Study On The Immobilization Of Carbonyl Reductase By PHA Magnetic Nanoparticles And Its Biocatalytic Properties

Chiral alcohol drugs play an important role in the fields of medical and health,biomedicine,and life and health due to their unique medicinal effects.Its preparation method has also become the focus of drug research.Tolvaptan（TVP）is a non-peptide vasopressin V2receptor antagonist which is of highly selective,and orally effective for the treatment of hyponatremia in heart failure.The optically pure tolvaptan is of less toxic,side effects to human body and higher metabolic stability.Optically pure tolvaptan was synthesized by asymmetric reduction of 7-chloro-1-[2-methyl-4-[（2-methylbenzoyl）amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1h-1-benzazepine（PK1）by biological method.It has the advantages of high stereoselectivity,environmental friendliness and low energy consumption.However,only S-TVP has been synthesized by biocatalysis,and no R-TVP has been reported.Therefore,in this study,to establish a biocatalytic synthesis route for R-TVP,a carbonyl reductase（RLSR5）with R-TVP synthesis ability was screened from rabbit livers through protein purification technology and genetic engineering methods.In order to further enhance the stability and reproducibility of free enzymes,polyhydroxyalkanoate（PHA）magnetic nanospheres immobilized RLSR5 was successfully synthesized to achieve the efficient synthesis of chiral tolvaptan.The main results are as follows:（1）The active crude protein and protein molecular weight of 23-53 kDa was isolated and purified from rabbit liver crude enzyme by ammonium sulfate fractional precipitation,DEAE weak anion exchange chromatography,Hi Trap Sephadex 6B molecular exclusion chromatography,ultrafiltration centrifugal interception and other methods to separate.The crude protein was detected by protein mass spectrometry,and the results were compared with the NCBI database to obtain 7 carbonyl reductases gene sequences.Primer Premier 5 was used to design primers.The carbonyl reductase gene fragment was amplified by PCR using rabbit liver genome as template,and three recombinant expression strains were successfully constructed.The whole cell catalytic ability of recombinant strain for PK1 was detected by HPLC,and the carbonyl reductase RLSR5 with higher catalytic ability was screened out.（2）The recombinant strain E.coli BL21（DE3）/p ET28a-RLSR5 was induced to express and a single carbonyl reductase RLSR5 with a molecular weight of 40 kDa was purified by Ni column affinity.The enzyme activity was 2.12 U,and the specific enzyme activity was26.5 U/m L.The coenzyme dependence,the effect of reaction temperature,pH,and metal ions on the enzymatic properties of carbonyl reductase were studied.It was concluded that the enzyme was NADPH-dependent,the optimal reaction temperature was 30°C,and the optimal reaction pH was 6.0.The enzyme maintains its maximum activity at a temperature of20°C and has better stability in a buffer system with a pH of 7.0.The 2m M metal ion influence experiment showed that the introduction of Zn²⁺ions inhibited the enzyme activity,and Mg²⁺had the strongest promotion effect on the enzyme activity.（3）Using poly（3-hydroxybutyrate-co-3-hydroxyhexanoate）（PHBHHx）and Fe₃O₄nanoparticles as raw materials,PHA magnetic nanoparticles with smooth surface,spherical shape and particle size of 500 nm were synthesized by solvent evaporation method.Based on the fact that the binding protein PhaP has strong adsorption capacity with PHA magnetic nanospheres surface,the fusion protein PhaP-linker-RLSR5 was immobilized on the surface of PHA magnetic nanospheres with a loading capacity of 203.5 mg/g.When the substrate concentration was 2.23 m M and the reaction time was 12 h at 37℃,the conversion of R-TVP was 97%and the e.e.value was 99%.Compared with the free enzyme,the immobilized enzyme had the best stability in pH 8.0 buffer system,maintained high activity and improved RLSR5 alkali resistance.The stability of the enzyme was good at 30℃,and the remaining enzyme activity reached 93%.After repeated use for 10 times,the immobilized carbonyl reductase still maintained 78.62%of the remaining enzyme activity.This immobilization method effectively improved the disadvantages of poor stability,poor tolerance and non-reusability of free enzyme.