Immobilization Of Aldo-keto Reductase And Alcohol Dehydrogenase For Biocatalytic Synthesis Of The Chiral Alcohol Intermediate Of Crizotinib

Enzyme catalysis has received increasing attention in the biotechnology pharmaceutical industry due to its environmentally friendly characteristics.However,enzyme catalysis is affected by its own natural properties,it is highly susceptible to external factors and loses its activity during the catalytic process,and besides,free enzymes are difficult to separate from reaction mixture and reuse,which results in the high cost for biocatalysis.The immobilized enzyme can overcome these problems of free enzymes because of its better environmental tolerance and convenient separation.In order to explore the enzymatic chiral synthesis method for the intermediate?S?-2,6-dichloro-3-fluorophenylethanol of anti-cancer drug crizotinib,the aldo-keto reductase and alcohol dehydrogenase were co-immobilized on the carrier for chiral catalytic biosynthesis to achieve the green and energy efficient preparation of target products.The epoxy resin and inorganic salt calcium phosphate were used as supports to pprepare the immobilized the aldo-keto reductase and alcohol dehydrogenase.Typically,scanning electron microscope?SEM?,fourier transform infrared spectroscopy?FT-IR?,X-ray diffractometry?XRD?and other instruments were used to characterize the obtained enzyme-calcium phosphate composite hybrid nanoflowers.The catalytic properties of immobilized enzymes prepared using the two kind of support,organic and inorganic,were investigated and examined,respectively.The two immobilized enzyme prepparations were also reused to catalyze the synthesis of crizotinib intermediate?S?-2,6-dichloro-3-fluorophenylethanol.Firstly,the foreign genes akr and adh for aldo-keto reductase?AKR?and alcohol dehydrogenase?ADH?were expressed in E.coli.,and the protein expression conditions were optimized.The final expression temperature was 23�C and the rotation speed was 220 rpm for AKR.The final concentration of IPTG was 0.3 mM and the induction time was 15 h.The optimal expression conditions for ADH were18�C,220 rpm and a final concentration of 0.5 mM IPTG.After purification by nickel column,a certain amount of free AKR and ADH were obtained and used in the bi-enzymatic coupling catalytic synthesis of target product.Under the optimal conditions using free enzymes,the yields and enantioselectivity?ee value?could reach40%and 99.98%,respectively.However,due to the extremely poor thermal stability of free enzymes,especially ADH,even at 30�C for 8 h,ADH and AKR lose half of their activities within 8 h.While catalyzing at higher temperature 60�C,both enzymes lost all of their activities and no product was observed by HPLC.Secondly,using the epoxy resin support,we could obtain the co-immobilized AKR and ADH with better thermal stability and reusability stability.The recovery rate of the enzymes activity could reach 80%or more.In addition,after the temperature is kept at 60�C for 8 hours,the epoxy resin immobilized enzyme still maintains 50%of the initial activity and only 20%of the free enzyme.Cyclic catalysis results show that the epoxy resin immobilized enzyme can guarantee more than 70%of the catalytic efficiency in 5 catalytic cycles.However,the yield of the epoxy resin immobilized enzyme decreased by 58%in the sixth catalytic cycle,and the stability was lacking,which may be related to the property of the epoxy resin carrier as an organic polymer carrier and the randomness of covalent attachment of the enzyme to the carrier related.In view of this,based on the biomineralization theory,the inorganic phosphate and the enzyme protein are co-crystallized to prepare the enzyme-calcium phosphate composite nanoflower,thereby realizing the immobilization of the enzyme in the inorganic carrier.After immobilization of keto-reductase?AKR?and alcohol dehydrogenase?ADH?by this method,the activity of both was significantly improved.Compared with the free enzyme activity of AKR and ADH,the specific activity of nanoflower immobilized enzyme?U/mg?increased by 3.3 times and 2.1 times respectively.In addition,the thermal stability of the immobilized enzyme was also significantly improved:after incubation for 8 hours at 60�C,the residual activity of the two enzymes in the enzyme-calcium phosphate composite nanoflower exceeded 80%of the initial activity,while the free enzyme only 20%of the initial activity was retained.The nanoflowers were analyzed by X-ray diffractometry and found to have a main structure of CaHPO₄and its derivatives.Analysis of nanoflowers by Fourier transform infrared spectroscopy revealed that the enzymes showed no change in the amide I band and the II band of the active center before and after immobilization,meaning that the structure of the enzyme did not change substantially.Finally,we used a scanning electron microscope nanoflower to analyze and confirm that its size and structure are similar to those of the previously reported structure.Therefore,this study considers that the immobilization method of enzyme-calcium phosphate crystals is effective.Finally,this paper focuses on the catalytic performance of enzyme-calcium phosphate composite nanoflowers.It was found that when the immobilization ratio of AKR and ADH in the enzyme crystal was 3:1,the highest catalytic activity for the synthesis of chiral ethanol,the catalytic yield was as high as 90.8%,and the final product?S?-2,6-dichloro-3-fluorophenylethanol showed a high and an ee value of99.98%.And the batch catalysis experiments of nanoflower immobilized enzymes proved that all of the initial activities were basically maintained within 16 cycles?12 h each cycle?,and this later planned amplification experiment laid a good foundation.In conclusion,based on the successful coupling of ADH and AKR double enzyme free enzyme to the catalytic synthesis of?S?-2,6-dichloro-3-fluorophenylethanol,the epoxy resin immobilization method and the enzyme-calcium phosphate composite nanometer.The nanoflower immobilization method was introduced into the immobilization process,and both methods made the stability of the enzyme protein significantly improved,and the latter advantage was more obvious.The enzyme-phosphate complex nanoflower immobilized enzyme with high activity,high stability and high environmental tolerance is expected to alleviate the high pollution of chemical synthesis and the high cost of free enzyme catalysis in the catalytic synthesis of target chiral intermediates.At the same time,the comparison of the immobilization methods of epoxy resin immobilization method and enzyme-calcium phosphate composite nanoflower can further enrich the theoretical and applied research of enzyme immobilization and catalysis.