CN114836486B

CN114836486B - Method for synthesizing chiral beta-amino alcohol by enzyme catalysis

Info

Publication number: CN114836486B
Application number: CN202210565012.3A
Authority: CN
Inventors: 李爱涛; 王斌举; 廖浪星; 胡瑞文; 龚安界; 叶龙武; 郑颜欣; 吴鹏
Original assignee: Hubei University; Xiamen University
Current assignee: Hubei University; Xiamen University
Priority date: 2022-05-23
Filing date: 2022-05-23
Publication date: 2023-05-12
Anticipated expiration: 2042-05-23
Also published as: CN114836486A

Abstract

The invention provides a method for preparing chiral beta-amino alcohol by utilizing monooxygenase catalysis, which comprises the following steps: asymmetric alkene and amine are used as substrates, and monooxygenase is used for catalyzing amino hydroxylation reaction to obtain chiral beta-amino alcohol.

Description

Method for synthesizing chiral beta-amino alcohol by enzyme catalysis

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing chiral beta-amino alcohol by catalyzing asymmetric olefin and amine to generate amino hydroxylation reaction by utilizing monooxygenase.

Background

Beta-amino alcohol is an organic synthesis intermediate with wide application, chiral beta-amino alcohol can be used for preparing a plurality of important compounds, such as chiral medicines, amino acids, chiral auxiliary agents and the like, plays a very important role in medicinal chemistry and biology, and is widely applied to a plurality of clinical medicines, such as antihypertensive medicines, antidiabetic medicines, antiasthmatic medicines, antimalarial medicines and the like, and contains beta-amino alcohol structural units. More than 75% of the organic molecules of the drug or drug intermediate contain amino functionality. The chiral amino alcohol with amino and hydroxyl functional groups shows good chiral inducibility in the field of asymmetric catalysis. Up to now, more than 300,000 compounds containing this unit in molecular structure have been reported, including more than 2000 natural compounds, more than 80 approved drugs and more than 100 candidate drugs (ghis lieri, d.; turner, n.j. Biocatanic Approaches to the Synthesis of Enantiomerically Pure Chiral amino s. Top. Catalyst. 2014,57, 284-300.). For example, (S) -2-amino-1-butanol is an intermediate in the synthesis of Ethambutol (EMB), an important antitubercular drug; indolin-2-one derivatives prepared with (S) -2-amino-2-phenylethanol as building blocks, which are inhibitors of P21-activated kinase (PAK 4); (S) -2-amino-2- (4-bromophenyl) ethanol is an intermediate for the synthesis of 4,4' -bis (biphenyl) -substituted bis (oxazoline) (BOX) ligands, one of the most widely studied ligands in asymmetric catalysis. Chiral beta-amino alcohols can also be used as enantioselective ligands for organometallic catalysts for the catalytic synthesis of some important optically active compounds. The chiral beta-amino alcohol has N atom and O atom with good coordination ability, can form a complex with a plurality of elements (such as B, li, zn and the like) to form a chiral catalyst with excellent performance, and has high stereoselectivity and catalytic activity.

Over the past decades, β -amino alcohols have been synthesized by organic chemical methods. One of the most practical and widely used methods for synthesizing beta-amino alcohols is the direct ammonolysis of epoxides with excess amine (Hanson, R.M. the Synthetic Methodology of Nonracemic Glycidol and Related 2,3-Epoxy alcohol. Chem. Rev.1991,91, 437-475.). In addition, various chemical methods using Lewis acids or different organic solvents have been developed to increase the efficiency of the ammonolysis reaction of epoxy compounds (Yamamoto, Y.; asao, N.; meguro, M.; tsukade, N.; nemoto, H.; adayari, N.; wilson, J.G.; nakamura, H. Regio-and Stereo-selective Ring Opening of Epoxides with Amide Cuprate Reagents.J.Chem.Soc., chem.Commun.1993,1201-1203.). However, these methods have some limitations such as high temperature conditions, the use of stoichiometric or moisture sensitive catalysts, and hazardous organic solvents. To solve these problems, a method of ammonolyzing an epoxide in water without using a catalyst and achieving a high yield has been developed (Azizi, n.; saidi, m.r. highly chemoselective addition of amines to epoxides in water. Org. Lett.2005,7 (17), 3649-3651.), but the method still relies on the use of a highly reactive epoxide, resulting in drawbacks in selectivity and late-stage applicability of the method. Furthermore, epoxides, especially chiral epoxides, are difficult to prepare, which often involves using metal catalysts under severe conditions, with unsatisfactory yields observed for many types of olefins (e.g., terminal olefins) (Zhang, w.; loebach, j.l.; wilson, s.r.; jacobsen, e.n. engineering epoxidation of unfunctionalized olefins catalyzed by salen manganese complexes.j. Am. Chem. Soc.1990,112, 2801.).

The chiral beta-amino alcohol chemical synthesis method in the prior art has the problems of complicated steps, poor operation safety, low product yield and the like, and the problems of poor selectivity and difficult preparation caused by the use of high-reactivity epoxide. In recent years, since enzymes are green catalysts, the reaction conditions are mild, and the possibility of preparing beta-amino alcohols including chiral beta-amino alcohols by biocatalysis has been explored. Patent document CN109706194a reports that lipase RM IM catalyzes the ring-opening reaction of aniline compounds and styrene oxide to synthesize phenethyl alcohol type β -amino alcohols, CN109735582a reports that lipase RM IM catalyzes the ring-opening reaction of aniline compounds and cyclohexene oxide to synthesize cyclohexenyl alcohol type β -amino alcohols, but the above synthesized phenethyl alcohol type β -amino alcohols and cyclohexanol type β -amino alcohols are all racemates. CN110172484a reports a method for preparing chiral beta-amino alcohol by cascade biocatalysis of alkene asymmetric amine hydroxylation, which requires combined catalysis by using 4 enzymes of alkene monooxygenase SMO, epoxide hydrolase EH, alcohol dehydrogenase ADH and transaminase TA, which requires that the enzyme dosage and enzyme activity of the 4 enzymes are well matched so as not to cause waste of expensive enzyme preparation and overhigh economic cost, and requires that the stereospecificity of alkene monooxygenase, epoxide hydrolase and alcohol dehydrogenase be consistent, and is (R) -selectivity or (S) -selectivity, and even requires that substrates such as amino donor are chiral compound (R) -1-phenethylamine, which is a possibility that the technical scheme does not have industrial application due to extremely severe material conditions.

There is therefore a need for a green, safe and efficient process for the preparation of highly enantioselective beta-amino alcohols, with reduced production costs and economic utility.

Disclosure of Invention

The inventor explores the industrial way of preparing chiral beta-amino alcohol by a biocatalysis method, and another way is to try to catalyze asymmetric hydroxylation reaction of asymmetric olefin and amine by singly adopting monooxygenase, and synthesize chiral beta-amino alcohol in a one-pot one-step mode. It has also been unexpectedly found that different species of monooxygenases have different stereoselectivity, giving optically active β -aminoalcohol enantiomers in either the (R) -or (S) -configuration, respectively. Specifically, the invention comprises the following technical scheme.

A method for preparing chiral beta-amino alcohol by enzyme catalysis, which comprises the following steps: asymmetric alkene and amine are used as substrates, and a monooxygenase is used for catalyzing an asymmetric amino hydroxylation reaction to obtain chiral beta-amino alcohol.

The term "asymmetric" refers to an olefin having a c=c double bond in its molecular structure and asymmetric structures on both sides, such as propylene, 1-butene, styrene, etc.

The asymmetric olefin beta-amino alcohol can be 2-phenyl-2- (N-phenyl) amino ethanol or 2-substituted phenyl-2- (N-phenyl) amino ethanol, and the steps are as follows:

using styrene/substituted styrene shown in a formula I and aniline as substrates, and catalyzing asymmetric amino hydroxylation reaction by monooxygenase to obtain (S) -configuration beta-amino alcohol shown in a formula II or (R) -configuration beta-amino alcohol shown in a formula III:

wherein R is selected from H, halogen and C1-C4 alkyl. When R is a substituent halogen or C1-C4 alkyl, it may be in the ortho, meta or para position.

In the above method, the monooxygenase used for catalyzing and preparing the (S) -configuration beta-amino alcohol shown in the formula II is wild-type cytochrome P450-BM3 WT with the amino acid sequence of SEQ ID NO. 1, cytochrome mutant P450-BM3F87G with the amino acid sequence of SEQ ID NO. 2, or cytochrome P450pyrTM with the amino acid sequence of SEQ ID NO. 3; the enzyme used for catalyzing and preparing the (R) -configuration beta-amino alcohol shown in III is ethylene monooxygenase styAB with amino acid sequences of SEQ ID NO. 4 and SEQ ID NO. 5, wherein styA is SEQ ID NO. 4 and styB is SEQ ID NO. 5, namely the ethylene monooxygenase styAB consists of two polypeptides styA and styB.

Wherein the wild-type cytochrome P450-BM3 is derived from Bacillus megaterium (Bacillus megaterium), NCBI accession number P14779.2, described in literature (K.L.Tee, U.Schwaneberg, angew.Chem.Int.Ed.2006,45, 5380-5383); the cytochrome P450pyrTM is derived from Sphingomonas sp.HXN-200, described in literature (W.Zhang, W.L.Tang, Z.S.Wang and Z.Li, adv.Synth.Catal.2010,352,3380.A.Li, J.Liu, S.Q.Pham, Z.Li, chem.Commun.2013,9, 11572-11574.); the ethylene monooxygenase styAB is derived from Pseudomonas sp.strain VLB120, and NCBI accession numbers are StyA: WP_019436615.1, styB: WP_019436616.1, described in literature (S.Panke, B.Witholt, A.Schmid, M.G.Wubbolts, appl.Environ.Microbiol.1998,64, 2032-2043.).

The halogen is selected from fluorine F and chlorine Cl.

The C1-C4 alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, preferably methyl or ethyl, more preferably methyl.

When the substituent R is selected from halogen and C1-C4 alkyl, R is preferably located at the para position, namely the olefin shown in the formula I is para-substituted styrene, and the reaction is as follows:

for example, the para-substituted styrene may be selected from 4-fluoro-styrene, 4-chlorostyrene, 4-methyl-styrene.

In the reaction system, the monooxygenase can be in the form of enzyme or in the form of an expressed microorganism thallus thereof.

The enzyme is in the form of free enzyme or immobilized enzyme.

The microorganism can be selected from Bacillus subtilis, lactobacillus brevis, escherichia coli, candida magnolia, pichia pastoris, and Saccharomyces cerevisiae. Preferably, the microorganism is E.coli, for example E.coli BL21 (DE 3).

When the monooxygenase is in the form of microbial cells, glucose is also added into the reaction system as an energy source for NADPH regeneration, and the cyclic regeneration of the cofactor can be realized without externally adding NADPH.

The pH of the reaction system may be from 6.5 to 9.5, preferably from pH7.0 to 8.8, for example from pH7.4 to 8.5; the reaction temperature may be 25-45 ℃.

The reaction system may be phosphate buffer.

Compared with the cascade multienzyme catalysis in the prior art, the chiral beta-amino alcohol synthesis process provided by the invention can be completed by catalyzing only one monooxygenase, has no strict requirements on material conditions, does not require substrates and is also chiral compound, so that the process is simplified, the reaction conditions are easy to control, and the production cost is also obviously reduced. Meanwhile, the chiral 2-phenyl-2- (N-phenyl) amino ethanol and the 2-substituted phenyl-2- (N-phenyl) amino ethanol prepared by the method have high optical purity, the ee value reaches more than 90%, and the method has industrial development potential.

Drawings

FIG. 1 shows a graph of a mixed cluster continuum spectrum (HCC) model of asymmetric epoxidation and ammonolysis mechanisms in the reaction of styrene with aniline using an olefin monooxygenase in accordance with the present invention. Density Functional Theory (DFT) calculations using Gaussian 16 software, geometry optimization and frequency calculations were performed in combination with SMD continuous solvation models at the theoretical level of B3LYP [8-10]/6-31G (d), using water as the solvent; the dispersion correction calculated using the D3 method of grime is contained in all calculated species; further refinement of energy for all atoms using the larger basal group 6-311++ G, the final gibbs free energy included B3LYP/6-311++ G levels of electron energy and B3LYP/6-31G (d) levels of entropy correction. Wherein 1d is by-product 1-phenyl-2- (N-phenyl) aminoethanol, RC is the reactant complex, TS1 is transition state 1, TS1a is transition state 1a, IC1 is intermediate complex 1, and IC1a is intermediate complex 1a. By analogy, TS represents the transition state and IC represents the intermediate complex. The complexing heat absorption of (R) -styrene oxide and aniline was 5.2kcal/mol, resulting in reactant complex RC. Starting from RC, two competing pathways may occur: one is that the amino group of aniline nucleophilically attacks the C1 site of the epoxide through TS1a (red), proceeding to the (S) -1d pathway; the other is the attack of the amino group of the aniline on the C2 site of the epoxide, which goes to the (S) -1C path by the TS1 (black) reaction. Both routes will provide zwitterionic intermediates IC1a/IC1, in which the anion O is stabilised by a strong H bond with adjacent water, whereas the positive charge of the amino group can be delocalised over the phenyl group; the anion O can then extract a proton from the adjacent water (IC1a→T2a/IC1→T2), which involves a slight barrier of about 1kcal/mol and results in strong H-bonding of the transient OH-intermediate to the surrounding water; subsequent proton transfer from positively charged amino groups to OH-is also easy (IC2→Ts3/IC2a→T3a) to form the products of β -aminoalcohol (S) -1c or (S) -1 d. Examination of the free energy curve shows that the step of determination is a nucleophilic attack step, TS1 has an energy 2.7kcal/mol lower than TS1a, and the reaction will result in the main product of beta-amino alcohol (S) -1 c; likewise, the reaction of (S) -styrene oxide with aniline will give the main product of (R) -1 c. The results indicate that the chirality of amino alcohol is determined by the chirality of styrene oxide and that water plays a key catalytic role in mediating the ammonolysis of styrene oxide and aniline. FIG. 1 is the Gibbs spectrum (kcal/mol) of aniline added to (S) -styrene oxide.

FIG. 2 is an HPLC chromatogram of a monooxygenase expressing strain, E.coli (P450-BM 3F 87G) or E.coli (styrAB) catalyzing the production of chiral β -amino alcohols from styrene and aniline. Wherein, a, (R) -1c (i.e., (R) -2-phenyl-2- (N-phenyl) aminoethanol); B. standard for (S) -1c (i.e., (S) -2-phenyl-2- (N-phenyl) aminoethanol); C. coli (P450-BM 3F 87G) catalysis product; D. coli (styrb) catalyzed products; b. and (3) aniline.

FIG. 3 shows the product (R) -1c ¹ H NMR(500MHz,CDCl ₃ ) A spectrogram.

FIG. 4 shows the product (R) -1c ¹³ C NMR(125MHz,CDCl ₃ ) A spectrogram.

FIG. 5 shows the product (S) -1c ¹ H NMR(500MHz,CDCl ₃ ) A spectrogram.

FIG. 6 shows the product (S) -1c ¹³ C NMR(125MHz,CDCl ₃ ) A spectrogram.

Detailed Description

The invention uses monooxygenase as one enzyme to catalyze the asymmetric amino hydroxylation reaction of styrene or para-substituted styrene and aniline, thereby synthesizing enantioselective beta-amino alcohol. Monooxygenases of different origins have respectively different characteristics for catalyzing the reaction of substrate compounds, since enzyme proteins generally have tertiary or quaternary structures, are selective for the size and structure of substrate molecules, and many enzymes are also stereospecific. For amino hydroxylation reaction products with chiral centers, the enzyme differences may also be reflected in chiral differences (R-configuration or S-configuration) and optically active purity of the catalytic products, such as high optically active purity of the catalytic products of enzymes with high stereoselectivity, but many of the same enzymes tend to be racemates due to low or even no stereoselectivity, i.e. the high optically active purity of the catalytic reduction products is very low or even absent.

It was found experimentally that the stereoselectivity of the wild type cytochrome P450-BM3 WT with amino acid sequence SEQ ID NO. 1, the cytochrome mutant P450-BM3F87G with amino acid sequence SEQ ID NO. 2, or the cytochrome P450pyrTM with amino acid sequence SEQ ID NO. 3 was identical, contrary to the stereoselectivity of the ethylenemonooxygenase styrAB with amino acid sequence SEQ ID NO. 4, resulting in a conformational difference of the product beta-amino alcohols (compounds II and III). FIG. 1 is a mixed cluster continuum (HCC) model we calculate the proposed mechanism of asymmetric epoxidation and ammonolysis of olefin monooxygenase styrenes in the reaction of styrenes with anilines. The alkene monooxygenase styAB performs an asymmetric epoxidation of the alkene substrate styrene, followed by ammonolysis of the nascent epoxide intermediate with aniline in aqueous solution. The mixed cluster continuum (HCC) model reveals the key role of epoxide and aniline in mediating this reaction by the mechanism of ammonolysis of water.

Compared with the traditional method for synthesizing the beta-amino alcohol by directly ammonolyzing the epoxide by using excessive amine, which has the problem that chiral epoxide is difficult to prepare, the biocatalysis method for synthesizing the enantiomerically pure beta-amino alcohol by catalyzing styrene or para-substituted styrene and aniline through monooxygenase has high yield and enantioselectivity, and simultaneously, the substrate is cheap and easy to obtain, and the raw material cost can be greatly reduced, so that the method is better used in the industrial production of the enantiomerically pure (S) -type or (R) -type beta-amino alcohol (compounds II and III).

The term "compound represented by formula X" is sometimes expressed herein as "formula X", "bottom (product) X" or "compound X", as will be understood by those skilled in the art. For example, both the compound of formula I and the substrate I refer to the same compound olefin.

As regards the different reaction products of R in the formulae II or III, for simplicity of description, the product of the styrene and aniline synthesis, β -aminoalcohol, 2-phenyl-2- (N-phenyl) aminoethanol, can be described herein as 1c; the product of the synthesis of 4-fluorostyrene and aniline, beta-amino alcohol, 2- (4-fluorophenyl) -2- (N-phenyl) aminoethanol, is described as 2c; the product of the synthesis of 4-chlorostyrene and aniline, beta-amino alcohol, 2- (4-chlorophenyl) -2- (N-phenyl) aminoethanol, was described as 3c; the product of the synthesis of 4-methylstyrene and aniline, β -aminoalcohol, 2- (4-methylphenyl) -2- (N-phenyl) aminoethanol, is described as 4c.

Correspondingly, for simplicity of description, the reaction substrates styrene, 4-fluorostyrene, 4-chlorostyrene and 4-methylstyrene may be described herein as 1a, 2a, 3a and 4a, respectively.

Monooxygenase P450-BM3 WT, P450-BM3F87G, P pyrTM and ethylene monooxygenase styAB can be obtained by fermentation methods of genetically engineered bacteria, and based on their amino acid sequences, the coding genes thereof, expression cassettes and plasmids containing these genes, and transformants containing the plasmids can be readily obtained by the skilled artisan.

These genes, expression cassettes, plasmids, transformants can be readily obtained by means of genetic engineering construction methods known to those skilled in the art.

In a reaction system for catalyzing the asymmetric amino hydroxylation of the substrate alkene and amine, the monooxygenase can be in the form of enzyme or in the form of thalli. The enzyme forms include free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier immobilized enzyme, etc.; the forms of the bacterial cells include viable bacterial cells and dead bacterial cells.

Compared with the free enzyme method, the immobilized enzyme technology has the advantages of simplified production process, improved production efficiency and the like. Meanwhile, as the enzyme can be used for multiple times and the stability of the enzyme is improved, the productivity of unit enzyme is effectively improved; and secondly, the immobilized enzyme is easy to separate from a substrate and a product, so that the purification process is simplified, the yield is higher, and the product quality is better.

Those skilled in the art will readily understand that the bacterial cells themselves are a natural enzyme-immobilized form and can be used as an enzyme preparation for catalytic reactions without the need for disruption, or even extraction and purification. Since the reaction substrate and the reaction product can conveniently pass through the biological barrier-cell membrane of the cell, the cell does not need to be subjected to disruption treatment, which is economically advantageous.

On the other hand, compared with the catalysis of the separated enzyme, the invention can continuously and inexhaustibly provide the enzyme or supply by utilizing the simple fermentation of the microorganism, does not need the operations of further extracting, purifying and separating the enzyme and the like, has obvious economic benefit and creates conditions for industrial application.

In one embodiment, recombinant E.coli (P450-BM 3 WT), E.coli (P450-BM 3F 87G), E.coli (P450 pyr), E.coli (styrAB) and E.coli (styrAB) expressing cytochrome P450-BM3 WT monooxygenase, mutant P450-BM3F87G, monooxygenase P450pyr or ethylenemonooxygenase styAB can be directly used as biocatalysts to catalyze the reaction of styrene/substituted styrene and aniline in phosphate buffer with glucose as energy source to produce beta-amino alcohol with S-configuration or R-configuration. For example, the specific steps are:

(1) Recombinant strains E.coli (P450-BM 3 WT), E.coli (P450-BM 3F 87G), E.coli (P450 pyrTM) and E.coli (styrAB) were constructed, cultured and fermented, cells were collected by centrifugation and resuspended in phosphate buffer (pH 7.4-8.5,200mM or so) and stored cold for later use.

(2) One-pot one-step whole cell catalytic reaction: the cell density in the reaction system was OD600 of about 40, about 25mM glucose was used as an energy source for NADPH regeneration, and styrene and aniline were added at a final concentration of about 2mM, and the reaction was terminated after reacting at 25-40℃for 12-24 hours.

Alternatively, whole cell catalytic reaction in one pot two-step method: the cell density in the reaction system is OD600 about 40, about 25mM glucose is used as energy source for NADPH regeneration, and styrene or substituted styrene with the final concentration of about 5mM is added for reaction for 1 hour at the temperature of 25-40 ℃; then 10-40mM aniline was added and the reaction was ended after 11-23 hours more.

In a preferred embodiment, the enzyme or the expression strain thereof is reacted with the (substituted) styrene having a low polarity for a certain period of time to completely catalyze the styrene into styrene oxide, and then aniline having a high polarity is added, which is advantageous for the reaction to proceed and helps to improve the conversion.

In the above reaction, glucose is added as an energy source for regenerating NADPH, thereby providing NADPH necessary for monooxygenase reaction, and recycling of cofactor can be realized without externally adding NADPH.

Alternatively, in the solution of the present invention, monooxygenase may also be used in combination with Glucose Dehydrogenase (GDH). Wherein glucose dehydrogenase is used to catalyze the oxidation of glucose while NADP is added ⁺ Reducing NADPH, or NAD+ to NADH, thereby regenerating NADPH or NADH.

The invention develops a method for synthesizing chiral beta-amino alcohol by a single oxygenase biocatalysis reaction, which has the following advantages: only one monooxygenase is needed to participate in the reaction, and the whole-cell catalyst has simple and efficient construction and preparation processes and low cost of enzyme preparation; the substrate styrene, substituted styrene and aniline are bulk chemical products, and are cheap and easy to obtain, and the raw material cost is low; the yield and the enantiomer purity of the beta-amino alcohol product are high. The invention produces the enantiomer pure beta-amino alcohol with high added value through the low-cost catalyst and the low-cost substrate, has obvious advantages and has industrial application potential.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The examples relate to the amounts, amounts and concentrations of various substances, wherein the percentages refer to percentages by mass unless otherwise specified.

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing were completed by the Committee Bioengineering Co., ltd.

The molecular biology experiments in the examples include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., and are mainly performed by referring to "molecular cloning experiment guidelines (third edition), J.Sam Broker, D.W. Lassel, huang Peitang, et al, science Press, beijing, 2002). The specific experimental conditions can be determined by simple experiments, if necessary.

The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.

Example 1: construction of recombinant E.coli expressing monooxygenase

1. For monooxygenases P450-BM3 WT, P450-BM3F87G, P450pyr and styrAB, engineering bacteria expressing E.coli were constructed. For example, according to the amino acid sequence SEQ ID NO. 1 (NCBI accession number P14779.2) of cytochrome P450-BM3 WT derived from microbial Bacillus megaterium (Bacillus megaterium) obtained in NCBI database, codon optimization of E.coli preference was performed, and the optimized gene sequence was SEQ ID NO. 6. The gene sequence is synthesized through complete gene, enzyme cutting sites NdeI and BamHI are designed at two ends, the enzyme cutting sites NdeI and BamHI are subcloned on an expression vector pRSF-Duet (purchased from Novagen), the enzyme cutting sites NdeI and BamHI are adopted, and the expression of P450-BM3 WT gene is placed under the control of T7 promoter and lacI repressor gene, so that recombinant plasmid pRSF-Duet-P450-BM3 WT is obtained. The plasmid pRSF-Duet-P450-BM3 WT is transformed into E.coli BL21 (DE 3) competence by using a calcium chloride method, positive clones are screened, and the correct inserted genes are verified by RT-PCR, so that recombinant E.coli (P450-BM 3 WT) expressing monooxygenase SEQ ID NO:1 is obtained.

2. Recombinant E.coli (P450-BM 3F 87G), E.coli (P450 pyrTM), E.coli (styAB) expressing cytochrome mutant P450-BM3F87G (SEQ ID NO: 2), cytochrome P450pyrTM (SEQ ID NO: 3), ethylene monooxygenase styAB (SEQ ID NO:4, SEQ ID NO: 5) were constructed in the same manner as described above.

Example 2: preparation of recombinant escherichia coli whole-cell catalyst

Recombinant E.coli (P450-BM 3 WT), E.coli (P450-BM 3F 87G), E.coli (P450 pyrTM), E.coli (styAB) constructed in example 1 were inoculated into 50mL of TB medium containing 50. Mu.g/mL kanamycin or 50. Mu.g/mL kanamycin+100. Mu.g/mL ampicillin, respectively, and cultured at 37℃and 220rpm for 6 to 8 hours to give precultures. The preculture (800. Mu.L) was transferred to 50mL TB medium containing 50. Mu.g/mL kanamycin or 100. Mu.g/mL ampicillin. Cells were cultured at 37℃and 220rpm to an OD600 of 0.6-0.8 and then induced by addition of IPTG to a final concentration of 0.2 mM. The cells were grown at 25℃for a further 14-17 hours. After the incubation, bacterial sludge was harvested by centrifugation at 4000rpm at 4℃for 10 minutes, washed twice with potassium phosphate buffer (200 mM, pH 8.5) and used as whole cell catalyst for subsequent bioconversion.

Example 3: synthesis of beta-amino alcohol by whole cell catalysis of styrene and aniline

1. Coli cells E.coli expressing P450-BM3 WT (P450-BM 3 WT) were resuspended in potassium phosphate buffer (200 mM, pH 8.5) containing 25mM glucose, the OD600 = 40 of the cell suspension, 2mL of the cell suspension was transferred to a 25mL screw-capped shake flask, the reaction was initiated by adding styrene and aniline at final concentrations of 2mM each, and the reaction was performed at 25℃and 220rpm for 24 hours. After the reaction was completed, the product was extracted with 60mL of ethyl acetate, and then the organic phases were combined and taken up with Na ₂ SO ₄ Drying for 6 hours. The organic solvent was then evaporated and the residue was purified by silica gel column chromatography to give purified product 1c. Nuclear magnetic resonance is used to identify the product structure.

Nuclear magnetic resonance analysis: the spectra were recorded on a Bruker AV-400 spectrometer, which was in chloroform-d 3. For the following ¹ H NMR spectra, chemical shifts are reported in ppm with an internal TMS signal of 0.0ppm as standard. For the following ¹³ C NMR spectrum, chemical shift reported in ppmThe internal chloroform signal was 77.0ppm as a standard. Data are reported as (s=singlet, d=doublet, t=triplet, q=quartet, m=multimodal or unresolved, coupling constant in Hz, integral).

The beta-amino alcohol which is the reaction product of the catalysis of monooxygenase P450-BM3 WT is (R) -1c, i.e. (R) -2-phenyl-2- (N-phenyl) aminoethanol, the HPLC chromatogram of which is shown in FIG. 2 ¹ The H NMR spectrum is shown in FIG. 3, which shows ¹³ The C NMR spectrum is shown in FIG. 4.

[α] _D ²⁰ ＝-19.45°(c＝1.0,CHCl3)。

¹ H NMR(500MHz,CDCl ₃ )δ7.31–7.22(m,4H),7.22–7.15(m,1H),7.07–7.02(m,2H),6.64(t,J＝7.5Hz,1H),6.50(d,J＝7.5Hz,2H),4.36(dd,J＝7.5,4.5Hz,1H),3.76(dd,J＝11.0,4.0Hz,1H),3.56(dd,J＝11.0,7.5Hz,1H)。

¹³ C NMR(125MHz,CDCl ₃ )δ147.2,140.0,129.0,128.6,127.3,126.6,117.7,113.7,67.0,59.7。

[α] _D ²⁰ ＝17.63°(c＝1.0,CHCl3)。

¹ H NMR(500MHz,CDCl ₃ )δ7.34–7.25(m,4H),7.23–7.17(m,1H),7.10–7.03(m,2H),6.65(t,J＝7.5Hz,1H),6.52(d,J＝8.5Hz,2H),4.39(dd,J＝7.5,4.0Hz,1H),3.80(dd,J＝11.5,4.5Hz,1H),3.60(dd,J＝11.0,7.0Hz,1H)。

¹³ C NMR(125MHz,CDCl ₃ )δ147.2,140.1,129.0,128.6,127.4,126.6,117.7,113.8,67.1,59.7。

2. Referring to the method of step 1 above, E.coli (styAB) cells expressing ethylene monooxygenase styAB were resuspended in potassium phosphate buffer (200 mM, pH 8.5) containing 25mM glucose,OD600 = 40 of cell suspension 2mL of cell suspension was transferred to 25mL shake flask with screw cap, the reaction was initiated by adding styrene and aniline, both in final concentration of 2mM, and the reaction was carried out at 25 ℃ and 220rpm for 24 hours. After the reaction was completed, the product was extracted with 60mL of ethyl acetate, and then the organic phases were combined and taken up with Na ₂ SO ₄ Drying for 6 hours. The organic solvent was then evaporated and the residue was purified by silica gel column chromatography to give purified product 1c. Nuclear magnetic resonance is used to identify the product structure. The reaction product β -aminoalcohol is (S) -1c, (S) -2-phenyl-2- (N-phenyl) aminoethanol, which has an HPLC chromatogram as shown in FIG. 2 ¹ The H NMR spectrum is shown in FIG. 5, which shows ¹³ The C NMR spectrum is shown in FIG. 6.

Example 4: P450-BM3 WT whole cell catalytic synthesis of enantiomerically pure beta-amino alcohol from styrene and aniline

Coli cells E.coli expressing P450-BM3 WT (P450-BM 3 WT) were resuspended in potassium phosphate buffer (200 mM, pH 8.5) containing 25mM glucose, the OD600 = 40 of the cell suspension, 2mL of the cell suspension was transferred to a 25mL screw-capped shake flask, the reaction was initiated by adding styrene and aniline at final concentrations of 2mM each, and the reaction was performed at 25℃and 220rpm for 24 hours. 8mL of acetonitrile was added directly to the flask and mixed with the reaction mixture to achieve uniform mixing of the substrate and product. The reaction mixture was centrifuged at 13000rpm for 3min at 1mL, and the supernatant was analyzed by reverse phase HPLC to determine the concentration of the product. 0.2mL of the sample was taken and mixed with 0.8mL of n-hexane, and the organic phase was separated by centrifugation at 13000rpm for 3 minutes and used for chiral HPLC analysis to determine the ee value of the product.

High performance liquid chromatography: the automated Shimadzu LC-2010HPLC system (japan) was equipped with 4 MTP frames using a reversed phase Zorbax Eclipse XDB-C18 chromatographic column (250×4.6mm,5 μm) at 40 ℃, mobile phase: acetonitrile: ultrapure water (0.1% tfa) =45: 55, flow rate 1.2mL/min; ultraviolet detection, detection wavelength is 210nm, and retention time of the product 1c is 7.5min. Product ee was determined on Shimadzu LC-2010HPLC system (japan) using chiral column (250×4.6mm,5 μm) at 35 ℃ and UV detection at 210nm, chiral column IC, mobile phase: n-hexane: isopropanol=95: 5, a flow rate of 0.8mL/min, a retention time of (R) -1c of 11.6min, and a retention time of (S) -1c of 19.2min.

Four product peaks were observed in the chiral HPLC chromatogram, and the ee value of (S) -form β -aminoalcohol was 27% compared to the standard of two chiral β -aminoalcohols 1c. Determination of beta-amino alcohol yield by reverse phase HPLC analysis, P450-BM3 WT had 1c as the main product, 34% yield, and product 1c: the 1d ratio is 92:8.

examples 5 to 7: synthesis of enantiomerically pure beta-amino alcohol (S) -1c by using P450-BM3F87G whole cell to catalyze styrene and aniline

1. One-pot one-step bioconversion: e.coli (P450-BM 3F 87G) cells expressing P450-BM3F87G were resuspended in potassium phosphate buffer (200 mM, pH 8.5) containing 25mM glucose, the OD600 = 40 of the cell suspension, 2mL of the cell suspension was transferred to a 25mL shake flask with a screw cap, and 5mM styrene and 10mM aniline were added to initiate the reaction, and the reaction was performed at 25℃and 220rpm for 24 hours. The yield of β -aminoalcohol was determined by reverse phase HPLC analysis, enantioselectivity was determined by chiral HPLC analysis and optical rotation measurement of the isolated product, resulting in (S) -1c with 90% ee value, but yield was 16%.

2. One-pot two-step bioconversion: 2mL of the cell suspension of E.coli P450-BM3F87G (P450-BM 3F 87G) was transferred to a 25mL shake flask with a screw cap, and styrene at a final concentration of 5mM was added first to react at 25℃and 220rpm for 1 hour, and aniline at a final concentration of 10mM was then added to continue the reaction for 23 hours. The yield of β -aminoalcohol was determined by reverse phase HPLC analysis, enantioselectivity was determined by chiral HPLC analysis and optical rotation measurement of isolated product, (S) -1c had 90% ee value, yield reached 79%.

3. The use of P450-BM3F87G E.coli (P450-BM 3F 87G) whole cells was used for one-pot two-step bioconversion, the amount of aniline used in the reaction was studied, 2mL of the cell suspension was transferred to a 25mL shake flask with a screw cap, styrene was added at a final concentration of 5mM at 25℃and 220rpm for 1 hour, and aniline was then added at a final concentration of 10, 20, 30, 40mM, respectively, for further reaction for 23 hours. The beta-amino alcohol yield was determined by reverse phase HPLC analysis and the enantioselectivity was determined by chiral HPLC analysis and optical rotation measurement of the isolated product. The product concentration increased with increasing aniline concentration, the highest yield at a 1:6 ratio of styrene to aniline, a 90% yield of product (S) -1c, and a 90% enantioselectivity.

The above examples show that the reaction of the enzyme P450-BM3F87G with a weak polar styrene for a certain period of time, the complete catalytic conversion of styrene to styrene oxide followed by the addition of a strong polar aniline is advantageous for the reaction and helps to increase the conversion.

Example 8: synthesis of enantiomerically pure beta-amino alcohol (R) -1c from styrene and aniline by styrAB whole cell catalysis

Coli e.coli (styrb) cells expressing styrb were resuspended in potassium phosphate buffer (200 mM, ph 8.5) containing 25mM glucose, the OD600 = 40 of the cell suspension, 2mL of the cell suspension was transferred to a 25mL shake flask with a screw cap, the reaction was initiated by adding styrene at a final concentration of 5mM, and the reaction was performed at 25 ℃ and 220rpm, after 1 hour the reaction was continued with the addition of aniline at a final concentration of 30mM for 23 hours.

The beta-amino alcohol yield was determined by reverse phase HPLC analysis and the enantioselectivity was determined by chiral HPLC analysis and optical rotation measurement of the isolated product. The yield of the styrab whole cell-catalyzed product (R) -1c was 90% and the enantioselectivity was 99%.

Examples 9 to 13: synthesis of beta-amino alcohol from para-substituted styrene and aniline

E.coli (P450 pyr) and E.coli (styrB) cells expressing P450pryTM or styrB, respectively, were resuspended in potassium phosphate buffer (200 mM, pH 8.5) containing 25mM glucose, the OD600 = 40 of the cell suspension, then 20mL of the reaction mixture was transferred to a 250mL shake flask, and the reaction was initiated by adding 200. Mu.L of para-substituted styrene substrate 2a (4-fluorostyrene) or 3a (4-chlorostyrene) or 4a (4-methylstyrene) solution (500 mM substrate in mother liquor DMSO; final concentration in the reaction system was 5 mM) at 25℃and 220 rpm. After 1 hour of reaction, 200. Mu.L of aniline solution (3M substrate in DMSO as a mother solution; 30mM as a final concentration in the reaction system) was added, and the reaction was continued at 25℃and 220rpm for 23 hours. After the reaction was completed, the product was extracted three times with 60mL of ethyl acetate, and then the organic phases were combined and taken up with Na ₂ SO ₄ Drying for 6 hours, evaporating the organic solvent, purifying the residue by silica gel column chromatography to obtain purified product 2c or 3c or 4c.

Reverse phase HPLC analysis conditions were the same as in example 4 with a retention time of 8.4min for product 2c, 12.6min for product 3c and 10.8min for product 4c. Chiral HPLC analysis of products 2c and 3c the same conditions as in example 4 using chiral column IB, retention time of (S) -2c of 15.9min, retention time of (R) -2c of 19.6min; the retention time of (S) -3c was 15.8min, and the retention time of (R) -3c was 19.1min; the retention time of (S) -4c was 12.9min and the retention time of (R) -4c was 15.8min.

The products were identified by nuclear magnetism, yields were determined by reverse phase HPLC analysis, enantioselectivity was determined by chiral HPLC analysis and optical rotation measurement of the isolated products, and the results are shown in table 1.

TABLE 1 results of P450pryTM or styAB catalyzed reaction of para-substituted styrenes with anilines to synthesize beta-amino alcohols

As can be seen from Table 1, after 24 hours of reaction, the yield of P450pryTM whole cell catalytic substrate 2a was 93% and the enantioselectivity of the product (S) -2c was 99%; the yield of P450pryTM whole cell catalytic substrate 3a was 87% and the enantioselectivity of product (S) -3c was 90%; the yield of the styrab whole cell catalytic substrate 2a is 92%, and the enantioselectivity of the product (R) -2c is 99%; the yield of the styrab whole cell catalytic substrate 3a is 86%, and the enantioselectivity of the product (R) -3c is 99%; the yield of styrb whole cell catalytic substrate 4a was 97% and the enantioselectivity of the product (R) -4c was 95%. The whole cells of the two monooxygenases have extremely high enantioselectivity, and the para-substituted styrene and the aniline are catalyzed to generate enantiomerically pure (S) -type and (R) -type beta-amino alcohol respectively.

The experimental results show that the monooxygenase P450-BM3 WT, P450-BM3F87G, P450pyr and styrAB can independently catalyze the asymmetric amino hydroxylation reaction of styrene/substituted styrene and aniline to obtain chiral beta-amino alcohol, the stereoselectivity of the former three is the same, but the stereoselectivity of the styrAB is opposite, and the method has development and application prospects.

Sequence listing

<110> university of Hubei

XIAMEN University

<120> method for synthesizing chiral beta-amino alcohol by enzyme catalysis

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Met Ala Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys

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Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys

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Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg

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Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp

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Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg

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Asp Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn

85 90 95

Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala

100 105 110

Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val

115 120 125

Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu

130 135 140

Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn

145 150 155 160

Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr

165 170 175

Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala

180 185 190

Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu

195 200 205

Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg

210 215 220

Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn

225 230 235 240

Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg

245 250 255

Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly

260 265 270

Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu

275 280 285

Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro

290 295 300

Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn

305 310 315 320

Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala

325 330 335

Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp

340 345 350

Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp

355 360 365

Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser

370 375 380

Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala

385 390 395 400

Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly

405 410 415

Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu

420 425 430

Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys

435 440 445

Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr

450 455 460

Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn

465 470 475 480

Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly

485 490 495

Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro

500 505 510

Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly

515 520 525

Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn

530 535 540

Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val

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Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala

565 570 575

Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala

580 585 590

Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp

595 600 605

Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp

610 615 620

Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys

625 630 635 640

Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu

645 650 655

Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu

660 665 670

Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu

675 680 685

Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile

690 695 700

Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly

705 710 715 720

Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu

725 730 735

Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln

740 745 750

Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met

755 760 765

Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu

770 775 780

Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr

785 790 795 800

Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser

805 810 815

Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile

820 825 830

Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser

835 840 845

Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile

850 855 860

Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys

865 870 875 880

Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu

885 890 895

Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg

900 905 910

Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu

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Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr

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Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr

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Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val

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Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp

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Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro

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Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val

1010 1015 1020

Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly

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Arg Tyr Ala Lys Asp Val Trp Ala Gly

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Met Ala Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys

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Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys

20 25 30

Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg

35 40 45

Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp

50 55 60

Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg

65 70 75 80

Asp Phe Ala Gly Asp Gly Leu Gly Thr Ser Trp Thr His Glu Lys Asn

85 90 95

Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala

100 105 110

Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val

115 120 125

Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu

130 135 140

Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn

145 150 155 160

Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr

165 170 175

Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala

180 185 190

Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu

195 200 205

Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg

210 215 220

Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn

225 230 235 240

Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg

245 250 255

Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly

260 265 270

Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu

275 280 285

Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro

290 295 300

Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn

305 310 315 320

Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala

325 330 335

Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp

340 345 350

Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp

355 360 365

Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser

370 375 380

Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala

385 390 395 400

Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly

405 410 415

Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu

420 425 430

Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys

435 440 445

Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr

450 455 460

Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn

465 470 475 480

Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly

485 490 495

Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro

500 505 510

Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly

515 520 525

Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn

530 535 540

Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val

545 550 555 560

Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala

565 570 575

Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala

580 585 590

Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp

595 600 605

Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp

610 615 620

Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys

625 630 635 640

Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu

645 650 655

Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu

660 665 670

Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu

675 680 685

Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile

690 695 700

Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly

705 710 715 720

Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu

725 730 735

Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln

740 745 750

Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met

755 760 765

Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu

770 775 780

Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr

785 790 795 800

Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser

805 810 815

Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile

820 825 830

Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser

835 840 845

Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile

850 855 860

Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys

865 870 875 880

Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu

885 890 895

Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg

900 905 910

Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu

915 920 925

Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr

930 935 940

Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr

945 950 955 960

Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val

965 970 975

Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp

980 985 990

Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro

995 1000 1005

Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val

1010 1015 1020

Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly

1025 1030 1035 1040

Arg Tyr Ala Lys Asp Val Trp Ala Gly

1045

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<213> Sphingomonas sp. HXN-200

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Met Glu His Thr Gly Gln Ser Ala Ala Ala Thr Met Pro Leu Asp Ser

1 5 10 15

Ile Asp Val Ser Ile Pro Glu Leu Phe Tyr Asn Asp Ser Val Gly Glu

20 25 30

Tyr Phe Lys Arg Leu Arg Lys Asp Asp Pro Val His Tyr Cys Ala Asp

35 40 45

Ser Ala Phe Gly Pro Tyr Trp Ser Ile Thr Lys Tyr Asn Asp Ile Met

50 55 60

His Val Asp Thr Asn His Asp Ile Phe Ser Ser Asp Ser Gly Tyr Gly

65 70 75 80

Gly Ile His Ile Asp Asp Gly Ile Gln Lys Gly Gly Asp Gly Gly Leu

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Asp Leu Pro Asn Phe Ile Ala Met Asp Arg Pro Arg His Asp Glu Gln

100 105 110

Arg Lys Ala Val Ser Pro Ile Val Ala Pro Ala Asn Leu Ala Ala Leu

115 120 125

Glu Gly Thr Ile Arg Glu Arg Val Ser Lys Thr Leu Asp Gly Leu Pro

130 135 140

Val Gly Glu Glu Phe Asp Trp Val Asp Arg Val Ser Ile Glu Ile Thr

145 150 155 160

Thr Gln Met Leu Ala Thr Leu Phe Asp Phe Pro Phe Glu Glu Arg Arg

165 170 175

Lys Leu Thr Arg Trp Ser Asp Val Thr Thr Ala Ala Pro Gly Gly Gly

180 185 190

Val Val Glu Ser Trp Asp Gln Arg Lys Thr Glu Leu Leu Glu Cys Ala

195 200 205

Ala Tyr Phe Gln Val Leu Trp Asn Glu Arg Val Asn Lys Asp Pro Gly

210 215 220

Asn Asp Leu Ile Ser Met Leu Ala His Ser Pro Ala Thr Arg Asn Met

225 230 235 240

Thr Pro Glu Glu Tyr Leu Gly Asn Val Leu Leu Leu Ile Val Gly Gly

245 250 255

Asn Asp Thr Thr Arg Asn Ser Met Thr Gly Gly Val Leu Ala Leu His

260 265 270

Lys Asn Pro Asp Gln Phe Ala Lys Leu Lys Ala Asn Pro Ala Leu Val

275 280 285

Glu Thr Met Val Pro Glu Ile Ile Arg Trp Gln Thr Pro Leu Ala His

290 295 300

Gln Arg Arg Thr Ala Ile Ala Asp Ser Glu Leu Gly Gly Lys Thr Ile

305 310 315 320

Arg Lys Gly Asp Lys Val Val Met Trp Tyr Tyr Ser Gly Asn Arg Asp

325 330 335

Asp Glu Val Ile Asp Arg Pro Glu Glu Phe Ile Ile Asp Arg Pro Arg

340 345 350

Pro Arg Gln His Leu Ser Phe Gly Phe Gly Ile His Arg Cys Val Gly

355 360 365

Asn Arg Leu Ala Glu Met Gln Leu Arg Ile Leu Trp Glu Glu Ile Leu

370 375 380

Thr Arg Phe Ser Arg Ile Glu Val Met Ala Glu Pro Glu Arg Val Arg

385 390 395 400

Ser Asn Phe Val Arg Gly Tyr Ala Lys Met Met Val Arg Val His Ala

405 410 415

<210> 4

<211> 415

<212> PRT

<213> Pseudomonas sp. strain VLB120

<400> 4

Met Lys Lys Arg Ile Gly Ile Val Gly Ala Gly Thr Ala Gly Leu His

1 5 10 15

Leu Gly Leu Phe Leu Arg Gln His Asp Val Asp Val Thr Val Tyr Thr

20 25 30

Asp Arg Lys Pro Asp Glu Tyr Ser Gly Leu Arg Leu Leu Asn Thr Val

35 40 45

Ala His Asn Ala Val Thr Val Gln Arg Glu Val Ala Leu Asp Val Asn

50 55 60

Glu Trp Pro Ser Glu Glu Phe Gly Tyr Phe Gly His Tyr Tyr Tyr Val

65 70 75 80

Gly Gly Pro Gln Pro Met Arg Phe Tyr Gly Asp Leu Lys Ala Pro Ser

85 90 95

Arg Ala Val Asp Tyr Arg Leu Tyr Gln Pro Met Leu Met Arg Ala Leu

100 105 110

Glu Ala Arg Gly Gly Lys Phe Cys Tyr Asp Ala Val Ser Ala Glu Asp

115 120 125

Leu Glu Gly Leu Ser Glu Gln Tyr Asp Leu Leu Val Val Cys Thr Gly

130 135 140

Lys Tyr Ala Leu Gly Lys Val Phe Glu Lys Gln Ser Glu Asn Ser Pro

145 150 155 160

Phe Glu Lys Pro Gln Arg Ala Leu Cys Val Gly Leu Phe Lys Gly Ile

165 170 175

Lys Glu Ala Pro Ile Arg Ala Val Thr Met Ser Phe Ser Pro Gly His

180 185 190

Gly Glu Leu Ile Glu Ile Pro Thr Leu Ser Phe Asn Gly Met Ser Thr

195 200 205

Ala Leu Val Leu Glu Asn His Ile Gly Ser Asp Leu Glu Val Leu Ala

210 215 220

His Thr Lys Tyr Asp Asp Asp Pro Arg Ala Phe Leu Asp Leu Met Leu

225 230 235 240

Glu Lys Leu Gly Lys His His Pro Ser Val Ala Glu Arg Ile Asp Pro

245 250 255

Ala Glu Phe Asp Leu Ala Asn Ser Ser Leu Asp Ile Leu Gln Gly Gly

260 265 270

Val Val Pro Ala Phe Arg Asp Gly His Ala Thr Leu Asn Asn Gly Lys

275 280 285

Thr Ile Ile Gly Leu Gly Asp Ile Gln Ala Thr Val Asp Pro Val Leu

290 295 300

Gly Gln Gly Ala Asn Met Ala Ser Tyr Ala Ala Trp Ile Leu Gly Glu

305 310 315 320

Glu Ile Leu Ala His Ser Val Tyr Asp Leu Arg Phe Ser Glu His Leu

325 330 335

Glu Arg Arg Arg Gln Asp Arg Val Leu Cys Ala Thr Arg Trp Thr Asn

340 345 350

Phe Thr Leu Ser Ala Leu Ser Ala Leu Pro Pro Glu Phe Leu Ala Phe

355 360 365

Leu Gln Ile Leu Ser Gln Ser Arg Glu Met Ala Asp Glu Phe Thr Asp

370 375 380

Asn Phe Asn Tyr Pro Glu Arg Gln Trp Asp Arg Phe Ser Ser Pro Glu

385 390 395 400

Arg Ile Gly Gln Trp Cys Ser Gln Phe Ala Pro Thr Ile Ala Ala

405 410 415

<210> 5

<211> 170

<212> PRT

<213> Pseudomonas sp. strain VLB120

<400> 5

Met Thr Leu Lys Lys Asp Met Ala Val Asp Ile Asp Ser Thr Asn Phe

1 5 10 15

Arg Gln Ala Val Ala Leu Phe Ala Thr Gly Ile Ala Val Leu Ser Ala

20 25 30

Glu Thr Glu Glu Gly Asp Val His Gly Met Thr Val Asn Ser Phe Thr

35 40 45

Ser Ile Ser Leu Asp Pro Pro Thr Val Met Val Ser Leu Lys Ser Gly

50 55 60

Arg Met His Glu Leu Leu Thr Gln Gly Gly Arg Phe Gly Val Ser Leu

65 70 75 80

Leu Gly Glu Ser Gln Lys Val Phe Ser Ala Phe Phe Ser Lys Arg Ala

85 90 95

Met Asp Asp Thr Pro Pro Pro Ala Phe Thr Ile Gln Ala Gly Leu Pro

100 105 110

Thr Leu Gln Gly Ala Met Ala Trp Phe Glu Cys Glu Val Glu Ser Thr

115 120 125

Val Gln Val His Asp His Thr Leu Phe Ile Ala Arg Val Ser Ala Cys

130 135 140

Gly Thr Pro Glu Ala Asn Thr Pro Gln Pro Leu Leu Phe Phe Ala Ser

145 150 155 160

Arg Tyr His Gly Asn Pro Leu Pro Leu Asn

165 170

<210> 6

<211> 3150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atggcaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60

ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120

tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180

gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240

gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300

cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360

gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420

gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480

tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540

gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600

gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660

attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720

ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780

acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840

ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900

gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaaac 960

gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020

gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080

cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140

gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200

tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260

cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320

aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380

tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440

acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500

ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560

gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620

ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680

aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740

aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800

cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860

atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920

tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980

ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040

agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100

ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160

ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220

ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280

acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340

cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400

atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460

cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520

aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580

tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640

tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700

atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760

ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820

catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880

cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940

gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000

ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060

gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120

cgatacgcaa aagacgtgtg ggctgggtaa 3150

Claims

1. A method for preparing chiral beta-amino alcohol by enzyme catalysis, which comprises the following steps: using asymmetric alkene and amine as substrates, catalyzing amino hydroxylation reaction by monooxygenase to obtain chiral beta-amino alcohol, wherein

The beta-amino alcohol is 2-phenyl-2- (N-phenyl) amino ethanol or 2-substituted phenyl-2- (N-phenyl) amino ethanol, and the steps are as follows:

using styrene/substituted styrene shown in a formula I and aniline as substrates, and catalyzing amino hydroxylation reaction by monooxygenase to obtain (S) -configuration beta-amino alcohol shown in a formula II or (R) -configuration beta-amino alcohol shown in a formula III:

wherein R is selected from H, halogen, C1-C4 alkyl,

the monooxygenase used for catalyzing and preparing the (S) -configuration beta-amino alcohol shown in the formula II is wild cytochrome P450-BM3 WT with the amino acid sequence of SEQ ID NO. 1, cytochrome mutant P450-BM3F87G with the amino acid sequence of SEQ ID NO. 2 or cytochrome P450pyrTM with the amino acid sequence of SEQ ID NO. 3; the enzymes used for the catalytic preparation of the (R) -configured beta-amino alcohols shown in III are ethylene monooxygenase styAB with the amino acid sequences SEQ ID NO. 4 and SEQ ID NO. 5, respectively, wherein styA is SEQ ID NO. 4 and styB is SEQ ID NO. 5.

2. The method of claim 1, wherein the halogen is selected from the group consisting of fluorine F, chlorine Cl; the C1-C4 alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl.

3. The method of claim 1, wherein when the substituent R is selected from the group consisting of halogen, C1-C4 alkyl, R is in the para position, reacting:

4. the method of claim 1, wherein the monooxygenase is in the form of an enzyme or a microorganism expressing the same in the reaction system.

5. The method of claim 4, wherein the enzyme form is a free enzyme or an immobilized enzyme.

6. The method of claim 4, wherein the microorganism is selected from the group consisting of bacillus subtilis, lactobacillus brevis, escherichia coli, candida magnolol, pichia pastoris, and saccharomyces cerevisiae.

7. The method according to claim 1, wherein when the monooxygenase is in the form of microbial cells, glucose is further added to the reaction system.

8. The method of claim 1, wherein the reaction system has a pH of 7.4 to 8.5.