Construction Of Cascade Catalytic System Based On Old Yellow Enzyme And Its Catalytic Asymmetric Reduction Of Conjugated Enones

Old yellow enzyme is a class of NAD（P）H-dependent oxidoreductases that can catalyze the asymmetric hydrogenation ofα,β-unsaturated aldehydes,ketones,esters,nitrile and nitro compounds and their derivatives.The reduction reaction of the C=C bond can generate up to two chiral centers and is widely used in the production of fine chemicals and pharmaceuticals.The application of these enzymes is often limited by the expensive coenzyme NAD（P）H,which can be regenerated by coupling with glucose dehydrogenase.In this paper,a coenzyme regeneration system and a multi-enzyme cascade catalytic system were constructed based on the old yellow enzyme to catalyze the asymmetric reduction of enone to synthesize（3S）-3-methylcyclohexanone and trans（1S,3S）-3-methylcyclohexanol,respectively.At the same time,in order to solve the problems of poor stability of free enzymes and difficult to reuse,the immobilized enzyme technology was applied.Combined with IMAC technology,the prepared carrier of SiO₂were modified by Ni-NTA,through the affinity between His-tag and Ni²⁺realizes one-step separation,purification and immobilization of the enzymes with His-tag.The main research contents are as follows:（1）The gene sequence of old yellow enzyme 1（OYE1）derived from Saccharomyces pastorianus was obtained from the gene bank,and the gene recombination system was constructed with p ET-28b（+）as the expression vector and E.coli BL21（DE3）as the expression host strains,and then verified the successful construction of recombinant plasmids and the soluble expression of the target protein,respectively.The fermentation conditions were optimized as inoculated into LB liquid medium containing kanamycin（final concentration of 50μg/m L）,1%inoculum volume,cultured at 37°C until the OD₆₀₀value was 0.6,0.4m M inducer isopropyl-β-D-thiogalactoside（IPTG）was added,and induced for another18 hours,the enzyme activity of OYE1 was up to 462.68 U/g_protein.The fermentation broth containing OYE1 was further separated and purified,and the enzymatic properties of OYE1 were explored.The optimum temperature was 35°C and the optimum p H was 9.5.（2）SiO₂ nanoflowers with uniform morphology and particle size were prepared and then functionalized with Ni-NTA to obtain functionalized immobilized enzyme carrier Ni-NTA@SiO₂.SDS-PAGE experiments showed that Ni-NTA@SiO₂had good selectivity to His-tag target proteins OYE1 and GDH.Subsequently,the immobilized enzymes（OYE1&GDH@Ni-NTA@SiO₂）were used to catalyze the asymmetric hydrogenation of3-methyl-2-cyclohexen-1-one in phosphate buffer at p H 7.5.Adding concentration of 0.2m M of NADP⁺,20 m M of glucose and 10 m M of substrate,and cultured at 30°C for 24 h,the conversion and selectivity of 3-methyl-2-cyclohexen-1-one were both>99%.In addition,OYE1&GDH@Ni-NTA@SiO₂exhibited better thermal stability,p H stability,storage stability and reusability than free enzymes.After the OYE1&GDH@Ni-NTA@SiO₂were reused 7 times,the conversion of3-methyl-2-cyclohexen-1-one could still be maintained at 46.02%.（3）A multi-enzyme one-pot cascade catalytic system was developed to catalyze the3-methyl-2-cyclohexen-1-one to high value-added chiral alcohol compounds using the old yellow enzyme and alcohol dehydrogenase（ADH-A）cascade reaction,and coupled with GDH to achieve in situ regeneration of coenzymes（NADPH and NADH）.Biocatalyst OYE1&ADH-A&GDH@Ni-NTA@SiO₂catalyze the asymmetric reduction of3-methyl-2-cyclohexen-1-one to trans（1S,3S）-3-methylcyclohexanol in a"one-pot"reaction.After optimizing the immobilization and reaction conditions,the yield of trans-（1S,3S）-3-methylcyclohexanol could be as high as 92.37%（ee>99%）.Compared with free enzymes,OYE1&ADH-A&GDH@Ni-NTA@SiO₂improved the thermal stability,p H stability,storage stability and reusability of the enzymes.After 5 consecutive cycles,the yield of trans（1S,3S）-3-methylcyclohexanol remained at 45.27%and exhibited high stereoselectivity（ee>99%）.In addition,it is further confirmed that the biocatalyst OYE1&ADH-A&GDH@Ni-NTA@SiO₂has good catalytic performance for cyclic,chain and aromatic ketenes.