载体的多克隆位点，再将重组载体pET28a转化至大肠杆菌E.coli BL21(DE3)中，获得表达醛酮还原酶KmAKR重组大肠杆菌E.coli BL21(DE3)/pET28a-kmAKR；(1) Engineering bacteria: after artificially synthesizing the aldoketone reductase KmAKR gene (nucleotide sequence shown in SEQID NO.1) derived from Kluyveromyces marx, it is connected to the multiple cloning site of the pET28a vector, and then The recombinant vector pET28a was transformed into Escherichia coli E.coli BL21(DE3), and the recombinant E.coli BL21(DE3)/pET28a-kmAKR expressing aldehyde and ketone reductase KmAKR was obtained;
(2)粗酶液：将步骤(1)重组大肠杆菌接种至LB固体培养基，37℃培养12h，获得活化菌体；将活化菌体接种至LB液体培养基，37℃、200rpm培养12h，获得种子液；将种子液以体积浓度2％的接种量接种至含终浓度50μg/mL卡那霉素的LB液体培养基，37℃、200rpm培养2h至OD值0.6-0.8，再向培养液中加入终浓度为0.15mM异丙基硫代半乳糖苷(Isopropylβ-D-thiogalactoside，IPTG)，28℃，200rpm培养12h后，4℃、8000rpm下离心10min，获得相应的湿菌体细胞；按照每1g湿菌体悬浮于20mL的pH 7、20mM磷酸钠缓冲液中制备菌悬液，超声破碎30min(功率300W，破碎1s，暂停3s)，12000rpm、4℃离心10min，收集上清，0.45μm微膜过滤去除细胞碎片等不溶性物质，滤液即为醛酮还原酶粗酶液；(2) Crude enzyme solution: Inoculate the recombinant Escherichia coli in step (1) into LB solid medium, culture at 37°C for 12 hours to obtain activated cells; inoculate the activated cells into LB liquid medium, cultivate at 37°C and 200rpm for 12 hours, Obtain the seed solution; inoculate the seed solution with an inoculum volume concentration of 2% into LB liquid medium containing a final concentration of 50 μg/mL kanamycin, cultivate at 37° C. and 200 rpm for 2 hours to an OD value of 0.6-0.8, and then add to the culture solution Add the final concentration of 0.15mM isopropylβ-D-thiogalactoside (IPTG), 28 ℃, 200rpm culture for 12h, 4 ℃, 8000rpm centrifuge 10min, to obtain the corresponding wet bacterial cells; Suspend 1g of wet bacteria in 20mL of pH 7, 20mM sodium phosphate buffer to prepare bacterial suspension, ultrasonically break for 30min (power 300W, break for 1s, pause for 3s), centrifuge at 12000rpm, 4°C for 10min, collect supernatant, 0.45μm Micromembrane filtration removes insoluble substances such as cell debris, and the filtrate is the crude enzyme solution of aldehyde and ketone reductase;
(3)纯酶液：镍柱(1.6cm×10cm，Bio-Rad，USA)用A液(含300mM NaCl和20mM咪唑的pH 7.0，20mM磷酸钠缓冲液)平衡后，上样步骤(2)粗酶液，上样量1-2个柱体积，然后用A液冲洗3-4个柱体积，去除杂质和结合较弱的蛋白；再用B液(含300mM NaCl，500mM咪唑的pH7.0、20mM磷酸钠缓冲液)洗脱目的蛋白并收集后置于冰上保存，再用50mM pH 7.0磷酸钠缓冲液4℃透析(透析袋的截留分子量为10kDa)12h，收集截留液，即为醛酮还原酶纯酶液。(3) Pure enzyme solution: Ni column (1.6cm×10cm, Bio-Rad, USA) is equilibrated with solution A (pH 7.0 containing 300mM NaCl and 20mM imidazole, 20mM sodium phosphate buffer), and the loading step (2) For crude enzyme solution, load 1-2 column volumes, then wash 3-4 column volumes with solution A to remove impurities and weakly bound proteins; then use solution B (pH7.0 containing 300mM NaCl, 500mM imidazole , 20mM sodium phosphate buffer) to elute the target protein and collect it, store it on ice, and then dialyze it with 50mM pH 7.0 sodium phosphate buffer at 4°C for 12h (the molecular weight cut-off of the dialysis bag is 10kDa) to collect the retentate, which is the aldehyde Ketoreductase pure enzyme solution.
所述葡萄糖脱氢酶纯酶液制备步骤和醛酮还原酶纯酶液的步骤相同，葡萄糖脱氢酶基因的核苷酸序列如SEQ ID NO.2所示。The preparation steps of the glucose dehydrogenase pure enzyme solution are the same as those of the aldehyde and ketone reductase pure enzyme solution, and the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.2.
优选的，所述共固定化酶按以下方法制备获得：Preferably, the co-immobilized enzyme is prepared by the following method:
(1)戊二醛修饰氨基树脂(1) Glutaraldehyde modified amino resin
将氨基树脂加入含体积浓度0.5-2％的戊二醛的磷酸钠缓冲液(0.2M，pH 7.0)中，37℃搅拌1h对氨基树脂进行活化，然后用0.2M，pH 7.0磷酸钠缓冲液冲洗树脂去掉未反应的戊二醛，获得戊二醛修饰的氨基树脂；Add the amino resin to sodium phosphate buffer (0.2M, pH 7.0) containing 0.5-2% glutaraldehyde by volume, stir at 37°C for 1 hour to activate the amino resin, and then use 0.2M, pH 7.0 sodium phosphate buffer Rinse the resin to remove unreacted glutaraldehyde to obtain a glutaraldehyde-modified amino resin;
(2)葡萄糖脱氢酶的固定化(2) Immobilization of glucose dehydrogenase
将葡萄糖脱氢酶BmGDH纯酶液添加到步骤(1)戊二醛修饰的氨基树脂中，在28℃、200rpm下孵育3小时，用0.2M、pH 7.0的磷酸钠缓冲液洗涤后，获得葡萄糖脱氢酶-树脂固定化复合物；Add glucose dehydrogenase BmGDH pure enzyme solution to step (1) glutaraldehyde-modified amino resin, incubate at 28°C, 200rpm for 3 hours, wash with 0.2M sodium phosphate buffer, pH 7.0, and obtain glucose Dehydrogenase-resin-immobilized complex;
(3)PEI修饰(3) PEI modification
将步骤(2)葡萄糖脱氢酶-树脂固定化复合物添加到体积浓度2～6％的PEI水溶液中，在28℃、200rpm下孵育过夜，并用0.2M、pH7.0的磷酸钠缓冲液洗涤，对氨基树脂表面进行PEI修饰，实现了树脂表面局部带正电的微环境，获得PEI修饰树脂；Add the glucose dehydrogenase-resin immobilized complex in step (2) to PEI aqueous solution with a volume concentration of 2-6%, incubate overnight at 28°C and 200rpm, and wash with 0.2M sodium phosphate buffer at pH 7.0 , carry out PEI modification on the surface of the amino resin, realize the microenvironment with local positive charge on the resin surface, and obtain the PEI modified resin;
(4)醛酮还原酶-葡萄糖脱氢酶共固定化酶(4) Aldehyde and ketone reductase-glucose dehydrogenase co-immobilized enzyme
将醛酮还原酶纯酶液加入步骤(3)PEI修饰树脂，在28℃、200rpm下孵育18小时，通过PEI和醛酮还原酶酶分子静电吸附作用，有效固定醛酮还原酶，用0.2M、pH7.0的磷酸钠缓冲液洗涤，获得醛酮还原酶-葡萄糖脱氢酶共固定化酶。Add the pure enzyme solution of aldehyde and ketone reductase to step (3) PEI modified resin, incubate at 28°C and 200rpm for 18 hours, through the electrostatic adsorption of PEI and aldehyde and ketone reductase enzyme molecules, effectively immobilize the aldehyde and ketone reductase, use 0.2M and washing with sodium phosphate buffer solution of pH 7.0 to obtain the co-immobilized enzyme of aldoketone reductase-glucose dehydrogenase.
本发明还提供一种所述醛酮还原酶-葡萄糖脱氢酶共固定化酶在不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯中的应用，所述应用的方法按如下步骤进行：以醛酮还原酶-葡萄糖脱氢酶共固定化酶为催化剂，以6-氰基-(5R)-羟基-3-羰基己酸叔丁酯为底物，以葡萄糖为辅助底物，添加NADPH，pH 4-10(优选pH为6)的缓冲液为反应介质，在25-65℃(优选40℃)下反应4h后，反应结束，反应液分离纯化，获得6-氰基-(3R,5R)-二羟基己酸叔丁酯。The present invention also provides a co-immobilized enzyme of aldehyde and ketone reductase-glucose dehydrogenase to prepare 6-cyano- The application in (3R,5R)-dihydroxyhexanoic acid tert-butyl ester, the method of described application is carried out as follows: with aldehyde ketone reductase-glucose dehydrogenase co-immobilized enzyme as catalyst, with 6-cyano- (5R)-Hydroxy-3-oxoylhexanoic acid tert-butyl ester as substrate, glucose as auxiliary substrate, NADPH, pH 4-10 (preferably pH 6) buffer as reaction medium, at 25-65°C (Preferably 40° C.) After reacting for 4 hours, the reaction is completed, and the reaction solution is separated and purified to obtain tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.
优选，所述底物添加终浓度10-100g/L，优选20g/L；所述葡萄糖添加终浓度10-100g/L，优选30g/L。所述固定化酶催化剂用量以缓冲液体积计为50-200g/L，优选-为150g/L。所述NADPH添加终浓度为1-3mM，优选1mM。Preferably, the substrate is added at a final concentration of 10-100 g/L, preferably 20 g/L; the glucose is added at a final concentration of 10-100 g/L, preferably 30 g/L. The amount of the immobilized enzyme catalyst is 50-200g/L in terms of buffer volume, preferably 150g/L. The final concentration of NADPH added is 1-3mM, preferably 1mM.
更优选，所述反应在连续流反应器中进行，所述连续流反应器包括反应液储罐、流量泵、填充式反应柱、液体收集罐，所述填充式反应柱设有进液口和出液口；所述反应液储罐通过流量泵与填充式反应柱进液口连接；所述填充式反应柱出液口分别与液体收集罐和反应稀释液连通，其中反应稀释液与高效液相色谱仪连通，液体收集罐与反应液储罐连通构成循环；More preferably, described reaction is carried out in continuous flow reactor, and described continuous flow reactor comprises reaction liquid storage tank, flow pump, packing type reaction column, liquid collection tank, and described packing type reaction column is provided with liquid inlet and Liquid outlet; the reaction liquid storage tank is connected to the liquid inlet of the packed reaction column through a flow pump; the liquid outlet of the packed reaction column is connected to the liquid collection tank and the reaction diluent respectively, wherein the reaction diluent and the high-efficiency liquid The phase chromatograph is connected, and the liquid collection tank is connected with the reaction liquid storage tank to form a cycle;
将醛酮还原酶-葡萄糖脱氢酶共固定化酶装载到填充式反应柱中，直至每个填充式反应柱都被装满，并将柱温保持在25-65℃；使用注射泵(SLP01-02A，兰格，中国)将含有底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯(5R)-1、NADP+和葡萄糖的pH4-10的缓冲液，在0.25-10mL/h流速下，连续流加到填充式反应柱中，反应完全后，反应液分离纯化，获得6-氰基-(3R,5R)-二羟基己酸叔丁酯。Load the ketone reductase-glucose dehydrogenase co-immobilized enzyme into the packed reaction columns until each packed reaction column is filled, and keep the column temperature at 25-65°C; use a syringe pump (SLP01 -02A, Lange, China) will contain the substrate 6-cyano-(5R)-hydroxy-3-oxoylhexanoic acid tert-butyl ester (5R)-1, NADP+ and glucose pH4-10 buffer, in At a flow rate of 0.25-10mL/h, it is continuously fed into a packed reaction column. After the reaction is complete, the reaction liquid is separated and purified to obtain tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.
优选的，所述填充式反应柱温度保持在40℃、缓冲液为pH 6.0、200mM的磷酸盐缓冲液，流速为5mL/h。Preferably, the temperature of the packed reaction column is kept at 40° C., the buffer is 200 mM phosphate buffer at pH 6.0, and the flow rate is 5 mL/h.
与现有技术相比，本发明的有益效果主要体现在：Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:
本发明选用表面功能化的树脂，采用化学交联和静电吸附的方法，醛酮还原酶和葡萄糖脱氢酶的共固定化酶，比游离酶的最适反应温度提高5℃，在弱酸性(pH 6)条件下，固定化酶相对活力比游离酶高12％，且共固化酶在使用8个批次后仍保留80.7％的相对活力。The present invention selects the resin of surface functionalization for use, adopts the method for chemical cross-linking and electrostatic adsorption, and the co-immobilization enzyme of aldehyde ketone reductase and glucose dehydrogenase improves 5 ℃ than the optimum reaction temperature of free enzyme, and is weakly acidic ( Under the condition of pH 6), the relative activity of the immobilized enzyme was 12% higher than that of the free enzyme, and the co-immobilized enzyme still retained 80.7% of the relative activity after using 8 batches.
本发明设计的固定化酶具备批次使用良好、酶活保留高的优势，构建的循环式连续流生物反应系统具备杂质少、固液分离容易、反应器易清洗、更换催化剂简便的优势，并验证了闭环、无废、绿色合成医药中间体的技术可行性，实现了辅酶循环，0.25-5mL/h流速下完全转化底物(20g/L)，7mL/h及以上流速下未反应的底物二次循环实现更高的转化率，5mL/h的流速下运行90小时的催化反应后保留了95％以上的转化率、实现了产物的连续稳定合成。对其他氧化还原酶的级联/偶联催化反应、固定化方法和载体的选择提供了参考，为工业上生物法合成药物成分提供新的思路和理论基础，对车间技术和设备的革新具有重要的借鉴意义，且具有可观的经济效益和广阔的应用前景。The immobilized enzyme designed in the present invention has the advantages of good batch use and high enzyme activity retention, and the constructed circular continuous flow bioreaction system has the advantages of less impurities, easy solid-liquid separation, easy cleaning of the reactor, and simple replacement of the catalyst, and The technical feasibility of closed-loop, waste-free and green synthetic pharmaceutical intermediates has been verified, and the coenzyme cycle has been realized. The substrate (20g/L) is completely converted at a flow rate of 0.25-5mL/h, and the unreacted substrate is converted at a flow rate of 7mL/h and above. The secondary cycle of the product achieves a higher conversion rate, and after the catalytic reaction runs for 90 hours at a flow rate of 5mL/h, the conversion rate of more than 95% is retained, and the continuous and stable synthesis of the product is realized. It provides a reference for the cascade/coupling catalytic reaction of other oxidoreductases, immobilization methods and carrier selection, and provides new ideas and theoretical basis for the industrial biosynthesis of pharmaceutical ingredients, which is of great importance to the innovation of workshop technology and equipment The reference significance, and has considerable economic benefits and broad application prospects.
(四)附图说明(4) Description of drawings
图1为醛酮还原酶-葡萄糖脱氢酶双酶偶联合成6-氰基-(3R,5R)-二羟基己酸叔丁酯的反应式。Fig. 1 is the reaction formula for synthesizing 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester through double enzyme coupling of aldehyde and ketone reductase-glucose dehydrogenase.
图2为实施例1醛酮还原酶纯酶液与葡萄糖脱氢酶纯酶液的SDS-PAGE电泳图。Fig. 2 is the SDS-PAGE electrophoresis diagram of the pure enzyme solution of aldehyde and ketone reductase and the pure enzyme solution of glucose dehydrogenase in Example 1.
图3为实施例2不同树脂的显微照片。Figure 3 is a photomicrograph of different resins of Example 2.
图4为实施例2不同树脂的化学结构示意图。4 is a schematic diagram of the chemical structures of different resins in Example 2.
图5为实施例3静电吸附不同来源醛酮还原酶的固定化酶活力柱形图。Fig. 5 is a bar graph of immobilized enzyme activity of aldehyde and ketone reductase from different sources electrostatically adsorbed in Example 3.
图6为实施例5戊二醛和聚乙烯亚胺浓度对固定化率的影响柱形图。Fig. 6 is a bar graph of the influence of the concentration of glutaraldehyde and polyethyleneimine on the immobilization rate in Example 5.
图7为实施例7葡萄糖脱氢酶浓度对固定化酶活力影响柱形图。Fig. 7 is a bar graph showing the effect of concentration of glucose dehydrogenase on the activity of immobilized enzyme in Example 7.
图8为实施例7醛酮还原酶浓度对固定化酶活力影响柱形图。Fig. 8 is a bar graph showing the effect of concentration of aldehyde and ketone reductase on the activity of immobilized enzyme in Example 7.
图9为实施例8树脂和酶-树脂固定化复合物的红外表征图。Fig. 9 is an infrared characterization diagram of the resin of Example 8 and the enzyme-resin immobilized complex.
图10为实施例8树脂及酶-树脂固定化复合物的热重表征图。Fig. 10 is a thermal gravimetric characterization diagram of the resin and the enzyme-resin immobilized complex in Example 8.
图11为实施例9pH对游离酶和固定化酶相对活性影响的曲线图。Fig. 11 is a graph showing the effect of pH on the relative activities of free enzyme and immobilized enzyme in Example 9.
图12为实施例10温度对游离酶和固定化酶相对活性影响的曲线图。Fig. 12 is a graph showing the effect of temperature on the relative activities of free enzyme and immobilized enzyme in Example 10.
图13为实施例11醛酮还原酶-葡萄糖脱氢酶共固定化酶的重复使用次数对相对活性影响的柱形图。Fig. 13 is a bar graph showing the effect of the repeated use times of the co-immobilized enzyme of aldehyde and ketone reductase-glucose dehydrogenase on the relative activity in Example 11.
图14为实施例12双酶共固化介导的循环式连续流反应器的示意图。Fig. 14 is a schematic diagram of a circulating continuous flow reactor mediated by dual enzyme co-curing in Example 12.
图15为实施例12连续流反应器不同流速催化合成6-氰基-(3R,5R)-二羟基己酸叔丁酯的转化率柱形图。Fig. 15 is a bar graph of the conversion rate of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate catalytically synthesized by the continuous flow reactor in Example 12 at different flow rates.
图16为实施例13醛酮还原酶-葡萄糖脱氢酶共固定化酶在釜式反应和连续流反应后的电镜表征图。Fig. 16 is an electron microscope characterization diagram of aldehyde and ketone reductase-glucose dehydrogenase co-immobilized enzyme in Example 13 after kettle reaction and continuous flow reaction.
(五)具体实施方式(5) Specific implementation methods
以下结合实例来进一步说明本发明，但本发明的保护并不仅限于此：The present invention is further described below in conjunction with examples, but protection of the present invention is not limited thereto:
LB液体培养基组成：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，溶剂为水，pH自然。LB liquid medium composition: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent is water, pH is natural.
LB固体培养基组成：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，琼脂18g/L，溶剂为水，pH自然。LB solid medium composition: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar 18g/L, solvent is water, pH is natural.
本发明实施例所用环氧树脂(epoxy resin)LXTE-600，氨基树脂(amino resin)LXTE-700，氨基环氧树脂(amino-epoxy resin)LXTE-706、羧基树脂(carboxyl resin)LXTE-800、离子交换树脂(ion exchange resin)LXTE-901、大孔树脂(macrosporousresin)LXTE-1000，以及氨基树脂(amino resin)LXTE-703、LXTE-704和LXTE-705均购自西安蓝晓公司；其中LXTE-703、LXTE-704和LXTE-705结构相同，只是树脂粒径分别为28.7，195.2，154.0μm.The epoxy resin (epoxy resin) LXTE-600 used in the embodiment of the present invention, amino resin (amino resin) LXTE-700, amino epoxy resin (amino-epoxy resin) LXTE-706, carboxyl resin (carboxyl resin) LXTE-800, Ion exchange resin (ion exchange resin) LXTE-901, macroporous resin (macrosporous resin) LXTE-1000, and amino resin (amino resin) LXTE-703, LXTE-704 and LXTE-705 were purchased from Xi'an Lanxiao Company; where LXTE -703, LXTE-704 and LXTE-705 have the same structure, but the resin particle size is 28.7, 195.2, 154.0μm respectively.
实施例1:醛酮还原酶纯酶液、葡萄糖脱氢酶纯酶液的制备Embodiment 1: the preparation of aldehyde and ketone reductase pure enzyme liquid, glucose dehydrogenase pure enzyme liquid
1、醛酮还原酶纯酶液1. Aldehyde and ketone reductase pure enzyme solution
(1)工程菌：将Genbank中源自马克斯克鲁维酵母(Kluyveromycesmarxianus)的醛酮还原酶KmAKR基因(核苷酸序列：SEQ ID NO.1)和源自多布克鲁维酵母(Kluyveromycesdobzhanskii)的醛酮还原酶KdAKR(核苷酸序列：SEQ ID NO.2)人工合成后，用相同的限制性内切酶(NcoI和XhoI)处理合成的目的基因片段和pET28a载体，并利用T4 DNA连接酶将经同样酶切处理的片段和载体pET28a进行连接，获得重组表达载体pET28a-kmakr与pET28a-kdakr。进一步将重组表达载体转入化至大肠杆菌E.coli BL21(DE3)，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下过夜培养，随机挑取单克隆抽提质粒进行测序鉴定，分别获得重组大肠杆菌E.coli BL21(DE3)/pET28a-kmakr与E.coliBL21(DE3)/pET28a-kdakr。(1) Engineering bacteria: the aldehyde and ketone reductase KmAKR gene (nucleotide sequence: SEQ ID NO.1) derived from Kluyveromyces marxianus (Kluyveromyces marxianus) in Genbank and derived from Kluyveromyces dobzhanskii (Kluyveromycesdobzhanskii) After artificial synthesis of aldehyde and ketone reductase KdAKR (nucleotide sequence: SEQ ID NO.2), treat the synthetic target gene fragment and pET28a vector with the same restriction enzymes (NcoI and XhoI), and use T4 DNA to connect The enzyme connects the fragment treated with the same restriction enzyme and the vector pET28a to obtain the recombinant expression vectors pET28a-kmakr and pET28a-kdakr. The recombinant expression vector was further transformed into Escherichia coli E.coli BL21(DE3), spread on an LB plate containing 50 μg/mL kanamycin resistance, cultivated overnight at 37°C, and randomly picked a single clone to extract the plasmid Sequence identification was carried out to obtain recombinant Escherichia coli E.coli BL21(DE3)/pET28a-kmakr and E.coliBL21(DE3)/pET28a-kdakr respectively.
(2)粗酶液：将步骤(1)获得的重组大肠杆菌分别接种至LB固体培养基，37℃培养12h，获得活化菌体；将活化菌体接种至LB液体培养基，37℃、200rpm培养8h，获得种子液；将种子液以体积浓度2％(v/v)的接种量接种至新鲜的含有终浓度50μg/mL卡那霉素抗性的LB液体培养基中，37℃、200rpm培养2h至OD值0.6-0.8，加入终浓度为0.15mM IPTG，28℃、200rpm培养12h。并在4℃、8000rpm下离心10min，收集湿菌体；按照每1g湿菌体悬浮于20mL的pH 7、20mM磷酸钠缓冲液中得到菌悬液，超声破碎30min(功率300W，破碎1s，暂停3s)，12000rpm、4℃离心10min，收集上清，0.45μm微膜过滤去除细胞碎片等不溶性物质，滤液即为醛酮还原酶粗酶液。(2) Crude enzyme solution: Inoculate the recombinant Escherichia coli obtained in step (1) into LB solid medium, culture at 37°C for 12 hours to obtain activated cells; inoculate the activated cells into LB liquid medium, 37°C, 200rpm Cultivate for 8 hours to obtain a seed solution; inoculate the seed solution with an inoculum volume concentration of 2% (v/v) into fresh LB liquid medium containing a final concentration of 50 μg/mL kanamycin resistance, 37 ° C, 200 rpm Cultivate for 2 hours until the OD value is 0.6-0.8, add IPTG at a final concentration of 0.15 mM, and cultivate at 28° C. and 200 rpm for 12 hours. Then centrifuge at 4°C and 8000rpm for 10min to collect the wet bacteria; suspend each 1g of wet bacteria in 20mL of pH 7, 20mM sodium phosphate buffer to obtain a bacterial suspension, and ultrasonically break for 30min (power 300W, break for 1s, pause 3s), centrifuge at 12000rpm, 4°C for 10min, collect the supernatant, filter through a 0.45μm micromembrane to remove insoluble substances such as cell debris, and the filtrate is the crude enzyme solution of aldehyde and ketone reductase.
(3)纯酶液：镍柱(1.6cm×10cm，Bio-Rad，USA)用A液(含300mM NaCl和20mM咪唑的pH 7.0，20mM磷酸钠缓冲液)平衡后，上样步骤(2)粗酶液，上样量为1-2个柱体积，用A液冲洗3-4个柱体积，去除杂质和结合较弱的蛋白；再用B液(含300mM NaCl，500mM咪唑的pH7.0、20mM磷酸钠缓冲液)洗脱目的蛋白并收集后置于冰上保存，再用50mM pH 7.0磷酸钠缓冲液4℃透析(透析袋的截留分子量为10kDa)12h，收集截留液，分别获得醛酮还原酶KmAKR纯酶液和醛酮还原酶KdAKR纯酶液。用十二烷基硫酸钠-聚丙烯酰凝胶电泳(SDS-PAGE)鉴定蛋白质大小，电泳图见图2所示，通过蛋白质定量试剂盒(BCA法)测定蛋白浓度，并用pH7.0、20mM磷酸钠缓冲液调整醛酮还原酶KmAKR纯酶液和醛酮还原酶KdAKR纯酶液的蛋白浓度均为0.5g/L。(3) Pure enzyme solution: Ni column (1.6cm×10cm, Bio-Rad, USA) is equilibrated with solution A (pH 7.0 containing 300mM NaCl and 20mM imidazole, 20mM sodium phosphate buffer), and the loading step (2) Crude enzyme solution, the sample volume is 1-2 column volumes, wash 3-4 column volumes with solution A to remove impurities and weakly bound proteins; then use solution B (containing 300mM NaCl, 500mM , 20mM sodium phosphate buffer) to elute the target protein and collect it, store it on ice, and then dialyze it with 50mM pH 7.0 sodium phosphate buffer at 4°C for 12h (the molecular weight cut-off of the dialysis bag is 10kDa), collect the retentate, and obtain the aldehyde Keto reductase KmAKR pure enzyme solution and aldehyde ketone reductase KdAKR pure enzyme solution. Use sodium dodecyl sulfate-polyacryl gel electrophoresis (SDS-PAGE) to identify the protein size, the electropherogram is shown in Figure 2, measure the protein concentration by the protein quantification kit (BCA method), and use pH7.0, 20mM Sodium phosphate buffer was used to adjust the protein concentration of aldehyde and ketone reductase KmAKR pure enzyme solution and aldehyde and ketone reductase KdAKR pure enzyme solution to 0.5 g/L.
2、葡萄糖脱氢酶纯酶液2. Glucose dehydrogenase pure enzyme solution
(1)工程菌：将Genbank中源自巨大芽孢杆菌(Bacillus megaterium de Bary)的葡萄糖脱氢酶BmGDH基因(核苷酸序列如SEQ ID NO.3所示)人工合成后，通过同源重组的方法连接至pET28b载体的多克隆位点，再将重组载体pET28b-bmgdh转化至大肠杆菌E.coliBL21(DE3)中，获得表达重组质粒的重组大肠杆菌E.coli BL21(DE3)/pET28b-bmgdh。(1) Engineering bacteria: After artificially synthesizing the glucose dehydrogenase BmGDH gene (nucleotide sequence shown in SEQ ID NO.3) derived from Bacillus megaterium de Bary in Genbank, through homologous recombination Methods Connect to the multiple cloning site of pET28b vector, and then transform the recombinant vector pET28b-bmgdh into Escherichia coli E.coli BL21(DE3) to obtain recombinant E.coli BL21(DE3)/pET28b-bmgdh expressing the recombinant plasmid.
(2)粗酶液：同醛酮还原酶粗酶液的方法制备。(2) Crude enzyme liquid: prepared by the same method as the crude enzyme liquid of aldehyde and ketone reductase.
(3)纯酶液：同醛酮还原酶纯酶液的方法制备，SDS-PAGE电泳图如图2，用pH 7.0、20mM磷酸钠缓冲液调整蛋白浓度为0.5g/L。(3) Pure enzyme solution: prepared by the same method as the pure enzyme solution of aldehyde and ketone reductase, the SDS-PAGE electrophoresis figure is shown in Figure 2, and the protein concentration was adjusted to 0.5g/L with pH 7.0 and 20mM sodium phosphate buffer.
图2显示，没有其他明显的杂蛋白条带，表明两个酶都得到了有效纯化，对比标准蛋白marker分子量，分析醛酮还原酶的分子量为～36kDa，葡萄糖脱氢酶的分子量为～29kDa，可以进行后续固定化酶的实验。Figure 2 shows that there are no other obvious foreign protein bands, indicating that the two enzymes have been effectively purified. Compared with the molecular weight of the standard protein marker, the molecular weight of aldehyde and ketone reductase is ~36kDa, and the molecular weight of glucose dehydrogenase is ~29kDa. Subsequent experiments with immobilized enzymes can be performed.
实施例2:不同树脂类型对醛酮还原酶KmAKR固定化的影响Embodiment 2: the impact of different resin types on the immobilization of aldehyde and ketone reductase KmAKR
1、不同树脂形态1. Different resin forms
为选择适合醛酮还原酶KmAKR固定化的树脂载体，对六种不同类型的树脂进行了基本形态和物理化学性能的表征，具体如下：In order to select a suitable resin carrier for the immobilization of aldehyde and ketone reductase KmAKR, the basic morphology and physicochemical properties of six different types of resins were characterized as follows:
树脂类型：环氧树脂(epoxy resin)LXTE-600，氨基树脂(amino resin)LXTE-700，氨基环氧树脂(amino-epoxy resin)LXTE-706、羧基树脂(carboxyl resin)LXTE-800、离子交换树脂(ion exchange resin)LXTE-901和大孔树脂(macrosporous resin)LXTE-1000。Resin type: epoxy resin (epoxy resin) LXTE-600, amino resin (amino resin) LXTE-700, amino-epoxy resin (amino-epoxy resin) LXTE-706, carboxyl resin (carboxyl resin) LXTE-800, ion exchange Resin (ion exchange resin) LXTE-901 and macroporous resin (macrosporous resin) LXTE-1000.
使用带有数码相机的显微镜(EVOS M5000)监测树脂的球形度和形状，结果见图3所示，几种树脂的化学结构示意图如图4。The sphericity and shape of the resins were monitored using a microscope (EVOS M5000) with a digital camera, the results are shown in Figure 3, and the chemical structures of several resins are shown in Figure 4.
2、不同种类树脂对KmAKR固定化评价2. Evaluation of different types of resins for KmAKR immobilization
将表1中的6种树脂分别用磷酸钠缓冲液(0.2M，pH 7.0)洗涤三次。将0.5g清洗后的树脂加入5mL实施例1方法制备的KmAKR纯酶液中(蛋白浓度为0.5g/L)，28℃，200rpm振荡18小时，用0.2M、pH 7.0的磷酸钠缓冲液洗涤三次，即获得醛酮还原酶-树脂固定化复合物，记为KmAKR@epoxy resin，KmAKR@amino resin，KmAKR@amino-epoxy resin，KmAKR@carboxyl resin，KmAKR@ion exchange resin，KmAKR@macrosporous resin。此外，采用数码相机的显微镜(EVOS M5000)附带的软件检测树脂尺寸。The six resins in Table 1 were washed three times with sodium phosphate buffer (0.2M, pH 7.0). Add 0.5 g of the cleaned resin to 5 mL of the KmAKR pure enzyme solution prepared by the method in Example 1 (the protein concentration is 0.5 g/L), shake at 200 rpm for 18 hours at 28 ° C, and wash with 0.2 M, pH 7.0 sodium phosphate buffer Three times, the aldehyde and ketone reductase-resin immobilized complex was obtained, which was recorded as KmAKR@epoxy resin, KmAKR@amino resin, KmAKR@amino-epoxy resin, KmAKR@carboxyl resin, KmAKR@ion exchange resin, KmAKR@macrosporous resin. In addition, the resin size was detected using the software attached to the microscope (EVOS M5000) of the digital camera.
酶活单位(U)定义：在35℃、pH 7.0条件下，催化每分钟产生1μmol产物6-氰基-(3R,5R)-二羟基己酸叔丁酯所需的酶量定义为1U。Definition of enzyme activity unit (U): at 35°C and pH 7.0, the amount of enzyme required to catalyze the production of 1 μmol of product 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester per minute is defined as 1U.
酶活检测方法：反应体系10mL：200mM、pH7.0磷酸钠缓冲液、1.0mM NADP+、20g/L6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、30g/L葡萄糖(作为BmGDH催化实现NADPH循环的底物)、5％(v/v)二甲基亚砜(DMSO)，醛酮还原酶-树脂固定化复合物(0.5g)。在200rpm，40℃条件下振荡孵育20min，然后取出100μL反应混合液，向其中加入900μL无水乙醇以淬灭反应。将该混合液高速离心(12,000rpm,5min)后，取上清液用0.22μm滤膜过滤后，使用高效液相色谱(HPLC)检测产物6-氰基-(3R,5R)-二羟基己酸叔丁酯的生成量。根据酶活定义，计算游离酶和固定化酶的活力。Enzyme activity detection method: Reaction system 10mL: 200mM, pH7.0 sodium phosphate buffer, 1.0mM NADP+ , 20g/L tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate, 30g/L glucose (as the substrate for NADPH cycle catalyzed by BmGDH), 5% (v/v) dimethylsulfoxide (DMSO), aldehyde and ketone reductase-resin-immobilized complex (0.5g). Shake and incubate at 200 rpm at 40° C. for 20 min, then take out 100 μL of the reaction mixture, and add 900 μL of absolute ethanol to it to quench the reaction. After the mixture was centrifuged at high speed (12,000rpm, 5min), the supernatant was filtered through a 0.22μm filter membrane, and the product 6-cyano-(3R,5R)-dihydroxyhexyl was detected by high performance liquid chromatography (HPLC). The formation of tert-butyl acid ester. According to the definition of enzyme activity, calculate the activity of free enzyme and immobilized enzyme.
液相检测条件：色谱柱C18(4.6×250mm，Acchrom,China)柱，流动相乙腈:水体积比为1:3，流速1.0mL/min，检测波长210nm，进样量10μL，柱温40℃。6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、6-氰基-(3R,5R)-二羟基己酸叔丁酯保留时间分别为：13.6min，10.3min。根据底物和产物对应的标准曲线，可以计算6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的消耗和6-氰基-(3R,5R)-二羟基己酸叔丁酯的生成量。Liquid phase detection conditions: chromatographic column C18 (4.6×250mm, Acchrom, China) column, the mobile phase acetonitrile:water volume ratio is 1:3, the flow rate is 1.0mL/min, the detection wavelength is 210nm, the injection volume is 10μL, and the column temperature is 40°C. The retention times of tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate and tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate were 13.6min and 10.3min, respectively. According to the standard curve corresponding to the substrate and product, the consumption of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate and the consumption of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate can be calculated The production of butyl esters.
固定化率、酶活回收率、固定化酶活力的计算公式如下：The calculation formulas of immobilization rate, enzyme activity recovery rate and immobilized enzyme activity are as follows:
表1、不同树脂对醛酮还原酶KmAKR的固定化效率影响Table 1. Effect of different resins on the immobilization efficiency of aldehyde and ketone reductase KmAKR
如表1显示，KmAKR@macrosporous resin的固定化率达到了100％，但由于在洗脱过程中，酶分子很容易从树脂的大孔结构中泄漏，所以没有检测到酶活。KmAKR@aminoepoxyresin和KmAKR@epoxy resin分别只显示了15.6％和几乎检测不到的酶活回收率。KmAKR@amino resin和KmAKR@ionexchange resin的固定化率分别为45.1％和61.1％，而且两者的酶活回收率分别为34.3％和38.0％，也相对高于其他组。优选氨基树脂LXTE-700和离子交换树脂LXTE-901作为固定化载体。As shown in Table 1, the immobilization rate of KmAKR@macrosporous resin reached 100%, but during the elution process, the enzyme molecules easily leaked from the macroporous structure of the resin, so no enzyme activity was detected. KmAKR@aminoepoxyresin and KmAKR@epoxy resin showed only 15.6% and almost undetectable activity recovery, respectively. The immobilization rates of KmAKR@amino resin and KmAKR@ionexchange resin were 45.1% and 61.1%, respectively, and the recovery rates of the two enzyme activities were 34.3% and 38.0%, respectively, which were relatively higher than other groups. Amino resin LXTE-700 and ion exchange resin LXTE-901 are preferred as immobilized carriers.
3、物理化学性能检测3. Physical and chemical performance testing
根据酶活回收率和固定化率比较(表1)，以及对合适填充的尺寸(＞300μm)的筛选，对优选的氨基树脂LXTE-700和离子交换树脂LXTE-901的比表面积用BET(Brunauer-Emmett-Teller)方法进行测定，孔性能特征(孔隙体积、孔隙宽度、平均孔隙)用BJH(Barret-Joyner-Halenda)方法检测，结果如表2所示，氨基树脂LXTE-700和离子交换树脂LXTE-901的平均孔径为25.11nm和112.22nm；孔体积分别为0.80和0.12cm3/g，研究结果表明合适的孔径和孔体积有利于促进底物对酶的活性部位的可及性。According to the comparison of enzyme activity recovery rate and immobilization rate (Table 1), and the screening of suitable filling size (> 300 μm), the specific surface area of the preferred amino resin LXTE-700 and ion exchange resin LXTE-901 was used by BET (Brunauer -Emmett-Teller) method is measured, and pore property characteristic (pore volume, pore width, average pore) detects with BJH (Barret-Joyner-Halenda) method, and the result is as shown in table 2, amino resin LXTE-700 and ion exchange resin The average pore diameters of LXTE-901 are 25.11nm and 112.22nm; the pore volumes are 0.80 and 0.12cm3 /g respectively. The research results show that appropriate pore diameter and pore volume are beneficial to promote the accessibility of the substrate to the active site of the enzyme.
表2氨基和离子交换树脂的表面积和孔性能表征(基于BET和BJH方法检测)Surface area and pore performance characterization of table 2 amino and ion exchange resin (based on BET and BJH method detection)
因此，综合考虑酶活回收率、树脂材料的直径和孔径大小，我们选择了氨基树脂LXTE-700进行后续的批次使用实验研究。Therefore, considering the recovery rate of enzyme activity, the diameter and pore size of the resin material, we chose the amino resin LXTE-700 for subsequent batch use experimental research.
实施例3：不同来源醛酮还原酶对于固定化酶酶活的影响Example 3: Effects of different sources of aldehyde and ketone reductase on the activity of immobilized enzymes
(1)将0.5g氨基树脂LXTE-700加入5mL含体积浓度0.5％的戊二醛的磷酸钠缓冲液(0.2M，pH 7.0)中，37℃搅拌1h对氨基树脂进行活化，然后用0.2M，pH 7.0磷酸钠缓冲液冲洗树脂去掉未反应的戊二醛，获得戊二醛修饰的氨基树脂(amino resin-GLA)。(1) Add 0.5g of amino resin LXTE-700 to 5mL sodium phosphate buffer (0.2M, pH 7.0) containing 0.5% glutaraldehyde by volume, stir at 37°C for 1h to activate the amino resin, and then use 0.2M , pH 7.0 sodium phosphate buffer to wash the resin to remove unreacted glutaraldehyde to obtain glutaraldehyde-modified amino resin (amino resin-GLA).
(2)将全部步骤(1)戊二醛修饰的氨基树脂添加到10mL体积浓度6％(v/v)的聚丙烯酰胺(PEI)水溶液中，在28℃、200rpm下孵育过夜，并用0.2M、pH 7.0的磷酸钠缓冲液洗涤，加入实施例1方法制备的5mL醛酮还原酶KmAKR纯酶液纯酶液(蛋白浓度0.5g/L)，在28℃、200rpm下孵育12小时，获得醛酮还原酶KmAKR固定化酶0.5g，记为KmAKR@aminoresin-PEI。(2) Add the glutaraldehyde-modified amino resin in all steps (1) to 10 mL of polyacrylamide (PEI) aqueous solution with a volume concentration of 6% (v/v), incubate overnight at 28°C and 200 rpm, and wash with 0.2M , pH 7.0 sodium phosphate buffer washing, add 5mL aldehyde and ketone reductase KmAKR pure enzyme liquid pure enzyme liquid (protein concentration 0.5g/L) prepared by the method of Example 1, incubate 12 hours at 28 ℃, 200rpm, obtain aldehyde Ketoreductase KmAKR immobilized enzyme 0.5g, recorded as KmAKR@aminoresin-PEI.
同上，将醛酮还原酶KmAKR纯酶液替换为同样方法制备的醛酮还原酶KdAKR纯酶液(蛋白浓度0.5g/L)，获得固定化酶KdAKR@aminoresin-PEI。As above, the pure enzyme solution of aldehyde and ketone reductase KmAKR was replaced with the pure enzyme solution of aldehyde and ketone reductase KdAKR (protein concentration 0.5g/L) prepared by the same method to obtain the immobilized enzyme KdAKR@aminoresin-PEI.
采用实施例2方法检测酶活，结果见图5，表明KmAKR@amino resin-PEI和KdAKR@amino resin-PEI固定化酶活力分别为13.6、3.9U/g。KmAKR@aminoresin-PEI的固定化酶活力是KdAKR@aminoresin-PEI的近2.5倍，因此选择醛酮还原酶KmAKR纯酶液用于后续实验。The enzyme activity was detected by the method in Example 2, and the results are shown in Figure 5, showing that the immobilized enzyme activities of KmAKR@amino resin-PEI and KdAKR@amino resin-PEI were 13.6 and 3.9 U/g, respectively. The immobilized enzyme activity of KmAKR@aminoresin-PEI is nearly 2.5 times that of KdAKR@aminoresin-PEI, so the pure enzyme solution of aldehyde and ketone reductase KmAKR was selected for subsequent experiments.
实施例4：醛酮还原酶-葡萄糖脱氢酶共固化酶的制备Embodiment 4: Preparation of aldehyde and ketone reductase-glucose dehydrogenase co-immobilization enzyme
戊二醛(GLA)和聚乙烯亚胺(PEI)功能化的氨基树脂用于葡萄糖脱氢酶和醛酮还原酶的共固定化研究。Amino resins functionalized with glutaraldehyde (GLA) and polyethyleneimine (PEI) were used for co-immobilization of glucose dehydrogenase and aldehyde-ketone reductase.
(1)戊二醛修饰氨基树脂(1) Glutaraldehyde modified amino resin
将0.5g氨基树脂LXTE-700(amino resin C6)加入5mL含体积浓度0.5％的戊二醛的磷酸钠缓冲液(0.2M，pH 7.0)中，37℃搅拌1h对氨基树脂进行活化，然后用0.2M，pH 7.0磷酸钠缓冲液冲洗树脂去掉未反应的戊二醛，获得戊二醛修饰的氨基树脂(amino resin-GLA)。Add 0.5 g of amino resin LXTE-700 (amino resin C6) into 5 mL of sodium phosphate buffer solution (0.2 M, pH 7.0) containing 0.5% glutaraldehyde by volume concentration, stir at 37 ° C for 1 h to activate the amino resin, and then use The resin was washed with 0.2 M, pH 7.0 sodium phosphate buffer to remove unreacted glutaraldehyde to obtain glutaraldehyde-modified amino resin (amino resin-GLA).
(2)葡萄糖脱氢酶的固定化(2) Immobilization of glucose dehydrogenase
将全部步骤(1)戊二醛修饰的氨基树脂中加入到实施例1方法制备的5mL葡萄糖脱氢酶BmGDH纯酶液(蛋白浓度0.5g/L)中，28℃、200rpm孵育3小时。用0.2M、pH 7.0的磷酸钠缓冲液洗涤三次后，获得葡萄糖脱氢酶酶-树脂固定化复合物，记为BmGDH@aminoresin-GLA。Add the glutaraldehyde-modified amino resin in all steps (1) to 5 mL of glucose dehydrogenase BmGDH pure enzyme solution (protein concentration 0.5 g/L) prepared by the method in Example 1, and incubate at 28° C. and 200 rpm for 3 hours. After washing three times with 0.2M sodium phosphate buffer at pH 7.0, the glucose dehydrogenase enzyme-resin-immobilized complex was obtained, which was designated as BmGDH@aminoresin-GLA.
(3)PEI修饰(3) PEI modification
将全部步骤(2)葡萄糖脱氢酶-树脂固定化复合物添加到10mL体积浓度6％(v/v)的PEI水溶液中，28℃、200rpm孵育过夜，并用0.2M、pH 7.0的磷酸钠缓冲液洗涤，从而实现对氨基树脂表面进行PEI修饰，使得树脂表面局部形成了带正电的微环境，获得PEI修饰树脂。同样条件下，将葡萄糖脱氢酶-树脂固定化复合物替换为amino resin C6，制备得到amino resin-PEI。Add the whole step (2) glucose dehydrogenase-resin immobilized complex to 10 mL of 6% (v/v) PEI aqueous solution, incubate overnight at 28°C, 200 rpm, and buffer with 0.2M sodium phosphate, pH 7.0 Liquid washing, so as to achieve PEI modification on the surface of the amino resin, so that a positively charged microenvironment is locally formed on the resin surface, and the PEI modified resin is obtained. Under the same conditions, the glucose dehydrogenase-resin immobilized complex was replaced by amino resin C6 to prepare amino resin-PEI.
(4)醛酮还原酶-葡萄糖脱氢酶共固定化酶(4) Aldehyde and ketone reductase-glucose dehydrogenase co-immobilized enzyme
将步骤(3)全部PEI修饰树脂加入到实施例1制备的5mL的醛酮还原酶KmAKR纯酶液(蛋白浓度0.5g/L)，在28℃、200rpm下孵育18小时，通过PEI和醛酮还原酶酶分子静电吸附作用，有效固定醛酮还原酶，用0.2M、pH 7.0的磷酸钠缓冲液洗涤3次，最终获得0.5g醛酮还原酶-葡萄糖脱氢酶共固定化酶，记为KmAKR&BmGDH@aminoresin(C6)-PEI。Add all PEI-modified resins in step (3) to 5 mL of aldehyde and ketone reductase KmAKR pure enzyme solution (protein concentration 0.5g/L) prepared in Example 1, incubate at 28°C and 200rpm for 18 hours, pass through PEI and aldehyde and ketone The electrostatic adsorption of the reductase enzyme molecule effectively immobilized the aldehyde and ketone reductase, washed 3 times with 0.2M, pH 7.0 sodium phosphate buffer, and finally obtained 0.5g of the aldehyde and ketone reductase-glucose dehydrogenase co-immobilized enzyme, which was denoted as KmAKR & BmGDH @aminoresin (C6) - PEI.
(5)酶活检测(5) Enzyme activity detection
采用实施例2方法检测酶活，反应混合物(10mL)由pH 7.0、200mM磷酸钠缓冲液、1.0mM NADP+、20g/L(5R)-1、30g/L葡萄糖、5％(v/v)二甲基亚砜(DMSO)和适量的游离酶(实施例1方法制备的纯酶液，以蛋白含量计为1.2mg KmAKR和0.4mgBmGDH)或0.5g共固定化酶组成。将反应混合物以35℃，200rpm振荡孵育20分钟，然后取出100μL反应混合物，向其中加入900μL无水乙醇以猝灭反应。将混合物以12000rpm离心5分钟，随后用0.22μm膜过滤上清液，并通过高效液相色谱法检测产物6-氰基-(3R,5R)-二羟基己酸叔丁酯生成量。游离/固定化KmAKR酶活性的一个单位被定义为催化每分钟产生1μmol产物6-氰基-(3R,5R)-二羟基己酸叔丁酯所需的游离/固定酶的量。结果显示：游离KmAKR酶活是23U/mg，0.5g/L BmGDH操作浓度下，固定化酶BmGDH@amino resin-GLA酶活是31U/g，0.5g/LKmAKR操作浓度下，KmAKR&BmGDH@amino resin(C6)-PEI酶活是19U/g。Using the method of Example 2 to detect enzyme activity, the reaction mixture (10mL) was composed of pH 7.0, 200mM sodium phosphate buffer, 1.0mM NADP+ , 20g/L (5R)-1, 30g/L glucose, 5% (v/v) Dimethyl sulfoxide (DMSO) and an appropriate amount of free enzyme (the pure enzyme solution prepared by the method of Example 1, calculated as protein content is 1.2 mg KmAKR and 0.4 mg BmGDH) or 0.5 g co-immobilized enzyme. The reaction mixture was incubated at 35° C. with shaking at 200 rpm for 20 minutes, then 100 μL of the reaction mixture was removed, and 900 μL of absolute ethanol was added thereto to quench the reaction. The mixture was centrifuged at 12000 rpm for 5 minutes, then the supernatant was filtered with a 0.22 μm membrane, and the amount of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate produced was detected by high performance liquid chromatography. One unit of free/immobilized KmAKR enzyme activity was defined as the amount of free/immobilized enzyme required to catalyze the production of 1 μmol of product tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate per minute. The results show that the enzyme activity of free KmAKR is 23U/mg, at the operating concentration of 0.5g/L BmGDH, the enzyme activity of the immobilized enzyme BmGDH@amino resin-GLA is 31U/g, at the operating concentration of 0.5g/L KmAKR, the activity of KmAKR&BmGDH@amino resin( C6)-PEI enzyme activity is 19U/g.
实施例5：树脂表面修饰的戊二醛和聚乙烯亚胺比例对固定化酶的影响Example 5: Effect of the ratio of glutaraldehyde and polyethyleneimine modified on the resin surface on the immobilized enzyme
1、戊二醛和聚乙烯亚胺(PEI)浓度对共固定化酶酶活回收率及固定化率的影响1. Effect of concentration of glutaraldehyde and polyethyleneimine (PEI) on recovery rate and immobilization rate of co-immobilized enzyme
通过调节戊二醛和聚乙烯亚胺的比例，分析在不同戊二醛(GLA)和聚乙烯亚胺(PEI)浓度下醛酮还原酶的固定化率和酶活回收率，筛选最佳的修饰比例，如图6所示，横坐标中0.5-0代表将实施例4步骤(3)省略，即不添加PEI；1-0代表将实施例4步骤(1)中戊二醛体积浓度改为1％，同时步骤(3)省略；2-0代表将实施例4步骤(1)中戊二醛体积浓度改为2％，同时步骤(3)省略；0-0代表将实施例4步骤(1)中戊二醛体积浓度改为0％，同时步骤(3)省略；依次类推，横坐标分别代表实施例4步骤(1)中戊二醛浓度和步骤(3)中PEI浓度。其他操作同实施例4。By adjusting the ratio of glutaraldehyde and polyethyleneimine, analyze the immobilization rate and enzyme activity recovery rate of aldehyde and ketone reductase under different concentrations of glutaraldehyde (GLA) and polyethyleneimine (PEI), and screen the best Modification ratio, as shown in Figure 6, in the abscissa, 0.5-0 represents that step (3) of embodiment 4 is omitted, that is, no PEI is added; 1-0 represents that the volume concentration of glutaraldehyde in step (1) of embodiment 4 is changed to 1%, while step (3) is omitted; 2-0 represents that the volume concentration of glutaraldehyde in step (1) of embodiment 4 is changed to 2%, and step (3) is omitted simultaneously; 0-0 represents that the step of embodiment 4 is changed to 2%. In (1), the glutaraldehyde volume concentration is changed to 0%, and step (3) is omitted simultaneously; By analogy, the abscissa represents respectively the concentration of glutaraldehyde in the step (1) of embodiment 4 and the concentration of PEI in the step (3). Other operations are the same as in Example 4.
如图6示，当PEI体积浓度为0％时，通过戊二醛共价固定的醛酮还原酶(席夫碱反应)，固定化率接近100％，但是该固定化方法下的酶回活收率很低，这表明戊二醛交联极容易导致醛酮还原酶失活。因此，通过引入强阳离子聚合物PEI上的氨基与戊二醛上的醛基反应，PEI功能化的氨基树脂总体呈电正性，通过醛酮还原酶(电负性)与载体之间的静电吸附(PEI和酶在不同pH下的zeta电位见表3)，在树脂表面实现了醛酮还原酶的有效固定。通过适当调节戊二醛和聚乙烯亚胺的添加浓度发现，当GLA和PEI浓度分别为0.5％和6％时，KmAKR@amino resin-PEI酶活回收率最高为39.7％(图6)。进一步增加聚乙烯亚胺浓度(＞6％)并不能提高固定化醛酮还原酶的酶活回收率，因此6％为最佳PEI浓度。As shown in Figure 6, when the volume concentration of PEI was 0%, the immobilization rate of aldehyde and ketone reductase (Schiff base reaction) covalently immobilized by glutaraldehyde was close to 100%, but the enzyme reactivation under this immobilization method The yield was very low, which indicated that glutaraldehyde cross-linking could easily lead to the inactivation of aldehyde and ketone reductase. Therefore, by introducing the amino group on the strong cationic polymer PEI to react with the aldehyde group on the glutaraldehyde, the amino resin functionalized by PEI is generally electropositive, and the static electricity between the aldehyde and ketone reductase (electronegative) and the carrier Adsorption (the zeta potentials of PEI and enzymes at different pHs are shown in Table 3), the effective immobilization of aldehyde and ketone reductase on the resin surface was realized. By properly adjusting the concentration of glutaraldehyde and polyethyleneimine, it was found that when the concentrations of GLA and PEI were 0.5% and 6%, respectively, the recovery rate of KmAKR@amino resin-PEI enzyme activity was 39.7% (Figure 6). Further increasing the concentration of polyethyleneimine (>6%) can not improve the recovery rate of the enzyme activity of immobilized aldehyde and ketone reductase, so 6% is the optimal PEI concentration.
2、不同pH下的酶和聚乙烯亚胺的Zeta电位2. Zeta potential of enzyme and polyethyleneimine at different pH
将游离的醛酮还原酶(实施例1方法制备的KmAKR，0.5g/L)和葡萄糖脱氢酶(实施例1方法制备的BmGDH，0.5g/L)以及PEI的水溶液(6％,v/v)，分别置于柠檬酸-Na2HPO4缓冲液(pH 4-6,200mM)、磷酸钠缓冲液(pH6-8,200mM)，Tris-HCl(pH 8-10，200mM)中，室温静置两分钟，使用Zetasizer Nano ZS仪器(Malvern，He-Ne激光，λ＝632nm)测量PEI和酶的zeta电位。在2分钟的平衡时间后，在25℃下进行测量，测量三次，结果见表3所示。The aqueous solution (6%, v/ v), respectively placed in citric acid-Na2 HPO4 buffer solution (pH 4-6, 200mM), sodium phosphate buffer solution (pH 6-8, 200mM), Tris-HCl (pH 8-10, 200mM), at room temperature For two minutes, the zeta potentials of PEI and enzymes were measured using a Zetasizer Nano ZS instrument (Malvern, He-Ne laser, λ = 632 nm). After the equilibration time of 2 minutes, the measurement was carried out at 25° C., and the measurement was performed three times. The results are shown in Table 3.
表3不同pH下的酶和聚乙烯亚胺的Zeta电位Zeta potentials of enzymes and polyethyleneimine at different pHs in table 3
实施例6：氨基树脂表面碳链长度对双酶固定化效率的影响Example 6: Effect of carbon chain length on the surface of amino resin on the immobilization efficiency of dual enzymes
将实施例4步骤(1)中氨基树脂LXTE-700(amino resin C6)分别替换为表4中不同型号的氨基树脂，其他操作相同，如表4，KmAKR&BmGDH@amino resin-PEI(C6)固定化率高于两个碳连接的氨基树脂KmAKR&BmGDH@amino resin(C2-1,C2-2,C2-3)。同时，KmAKR&BmGDH@amino resin(C6)的酶活回收(39.7％)也明显高于KmAKR&BmGDH@amino resin(C2)(均不到20％)组的。Replace the amino resin LXTE-700 (amino resin C6) in step (1) of Example 4 with amino resins of different types in Table 4, and the other operations are the same, as shown in Table 4, KmAKR&BmGDH@amino resin-PEI (C6) is immobilized The rate is higher than that of two carbon-linked amino resins KmAKR&BmGDH@amino resin (C2-1, C2-2, C2-3). At the same time, the enzyme activity recovery (39.7%) of KmAKR&BmGDH@amino resin (C6) was also significantly higher than that of KmAKR&BmGDH@amino resin (C2) (less than 20%) group.
氨基树脂(amino resin(C6),LXTE-700)，相比其他几种氨基树脂(amino resin(C2)LXTE-703、LXTE-704、LXTE-705)也具有作为填充材料更适合的尺寸(＞300μm)，LXTE-703,LXTE-704,LXTE-705的尺寸均小于300μm(表4)。Amino resin (amino resin (C6), LXTE-700), compared with several other amino resins (amino resin (C2) LXTE-703, LXTE-704, LXTE-705), also has a more suitable size as a filling material (> 300μm), LXTE-703, LXTE-704, LXTE-705 are all smaller than 300μm (Table 4).
因此KmAKR&BmGDH@amino resin(C6)-PEI是最佳的双酶-树脂固定化复合物，可以选择作为连续流反应器良好的填充介质。Therefore, KmAKR&BmGDH@amino resin(C6)-PEI is the best dual enzyme-resin immobilization complex, which can be selected as a good filling medium for continuous flow reactors.
表4不同碳链长度的氨基树脂对双酶固定化的效率影响Table 4 The effect of amino resins with different carbon chain lengths on the efficiency of dual enzyme immobilization
实施例7:酶浓度优化Embodiment 7: Enzyme concentration optimization
1、葡萄糖脱氢酶浓度1. Glucose dehydrogenase concentration
在固定化过程中，通过调节加入至戊二醛修饰的氨基树脂(Resin-GLA)中葡萄糖脱氢酶的操作浓度(体积均为5mL)，探究酶不同操作浓度下的固定化活力。During the immobilization process, by adjusting the operating concentration of glucose dehydrogenase added to glutaraldehyde-modified amino resin (Resin-GLA) (both volumes were 5 mL), the immobilization activity of the enzyme at different operating concentrations was explored.
将实施例4步骤(2)葡萄糖脱氢酶BmGDH纯酶液的蛋白浓度分别调整为0、0.1、0.25、0.5、1、2g/L，体积均为5mL，制备得到BmGDH@aminoresin-GLA，酶活力见图7所示，如图7所示，当葡萄糖脱氢酶浓度为0.25g/L时，最高固定化酶的活力为34U/g，更高的葡萄糖脱氢酶浓度并没有提升固定化酶的活力，是因为在0.25g/L操作浓度下，固定化葡萄糖脱氢酶已足够催化反应所需的辅因子再生。Adjust the protein concentration of glucose dehydrogenase BmGDH pure enzyme solution in step (2) of Example 4 to 0, 0.1, 0.25, 0.5, 1, 2g/L respectively, and the volume is 5mL to prepare BmGDH@aminoresin-GLA, the enzyme The activity is shown in Figure 7. As shown in Figure 7, when the concentration of glucose dehydrogenase is 0.25g/L, the highest activity of the immobilized enzyme is 34U/g, and the higher concentration of glucose dehydrogenase does not improve the immobilization The activity of the enzyme is because at the operating concentration of 0.25g/L, the immobilized glucose dehydrogenase is sufficient to catalyze the regeneration of the cofactor required for the reaction.
2、醛酮还原酶浓度2. Concentration of aldehyde and ketone reductase
将实施例4步骤(4)醛酮还原酶KmAKR纯酶液的蛋白浓度分别调整为0、0.25、0.5、2、4、5g/L，体积均为5mL，其他操作同实施例4，分别得到KmAKR&BmGDH@amino resin(C6)-PEI，酶活力见图8所示，醛酮还原酶的固定化活力随着操作浓度的增加而提高，浓度大于4g/L后，固定化酶的活力并没有显著提高，这主要是因为醛酮还原酶在树脂上的负载量达到了饱和，因此当最佳操作浓度为4g/L，最高的共固定化酶的活力达到73U/g。The protein concentration of step (4) aldehyde and ketone reductase KmAKR pure enzyme liquid of embodiment 4 is adjusted to 0, 0.25, 0.5, 2, 4, 5g/L respectively, and the volume is 5mL, other operation is the same as embodiment 4, obtains respectively KmAKR&BmGDH@amino resin(C6)-PEI, the enzyme activity is shown in Figure 8. The immobilized activity of aldehyde and ketone reductase increases with the increase of the operating concentration. When the concentration is greater than 4g/L, the activity of the immobilized enzyme is not significant This is mainly because the load of aldehyde and ketone reductase on the resin has reached saturation, so when the optimal operating concentration is 4g/L, the activity of the highest co-immobilized enzyme reaches 73U/g.
实施例8：树脂和共固定化酶的红外和热重表征Example 8: Infrared and Thermogravimetric Characterization of Resins and Co-immobilized Enzymes
将amino resin(C6)与实施例4方法制备的BmGDH@aminoresin(C6)-GLA和KmAKR&BmGDH@aminoresin(C6)-PEI，以及amino resin(C6)-PEI分别采用傅里叶变换红外光谱(FTIR)(Nicolet 6700FTIR-ATR分析仪，Thermo Fisher Scientific，USA)和热重分析(TGA)(TGA Q5000，TA Instruments，USA)表征。以1cm-1波数的分辨率记录了400至4000cm-1的FTIR光谱。样品的TGA检测在氮气流下进行，温度范围为30-800℃，频率为10℃/min。BmGDH@aminoresin(C6)-GLA and KmAKR&BmGDH@aminoresin(C6)-PEI prepared by amino resin (C6) and the method of Example 4, and amino resin (C6)-PEI were respectively adopted Fourier transform infrared spectroscopy (FTIR) (Nicolet 6700FTIR-ATR analyzer, Thermo Fisher Scientific, USA) and thermogravimetric analysis (TGA) (TGA Q5000, TA Instruments, USA) for characterization. FTIR spectra were recorded from 400 to 4000 cm-1 with a resolution of 1 cm-1 wavenumber. The TGA detection of the samples was carried out under nitrogen flow, the temperature range was 30-800°C, and the frequency was 10°C/min.
图9红外光谱(FTIR)图显示，共固定化酶存在明显的酰胺Ⅱ带，而单纯的树脂本身和PEI功能化的树脂并没有酰胺Ⅱ带，因此可以判断酶可以有效固定化于树脂表面，形成了葡萄糖脱氢酶-树脂固定化复合物(BmGDH@aminoresin-GLA)和双酶-树脂共固化复合物(KmAKR&BmGDH@aminoresin(C6)-PEI)。Figure 9 Infrared Spectrum (FTIR) shows that the co-immobilized enzyme has an obvious amide II band, while the pure resin itself and the PEI functionalized resin do not have an amide II band, so it can be judged that the enzyme can be effectively immobilized on the resin surface. Glucose dehydrogenase-resin-immobilized complex (BmGDH@aminoresin-GLA) and double enzyme-resin co-immobilized complex (KmAKR&BmGDH@aminoresin(C6)-PEI) were formed.
图10，TGA图显示，600℃以后酶-树脂固定化复合物质量百分比低于单纯的树脂本身，这主要是因为酶占据了固定化复合物的一定比例。红外及热重图谱都表明了，通过戊二醛交联，葡萄糖脱氢酶可以首先共价固定于氨基树脂上，醛酮还原酶可以通过静电吸附于PEI功能化的氨基树脂表面，实现了双酶共固化于同一树脂载体。Figure 10, TGA diagram shows that after 600°C, the mass percentage of the enzyme-resin immobilized complex is lower than that of the pure resin itself, mainly because the enzyme occupies a certain proportion of the immobilized complex. Both infrared and thermogravimetric spectra showed that glucose dehydrogenase can be covalently immobilized on the amino resin through glutaraldehyde crosslinking, and aldehyde and ketone reductase can be electrostatically adsorbed on the surface of PEI-functionalized amino resin, realizing the dual The enzymes are co-cured on the same resin carrier.
实施例9：游离/固定化酶pH优化Embodiment 9: free/immobilized enzyme pH optimization
将游离酶(实施例1方法制备的蛋白浓度0.25g/L的葡萄糖脱氢酶BmGDH纯酶液5mL，蛋白浓度4g/L的醛酮还原酶KmAKR纯酶液5mL)、实施例4方法制备的KmAKR&BmGDH@aminoresin(C6)-PEI，采用实施例2酶活检测方法，在不同pH条件(柠檬酸-Na2HPO4缓冲液(pH 4-6)、磷酸钠缓冲液(pH 6-8)，Tris-HCl(pH 8-10)，离子浓度均为200mM)下检测活力，以对应的最适pH下的活力为100％，计算并归一化其他pH下的相对活力，如图11示。游离酶在pH 6 -8范围内表现出较高活性，最适pH条件为200mM,pH 7磷酸钠缓冲液。对于共固定化酶，在pH 5-7范围内表现出较高活性(相对活力大于80％)，最适反应pH条件为200mM,pH 6磷酸钠缓冲液，与游离酶相比，固定化酶的最适pH偏低，阳离子聚合物PEI改变了共固定化酶催化的局部微环境，导致最适pH略向酸性偏移。因此我们将磷酸钠缓冲液(200mM，pH 6)，作为连续流反应器的最佳反应条件。Free enzyme (glucose dehydrogenase BmGDH pure enzyme solution 5mL of protein concentration 0.25g/L prepared by the method of embodiment 1, aldehyde and ketone reductase KmAKR pure enzyme solution 5mL of protein concentration 4g/L), prepared by the method of embodiment 4 KmAKR&BmGDH@aminoresin(C6)-PEI, using the enzyme activity detection method of Example 2, under different pH conditions (citric acid-Na2 HPO4 buffer solution (pH 4-6), sodium phosphate buffer solution (pH 6-8), Tris-HCl (pH 8-10), the ion concentration is 200mM), and the activity at the corresponding optimum pH is 100%, and the relative activity at other pHs is calculated and normalized, as shown in Figure 11. The free enzyme shows high activity in the range of pH 6-8, and the optimum pH condition is 200mM, pH 7 sodium phosphate buffer. For co-immobilized enzymes, it exhibits higher activity (relative activity greater than 80%) in the range of pH 5-7, and the optimum reaction pH condition is 200mM, pH 6 sodium phosphate buffer, compared with free enzymes, immobilized enzymes The optimum pH of the cationic polymer was low, and the cationic polymer PEI changed the local microenvironment catalyzed by the co-immobilized enzyme, resulting in a slight shift of the optimum pH towards acidity. So we use sodium phosphate buffer (200mM, pH 6) as the best reaction condition for continuous flow reactor.
实施例10、游离/固定化酶温度优化Embodiment 10, free/immobilized enzyme temperature optimization
将游离酶(实施例1方法制备的蛋白浓度0.25g/L的葡萄糖脱氢酶BmGDH纯酶液5mL，蛋白浓度4g/L的醛酮还原酶KmAKR纯酶液5mL)、按实施例4方法制备KmAKR&BmGDH@aminoresin(C6)-PEI，采用实施例2酶活检测方法，在反应温度25-65℃范围内进行测定，游离/固定化酶的相对活性是通过与它们的最高水平活性相比较来评估的，以对应的最适温度下的活力为100％，计算并归一化得到其他温度下的相对活力。结果如图12示，游离酶和共固定化酶均在35-45℃的范围内，表现了较高的相对活力(大于80％)，这表明共固定化酶并没有明显改变酶自身催化的温度范围，设计的固定化方法能有效的保留该温度范围内的催化活性，对酶的空间构象改变较少。对比最适温度，游离酶的最适反应温度为35℃，共固定化酶的最适反应温度为40℃，固定化酶的最适温度增加了5℃，推测是因为表面功能化的树脂载体对酶的保护作用和PEI良好的粘性对醛酮还原酶具有稳定的作用，使其在更高的温度下表现了更可观的活性。因此，将40℃作为构建的连续流反应器的最佳反应温度。Free enzyme (5 mL of pure enzyme solution of glucose dehydrogenase BmGDH with a protein concentration of 0.25 g/L prepared by the method of Example 1, 5 mL of pure enzyme solution of aldehyde and ketone reductase KmAKR with a protein concentration of 4 g/L) was prepared according to the method of Example 4 KmAKR&BmGDH@aminoresin(C6)-PEI, using the enzyme activity detection method in Example 2, measured at a reaction temperature of 25-65°C, the relative activity of free/immobilized enzymes was evaluated by comparing with their highest level of activity The activity at the corresponding optimum temperature is taken as 100%, and the relative activity at other temperatures is calculated and normalized. The results are shown in Figure 12. Both the free enzyme and the co-immobilized enzyme showed high relative activity (greater than 80%) in the range of 35-45°C, which indicated that the co-immobilized enzyme did not significantly change the catalytic activity of the enzyme itself. Temperature range, the designed immobilization method can effectively retain the catalytic activity in this temperature range, and change the spatial conformation of the enzyme less. Compared with the optimum temperature, the optimum reaction temperature of the free enzyme is 35°C, the optimum reaction temperature of the co-immobilized enzyme is 40°C, and the optimum temperature of the immobilized enzyme is increased by 5°C, presumably because of the surface functionalized resin carrier The protective effect on the enzyme and the good viscosity of PEI have a stabilizing effect on the aldehyde and ketone reductase, making it exhibit more considerable activity at higher temperatures. Therefore, 40°C was taken as the optimum reaction temperature for the constructed continuous flow reactor.
实施例11、共固定化酶的批次使用Example 11, batch use of co-immobilized enzymes
将实施例4方法制备的KmAKR&BmGDH@aminoresin(C6)-PEI，以及实施例2方法制得的KmAKR@amino resin(C6)、KmAKR@ion exchange resin，采用实施例2方法检测酶活，反应结束后，离心(12,000rpm,5min)，沉淀用200mM,pH 7的磷酸钠缓冲液冲洗3遍，作为催化剂进行下一轮反应。The KmAKR&BmGDH@aminoresin(C6)-PEI prepared by the method in Example 4, and the KmAKR@amino resin(C6) and KmAKR@ion exchange resin prepared by the method in Example 2 were used to detect the enzyme activity by the method in Example 2. After the reaction , centrifuged (12,000rpm, 5min), the precipitate was washed 3 times with 200mM, pH 7 sodium phosphate buffer, and used as a catalyst for the next round of reaction.
在每个循环中，酶-树脂固定化复合物对应的活性被归一化为它们在第一个循环中的相对活力。结果如图13示，经过PEI修饰的葡萄糖脱氢酶-醛酮还原酶双酶-树脂共固定化酶(KmAKR&BmGDH@aminoresin(C6)-PEI)相比不修饰的氨基树脂和离子交换树脂固定的醛酮还原酶(KmAKR@aminoresin，KmAKR@ionexchangeresin)，具有更好的重复批次使用稳定性，在重复使用10次后，其相对活力仍有80.7％。该结果显示，将KmAKR&BmGDH@aminoresin(C6)-PEI的共固定化酶作为填充材料，构建连续流反应器是可行且有效的。At each cycle, the corresponding activities of the enzyme-resin-immobilized complexes were normalized to their relative activities in the first cycle. The results are shown in Figure 13. Compared with unmodified amino resin and ion exchange resin immobilized enzyme (KmAKR&BmGDH@aminoresin(C6)-PEI) modified by PEI Aldehyde and ketone reductase (KmAKR@aminoresin, KmAKR@ionexchangeresin) has better stability in repeated batch use, and its relative activity is still 80.7% after repeated use 10 times. The results showed that it is feasible and effective to construct a continuous flow reactor using the co-immobilized enzyme of KmAKR&BmGDH@aminoresin(C6)-PEI as the filling material.
综上所述，我们通过在氨基树脂表面使用戊二醛交联葡萄糖脱氢酶，PEI功能化的葡萄糖脱氢酶-树脂固定化复合体，进一步吸附醛酮还原酶，成功实现了同载体、异方法共固化双酶，制备得到的醛酮还原酶-葡萄糖脱氢酶共固定化酶(KmAKR&BmGDH@aminoresin(C6)-PEI)，作为填充介质，构建双酶介导的连续流反应器，应用于后续的生物法不对称合成6-氰基-(3R,5R)-二羟基己酸叔丁酯的实验。To sum up, we successfully realized the same carrier, ketone reductase by using glutaraldehyde cross-linked glucose dehydrogenase on the surface of amino resin, PEI functionalized glucose dehydrogenase-resin immobilized complex, and further adsorbing aldehyde and ketone reductase. Different methods co-immobilize double enzymes, and the prepared aldehyde and ketone reductase-glucose dehydrogenase co-immobilized enzyme (KmAKR&BmGDH@aminoresin(C6)-PEI) is used as a filling medium to construct a double-enzyme-mediated continuous flow reactor. In the follow-up experiment of asymmetric synthesis of 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester.
实施例12、连续流反应器的构建Embodiment 12, the construction of continuous flow reactor
1、参照图14，将实施例4方法制备的共固定化酶KmAKR&BmGDH@amino(C6)-PEI作为填充材料，构建双酶介导的连续流反应器，所述连续流反应器包括反应液储罐、流量泵、填充式反应柱、液体收集罐，所述填充式反应柱设有进液口和出液口。所述反应液储罐通过流量泵与填充式反应柱进液口连接；所述填充式反应柱出液口分别与液体收集罐和反应稀释液连通，其中反应稀释液体与高效液相色谱仪连通，液体收集罐与反应液储罐连通构成循环。1. With reference to Figure 14, the co-immobilized enzyme KmAKR&BmGDH@amino(C6)-PEI prepared by the method in Example 4 is used as a filling material to construct a dual-enzyme-mediated continuous flow reactor, which includes a reaction liquid storage A tank, a flow pump, a packed reaction column, and a liquid collection tank, the packed reaction column is provided with a liquid inlet and a liquid outlet. The reaction liquid storage tank is connected to the liquid inlet of the packed reaction column through a flow pump; the liquid outlet of the packed reaction column is connected to the liquid collection tank and the reaction diluent respectively, wherein the reaction diluent is connected to the high performance liquid chromatograph , the liquid collection tank communicates with the reaction liquid storage tank to form a cycle.
2、将3.7g的共固定化酶装载到6mL容积的填充式反应柱中，直至填充式反应柱都被装满，并将柱温保持在40℃。使用注射泵(SLP01-02A，兰格，中国)将含有底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯(5R)-1(终浓度20g/L)、NADP+(终浓度1mM)和葡萄糖(终浓度30g/L)的pH 6.0、200mM的磷酸盐缓冲液，在不同的注射泵设置的流速下(0.25、0.5、1、2、5、7、10mL/h)，连续流加到填充柱中，从填充式反应柱出液口定期取出反应混合液，通过HPLC监测转化率和deP(产物的非对映体过量)。收集的反应液在不再次添加辅因子，作为进料混合液循环泵入填充柱，以验证闭环系统的可行性，为工业上实现自闭合、无废循环的技术革新进行了实验证明。结果见图15所示。2. Load 3.7 g of co-immobilized enzyme into a packed reaction column with a volume of 6 mL until the packed reaction column is full, and keep the column temperature at 40° C. Use a syringe pump (SLP01-02A, Lange, China) to inject tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (5R)-1 (final concentration 20 g/L), NADP+ (final concentration 1mM) and glucose (final concentration 30g/L) pH 6.0, 200mM phosphate buffer at different flow rates set by the syringe pump (0.25, 0.5, 1, 2, 5, 7, 10mL/ h), continuous flow is added to the packed column, and the reaction mixture is regularly taken out from the liquid outlet of the packed reaction column, and the conversion rate and deP (diastereomeric excess of the product) are monitored by HPLC. The collected reaction solution is pumped into the packed column as a feed mixed solution without adding cofactors again, to verify the feasibility of the closed-loop system, and to prove the technical innovation of self-closing and waste-free circulation in the industry. The results are shown in Figure 15.
3、考察了关键工艺参数，包括柱体积(1.5，3.5和6mL)和流速(0.25-10.0mL/h)，并检测相应工艺参数下的转化率，时空产率和操作稳定性。同样条件下，分别将0.8g的共固定化酶装载到1.5mL的填充式反应柱，2.0g的共固定化酶装载到3.5mL的填充式反应柱，其他操作相同，结果见表5。3. The key process parameters were investigated, including column volume (1.5, 3.5 and 6mL) and flow rate (0.25-10.0mL/h), and the conversion rate, space-time yield and operational stability under the corresponding process parameters were detected. Under the same conditions, 0.8 g of co-immobilized enzyme was loaded into a 1.5 mL packed reaction column, and 2.0 g of co-immobilized enzyme was loaded into a 3.5 mL packed reaction column. Other operations were the same, and the results are shown in Table 5.
图15和表5，显示了连续流反应最佳的柱体积为6mL，流速为5mL/h，当转化率＞99％，时空产率高达374.7g/L/d。将未反应完的混合液二次循环泵入，检测发现第二次反应的转化率明显高于第一次反应(图15)，这为更高浓度的底物在高流速下实现完全转化，提供可能性，提高了生产效率。Figure 15 and Table 5 show that the optimal column volume for the continuous flow reaction is 6 mL, the flow rate is 5 mL/h, and when the conversion rate is >99%, the space-time yield is as high as 374.7 g/L/d. The unreacted mixed solution was pumped into the secondary cycle, and it was found that the conversion rate of the second reaction was significantly higher than that of the first reaction (Figure 15), which means that the higher concentration of the substrate was completely converted at a high flow rate. Provides possibilities and increases productivity.
因此本发明设计的对传统树脂材料进一步表面修饰，结合化学和物理的方法实现了葡萄糖脱氢酶和醛酮还原酶的共固化，为氧化还原类酶的固定化及应用提供了方法创新和参考，构建的循环式双酶介导的连续流反应器，为工业上利用多酶偶联催化实现辅酶循环、无废、绿色、连续、自动化合成医药中间体的技术革新提供了实验基础，且具备更高效生产医药中间体、工艺放大的潜力。Therefore, the design of the present invention further modifies the surface of traditional resin materials, and combines chemical and physical methods to realize the co-curing of glucose dehydrogenase and aldehyde ketone reductase, providing method innovation and reference for the immobilization and application of redox enzymes , the cyclic dual-enzyme-mediated continuous flow reactor constructed provides an experimental basis for the industrial use of multi-enzyme coupling catalysis to realize the technological innovation of coenzyme cycle, waste-free, green, continuous, and automatic synthesis of pharmaceutical intermediates, and has Potential for more efficient production of pharmaceutical intermediates and process amplification.
表5不同柱体积和流速下的时空产率及转化率Table 5 Space-time yields and conversions under different column volumes and flow rates
实施例13：酶-树脂固定化复合物的电镜表征Example 13: Electron Microscopic Characterization of Enzyme-Resin Immobilized Complex
釜式反应模式：将含有1.5g醛酮还原酶-葡萄糖脱氢酶共固定化酶(KmAKR&BmGDH@aminoresin(C6)-PEI)、底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯(5R)-1(终浓度20g/L)、NADP+(终浓度1mM)和葡萄糖(终浓度30g/L)的pH 6.0、200mM的磷酸盐缓冲液，总体积10mL，投料到釜式反应器中，200rpm，40℃，反应4h。Kettle reaction mode: Co-immobilized enzyme containing 1.5g aldehyde and ketone reductase-glucose dehydrogenase (KmAKR&BmGDH@aminoresin(C6)-PEI), substrate 6-cyano-(5R)-hydroxy-3-carbonylhexyl Phosphate buffer solution of tert-butyl acid (5R)-1 (final concentration 20g/L), NADP+ (final concentration 1mM) and glucose (final concentration 30g/L), pH 6.0, 200mM, total volume 10mL, feeding into In the tank reactor, 200rpm, 40°C, reacted for 4h.
通过扫描电子显微镜(SEM)(S-4700(Ⅱ)，日立，日本)观察将实施例12步骤2流速5mL/h下反应90h后的以及釜式反应模式反应4h的的醛酮还原酶-葡萄糖脱氢酶共固定化酶(KmAKR&BmGDH@aminoresin(C6)-PEI)的显微形态。使用在15kV的加速电压下操作的检测器拍摄SEM图像。共固定化酶在釜式和连续流反应器中的形态表征如图16，在釜式模式下仅使用4小时后，共固定化酶表面出现明显的损坏和凹坑(图16)，这主要是由固体物质之间(树脂载体和反应器)的碰撞造成的。相反，共固定化酶在构建的连续流反应器连续流动反应长达90小时没有出现损坏现象，保留了共固定化酶的球形度、完整性和表面光滑程度，表明流体与固体载体之间的接触不易导致微球的磨损，有利于共固定化酶在连续流反应中的长期使用。因此，我们设计的双酶介导的连续流生物反应器，能够为生物催化、医药合成等领域提出新的思路和技术支持。By scanning electron microscope (SEM) (S-4700 (II), Hitachi, Japan) to observe the aldehyde and ketone reductase-glucose after reacting for 90h at the flow rate of 5mL/h in step 2 of Example 12 and reacting for 4h in the kettle reaction mode Microscopic morphology of dehydrogenase co-immobilized enzyme (KmAKR&BmGDH@aminoresin(C6)-PEI). SEM images were taken using a detector operated at an accelerating voltage of 15 kV. The morphological characterization of co-immobilized enzymes in kettle and continuous flow reactors is shown in Figure 16. After only 4 hours of use in kettle mode, obvious damage and pits appeared on the surface of co-immobilized enzymes (Figure 16), which mainly It is caused by the collision between solid substances (resin support and reactor). On the contrary, the continuous flow reaction of the co-immobilized enzymes in the constructed continuous flow reactor did not show damage for up to 90 hours, and the sphericity, integrity and surface smoothness of the co-immobilized enzymes were retained, indicating that the fluid-solid support The contact is not easy to cause wear of the microspheres, which is beneficial to the long-term use of co-immobilized enzymes in continuous flow reactions. Therefore, the dual-enzyme-mediated continuous flow bioreactor we designed can provide new ideas and technical support for biocatalysis, pharmaceutical synthesis and other fields.
上述仅为本发明的较佳实施例，并非限定发明的范围，本发明的上述实施例可做出各种变化。即凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰，皆落入本发明专利的权利要求保护范围。The above are only preferred embodiments of the present invention, and do not limit the scope of the invention. Various changes can be made to the above embodiments of the present invention. That is to say, all simple and equivalent changes and modifications made according to the claims and description of the application for the present invention fall within the protection scope of the claims of the patent of the present invention.
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