| Chiral epoxides are important building blocks for the synthesis of many medicines,pesticides and fine chemicals.For a long time,the development of efficient and green chiral epoxides synthesis methods has been a hot spot in synthetic methodology research.Asymmetric epoxidation of alkenes is the most effective method for preparing chiral epoxides.So far,many methods of chemical catalysis and enzymatic catalysis of asymmetric alkene epoxidation have been developed.Compared with chemical catalysis,enzyme catalysis has the advantages of mild reaction conditions,high catalytic efficiency,and high enantioselectivity,which is more in line with the current situation.The concept of "green" development.Styrene monooxygenase can catalyze the asymmetric epoxidation of styrene compounds to(S)-epoxides with high efficiency and high enantioselectivity.However,only six styrene monooxygenases can catalyze unconjugated terminal olefins.Symmetric epoxidation reactions,and these enzymatic reactions have either low selectivity or very poor activity,that is,no styrene monooxygenase can efficiently and highly enantioselectively catalyze the asymmetric epoxidation of unconjugated alkenes.In this paper,by mining the protein database,it is found that Hh APL annotated as alanine phosphoribitol ligase in Herbaspirillum huttiense has the ability to catalyze the epoxidation of allylbenzene,and the enzyme has excellent enantioselectivity and high catalytic activity in catalyzing the epoxidation reaction.,The protein was determined to be a new type of monooxygenase,named Herbaspirillum huttiense monooxygenase(HhMO).Through the optimization of the enzyme catalytic reaction system and the analysis of the catalytic specificity,an efficient styrene monooxygenase-catalyzed allylbenzene epoxidation reaction system was constructed.at the same time,this paper also found that HhMO catalyzes allyl benzene epoxidation.Grade alcohol epoxidation not only has high catalytic activity and excellent enantioselectivity,the catalytic reaction also has excellent diastereoselectivity.in addition,this paper also deeply analyzes the enzyme-catalyzed conjugated alkenes.It is found that HhMO catalyzed styrene and its derivatives also have high catalytic activity and excellent enantioselectivity.The main research conclusions are as follows:Firstly,based on the six styrene monooxygenases known to catalyze the epoxidation of unconjugated alkenes,their ancestor protein An Sty A was constructed.By characterizing the protein’s catalytic function,it was found that the ancestor protein catalyzed the asymmetry of allylbenzene epoxidation has only moderate enantioselectivity and high catalytic activity.Subsequently,the sequence alignment analysis in the NCBI protein database with An Sty A as the reference sequence,integrated the functions of the six known styrene monooxygenases in motif analysis,three candidate proteins with 30-50% similarity to An Sty A and different numbers of functional motifs were selected.the E.coli overexpression system of these three proteins was constructed and used as a catalyst to use allyl as a screening substrate,it was found that Hh APL from Herbaspirillum huttiense,annotated as Alanine Phosphoribitol Ligase,has excellent enantioselectivity and high catalytic activity for catalyzing allylbenzene.Based on the protein’s function as a monooxygenase,the Hh APL has excellent enantioselectivity and high catalytic activity.Named Herbaspirillum huttiense monooxygenase(HhMO).In order to construct an efficient HhMO-catalyzed allylbenzene epoxidation reaction system,this thesis conducted an in-depth exploration of the temperature,pH,and substrate cosolvent in the HhMO-catalyzed allylbenzene epoxidation system,and determined that the enzyme catalyzed The best reaction conditions for allylbenzene epoxidation are: potassium phosphate buffer 20 m L(0.1 M,pH 6.0),cell mass 2 g,substrate 10 mg,n-octane 5%(v/v),37 After reacting at ℃ for 24 hours,100%substrate conversion was achieved,and allyl benzene epoxides with >99% ee was obtained.Finally,an efficient enzyme-catalyzed allyl benzene epoxidation reaction system was established.In order to study the specificity of the substrate catalyzed by the enzyme,the type of substrate used for the epoxidation of unconjugated alkenes catalyzed by HhMO was analyzed in depth.HhMO catalyzes the epoxidation of allylbenzene and its derivatives with excellent enantioselectivity and high catalytic activity.the steric hindrance and electronic effects of the substituent groups on the substrate benzene ring have the enantioselectivity and the enantioselectivity of the enzyme catalyzed The catalytic activity has a greater impact.The enzyme catalyzes the substrates substituted at the meta or para position on the benzene ring with higher enantioselectivity and catalytic activity,and has higher catalytic activity for electron-rich alkenes,but for poor electronic alkenes do not have catalytic activity.in addition,HhMO catalyzes the epoxidation of allyl secondary alcohols with excellent enantioselectivity(>99% ee)and high catalytic activity(50% Yield),and the process also has very excellent diastereoselectivity(>99% de).HhMO can completely convert5 mg(S)-allyl secondary alcohol into the corresponding epoxides product within 20 minutes,and(R)-Allyl secondary alcohol substrates will not be converted into products,and the kinetic resolution of racemic substrates will be completed to obtain(R,R)-glycidol(>99% ee,>99% de),shows that HhMO can specifically recognize(S)-allyl secondary alcohols.In addition,this thesis measured the kinetic parameters of the epoxidation of allyl secondary alcohols catalyzed by HhMO,and found that the enzyme catalyzed the epoxidation of allyl secondary alcohols has a lower Km.The specific parameters are: the Km value is 2.273 ± 0.679 μM,kcat value is 0.0182 ± 0.006 min-1.The research results show that HhMO has excellent enantioselectivity and high catalytic activity for the epoxidation of allylbenzene derivatives.At the same time,HhMO can also catalyze the epoxidation of allyl secondary alcohols and their derivatives with excellent enantioselectivity(>99% ee)and excellent diastereoselectivity(>99% de)and high catalytic activity.In order to explore the key amino acid sites of HhMO’s chiral hydroxyl recognition for allyl secondary alcohols,this thesis compares the amino acid sequences of HhMO and Sty AB2 to determine that Thr207 of HhMO corresponds to the para-allyl secondary in Sty AB2.Alcohol chiral hydroxyl has a recognition effect on Thr200.Through point mutation of Thr207 of HhMO and verification of its catalytic function,the results show that Thr207 of HhMO does not recognize the chiral hydroxyl of allyl secondary alcohols and may have may have very different substrate binding pockets with other known monooxygenases.In addition,this thesis also conducted an in-depth analysis of the characteristics of HhMO-catalyzed styrene monooxygenase natural substrate epoxidation,and found that the enzyme catalyzed styrene and its derivatives has a moderate or higher enantioselectivity.HhMO catalyzes styrene only with moderate selectivity(80% ee)and low catalytic activity(25% Yield),but the enzyme catalyzes styrene such as methyl and methoxy substituted at the para and meta positions on the benzene ring When it is a derivative,it exhibits excellent enantioselectivity(>99% ee)and high catalytic activity(70% Yield).in addition,HhMO-catalyzed styrene epoxidation is also affected by the substituted groups on the benzene ring of the substrate.The steric hindrance and electronic effects of HhMO catalyzed by the meta-and para-substituted styrenes on the benzene ring have higher enantioselectivity and catalytic activity than the ortho-substituted styrenes on the benzene ring.HhMO has higher catalytic activity for electron-rich styrenes,and no catalytic activity for electron-poor styrenes.In addition,this paper also deeply analyzes the kinetic characteristics of HhMO catalyzed epoxidation of styrene.The specific parameters are: Km value is0.154±0.032 m M,kcat value is 3.496 ± 0.006 min-1.The research results show that HhMO catalyzes the epoxidation of styrene and its derivatives with more than moderate enantioselectivity and high catalytic activity.In summary,this thesis has successfully screened a new enzyme HhMO that exhibits excellent enantioselectivity and high catalytic activity for both unconjugated alkenes and conjugated alkenes by mining the protein database.The HhMO shows high catalytic efficiency for the asymmetric epoxidation of allyl secondary alcohols.This method provides a green synthetic route for the production of valuable optically pure glycidol derivatives in the fine chemical and pharmaceutical industries. |
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