| Chiral amines are important intermediates for many compounds with the pharmaceutical or biological activity,thus their efficient and green synthetic routes have been a research focus in recent years.Continuous flow biocatalysis has many advantages,such as fast catalytic efficiency,high space-time yield,and it could also couple with downstream analysis and purification processes and has been identified as the foremost key green research areas for sustainable manufacturing from pharmaceutical and fine chemicals industries.In this project,S-selective ?-transaminase ATA-W12 with high activity and high enantioselectivity was covalently immobilized on heterofunctional epoxy resins,and a continuous flow biocatalysis system was developed using immobilized ATA-W12 in a packed bed reactor.The main research contents of this article are listed as follows:(1)Screening and amination modification of epoxy resins.Based on the evaluation indexes of enzyme activity recovery rate and binding efficiency,four epoxy resins were pre-screened,and the best epoxy carrier was determined to be 107s.The epoxy resin 107s was modified with EDA,and the results of the contact angle showed that the hydrophilicity of the carrier was enhanced after modification;the modification reaction reached equilibrium after 4 hours,and the amino modification density maximum was 70 ?mol/gcarrier.SEM results showed that the amino modification would not affect the structure and morphology of the carrier.(2)Optimization of immobilization conditions and research on the properties of immobilized enzymes.Optimized the immobilization conditions of ATA-W12 immobilized on EDA-Epoxy Supports(EES),determined the optimal amino modification density of the carrier was 20 ?mol/gcarrier,and the protein loading was 7.2 mgprotein/gcarrier,immobilization for 6 h.Optimized and determined the optimal reaction conditions for immobilized enzyme ATAW12@EES was 100 mM pH 8.0 phosphate buffer,37?.ATAW12@EES maintained about 89%residual activity after 30 days at 4?,and retained about 85%residual activity after 20 cycles of batch reaction.It showed stronger tolerance than free enzyme under multiple pH conditions.Compared with free enzymes,the stability of the immobilized enzymes was significantly enhanced,which laid the foundation for its large-scale application.(3)Development of continuous flow biocatalysis system.ATAW12@EES was packed into a glass column(length 10 cm,i.d.10 mm),and the conditions of the flow catalysis process were optimized.The optimal conditions were determined as follows:the flow direction was downward,the packing amount of immobilized biocatalysts was 2.5 g,and the flow rate was 0.4 mL/min.Under this condition,the continuous asymmetric synthesis of(S)-1-Boc-3-aminopiperidine was carried out,and the time-space yield reached as high as 930.73 g·L-1·d-1.After 24 hours of continuous operation,the reaction conversion rate maintained at more than 90%,providing feasibility support for the application of?-transaminase in the efficient and continuous production of(S)-1-Boc-3-aminopiperidine.(4)Co-immobilization of ATA-W12 and cofactor PLP.Due to the high price of PLP and the difficulty of follow-up separation,we studied the co-immobilization of ATA-W12 and PLP.The epoxy resin was modified with IDA and CoCl2 for directional immobilization and purification of His tagged ATA-W12,and then the co-immobilization of PLP was through functional group modified by EDA/eA/PEI.The PEI modified carrier had a PLP loading of 9.37 ?mol/g,the recovery rate of the obtained co-immobilized enzyme(ATAW12@Co2+/PEI)activity was 106%,and about 90%of the residual enzyme activity was retained after 30 cycles of reuse.ATAW12@Co2+/PEI carried out the continuous deamination reaction of S(-)-?-methylbenzylamine at the flow rates of 0.5 mL/min and 1.0 mL/min.After continuous working for about 8 h and 4 h,respectively,there was no obvious loss of enzyme activity.There was no exogenous addition of cofactors during the reaction,which greatly reduced the economic cost. |
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