Construction Of Recyclable Magnetic Bifunctional Biocatalysts And Application To Synthesis Of Chiral Alcohol

(R)-3-quinuclidinol is an important intermediate for the synthesis of chiral drugs such as revastatol,adiponium bromide and solifenacin.Due to its mild reaction conditions,environmental friendliness,and high selectivity,the biocatalytic method is widely used for the synthesis of?R?-3-quinuclidinol.When using the enzyme-coupled method,the carbonyl reduction and coenzyme regeneration processes are carried out in different microbe cells,so the substrate,product and coenzyme must be exchanged between cells,and diffused inside and outside the cells,leading to prolonged biotransformation time and low efficiency,which is not conducive to industrialization.In response to this defect,this project intends to construct a double-active center,bifunctional hybrid enzyme by DNA recombination and protein engineering technology,to achieve asymmetric carbonyl reduction and coenzyme regeneration followed by recycling coenzyme,and enhance bioconversion efficiency.Furthermore,a highly efficient,recyclable magnetic bifunctional biocatalyst was constructed by the affinity immobilization technology,and a new green biocatalytic process for asymmetric synthesis of?R?-3-quinuclidinol was developped.1.Construction of a bifunctional hybrid enzyme expression vector and induction of target enzyme expression.Two full-length gene sequencesencodingcarbonylreductase?MlQR?andglucose dehydrogenase?GDH?are totally synthesized and linked by a flexible linker by gene recombination technology.The mlg gene fragment was ligated into the vector pET28a to form a recombinant expression vector pET28a-mlg.The expression vector was transformed into competent cells,and the optimized expression conditions were 16?,0.2 mM IPTG,inducation expression for 36 h,obtaining a highly active,soluble and highly expressed bifunctional hybrid enzyme?MLG?.MS confirmed that the amino acid sequence of MLG matched well with the amino acid sequence in the protein database.The activity of MLG was determined,in which the activity of carbonyl reductase was 3678 U/g and the activity of coenzyme regenerating enzyme was 5070 U/g.The kinetic constant was determined.The K_m value of MLG to substrate 3-quinuclinone was 12.06mM,K_cat/K_m was 0.39;the K_m value of MLG to substrate glucose was1.62 mM,and K_cat/K_m was 6.08.2.Recombinant whole cell catalyzed asymmetric synthesis of?R?-3-quinuclidinol.The optimized biotransformation conditions were30?,pH 7-8,0.2 mM coenzyme,glucose?1.5 times substrate equivalent?,and substrate/recombinant cell mass ratio of 24:1.When the substrate 3-quinuclidinone loading was 486 g/L,the biotransformation time was 5.5 h,the conversion was 100%,the yield was 90%,the ee value was 100%by GC,and the space time yield was 1505.5 g L^-11 d^-1.3.Construction of a recyclable magnetic bifunctional biocatalyst.The magnetic Fe₃O₄ nanoparticles were synthesized by co-precipitation method,coupling the chelating agent NTA to the surface of the magnetic nanoparticles by silane,and then loading Ni²⁺to form nano-affinity magnetic beads.The histidine-tagged bifunctional hybrid enzyme is immobilized by Ni²⁺affinity to form a recyclable magnetic bifunctional biocatalyst.Magnetic dual-functional biocatalysts were characterized by Malvern particle size analyzer,SEM,FT-IR,TG,XRD and PPMS.The magnetic bifunctional biocatalyst has a size of 20-40 nm,saturation magnetization is 38.96 emu/g,inductive of superparamagnetic property.The enzymatic properties of the magnetic bifunctional biocatalyst were evaluated.The results showed that the carbonyl reductase?MlQR?activity was 2866 U/g and the glucose dehydrogenase?GDH?activity was6195 U/g.The maximum enzyme loading of nano-affinity magnetic beads was 70.6 mg/g.The recovery rates of carbonyl reductase and glucose dehydrogenase activities in immobilized enzyme MLG were77.99%and 122.18%,respectively.4.Application of magnetic bifunctional biocatalysts in the synthesis of?R?-3-quinuclidinol.The biotransformation conditions were 30?,pH8.0,coenzyme 0.2 mM,glucose?1.5 times substrate equivalent?,bifunctionalbiocatalystandsubstratemassratioof1:70,biotransformation time of 5.5 h.The conversion was determined to be100%,the yield was 90%,and the ee value was 100%.Based on the 1:40mass ratio of bifunctional biocatalyst to substrate for bioconversion,magnetic bifunctional biocatalyst can be recycled 11 times.The relative yield of the product obtained per gram of magnetic bifunctional biocatalyst reached 312.37g.