Expression,Characterization And Application Of The Prenyltransferase NovQ

Prenyltransferase is a type of key enzyme involved in the synthesis of prenylated active compounds,which can be divided into membrane-bound and soluble types according to its existing form.The catalytic process of soluble prenyltransferase does not need to be attached to the biofilm,so it has a broader application prospect.The soluble prenyltransferase is mostly derived from Streptomyces.However,there will be some shortcomings after heterologous expression,such as low solubility,weak catalytic activity and poor resistance,especially in the synthesis process of high-value chemicals.E.coli has become a popular heterologous expression strain,which possesses remarkable preponderance such as fast bacterial growth,high protein expression,mild fermentation conditions and clear genetic background.In this paper,the soluble prenyltransferase NovQ derived from Streptomyces niveus is used as the research object,and E.coli as the host bacteria for the high-efficiency heterologous soluble expression;chemical synthesis method was used to synthesize enzymatic substrates;the activity of NovQ was improved by site-directed mutation and immobilization,which laid the foundation for further research.By optimizing the types of solubilizing tags,solubilizing expression methods,induced expression conditions and protein purification parameters,a simple and fast method for obtaining soluble NovQ pure enzyme was established.The results showed that the yield of MBP-NovQ expressed in E.coli Rosetta(DE3)was 140 mg/L after induction at 20? for 12 h,of which 86%were soluble components.Combining Ni-NTA affinity chromatography and TEV protease digestion,the purity of the obtained label-free soluble NovQ is over 90%.Since the substrate catalyzed by NovQ in vitro is unstable,we obtained target product by the chemical-enzyme coupling method.The results showed that DMAPP and menadione hydroquinol with higher purity can be synthesized using dimethylallyl bromide and menadione as raw materials.The oxidation results showed that menadione hydroquinol can be completely oxidized after being exposed to air for 1 h.Original NovQ can synthesize the only product MK-1 in vitro,and the HPLC peak time of MK-1 is 4.9 min.In addition,the catalytic sample was tested and identified by HPLC,UV spectroscopy,LC-MS and 1H-NMR,proving that vitamin K2 homolog MK-1 was synthesized in vitro.The key active site Glu281 of NovQ was obtained by computer simulation experiments,and used as a mutational target.Relative activity of E281Q was 2.05 times than that of original NovQ.The optimal catalytic conditions were 32?,pH 8,Mg2+concentration of 2.5 mM,menadione hydroquinol concentration of 0.5 mM,and DMAPP concentration of 0.25 mM.Subsequently,engineering bacteria S2 was constructed by exogenous introduction of MVA pathway,which achieved the whole cell synthesis of MK-1 combining the addition of aromatic substrates.Under the same catalytic conditions,MVA-O was used as the induction medium,and the yield of NovQ protein was 61 mg/L.After 24 h induction,the yield of MK-1 is 4.7 mg/L.In the immobilization experiment,CMNs were prepared using the "three-step method",and NovQ was further adsorbed to obtain NCMNs.FI-RT,XRD,TGA,VSM,XPS,and SEM were used to characterize CMNs and NCMNs,confirming the successful immobilization of NovQ and unchanged chemical properties of Fe3O4.Enzymatic experiments show that the resistance and stability of immobilized NovQ have been improved.When the concentration of menadione hydroquinol is higher than 2 mM,the activity of immobilized NovQ is 1.45 times than that of free enzyme;when the concentration of DMAPP is 1.5 mM,the relative activity of immobilized NovQ is 53%,which is 4.8 times than that of free enzyme;after 40 days storage at 4?,the residual activity exceeds 65%;after repeated 7 and 10 cycles,the residual activity of immobilized NovQ was 81%and 64%,respectively.