CN113999879A - A kind of method for peroxidase catalyzed oxidation of aromatic hydrocarbons and derivatives thereof - Google Patents

Abstract

The invention discloses a method for catalyzing and oxidizing aromatic hydrocarbons and derivatives thereof by hydrogen peroxide-dependent peroxidase. It uses aromatic hydrocarbons or their derivatives as reaction substrates, and in the presence of peroxides and peroxidase in a buffer reaction system or a pure aromatic hydrocarbon reaction system, the corresponding alcohol, aldehyde or ketone products are obtained. The present invention is an enzyme-catalyzed method for oxidizing aromatic hydrocarbons under normal temperature and pressure, and directly prepares high value-added compounds such as alcohols, aldehydes and ketones of aromatic hydrocarbons by a one-pot method, and does not use any other water or cosolvent in the preparation process. The solvent belongs to a green preparation method and has the advantages of simple preparation process, high yield and high selectivity.

Description

A kind of method for peroxidase catalyzed oxidation of aromatic hydrocarbons and derivatives thereof

technical field

The invention belongs to the technical field of biocatalysis, and in particular relates to a method for catalyzing and oxidizing aromatic hydrocarbons and derivatives thereof by a hydrogen peroxide-dependent peroxidase.

Background technique

Benzene, toluene, naphthalene, p-chlorotoluene and other compounds are oxidized to form phenol or benzyl alcohol/aldehyde, etc., which are used as fine organic chemical intermediates and are widely used in the fields of medicine, pesticides and dyes. Taking p-chlorobenzaldehyde as an example, in the pharmaceutical industry, p-chlorobenzaldehyde is subjected to condensation and mercaptopropionic acid cyclization to obtain fenalol, which can also be used to synthesize the drug chlorobenzaldehyde, sedative aminophenylbutyric acid, and veterinary drug hydrochloric acid. In the pesticide industry, p-chlorobenzaldehyde is an important intermediate for the synthesis of plant growth regulators uniconazole, chlorocinnamaldehyde and the herbicide Maidisan; in the dye industry, p-chlorobenzaldehyde is used as the intermediate It is a triphenylmethane type acid dye with good vividness and high coloring degree, which is widely used in wool spinning and silk printing and dyeing industries. In addition, p-chlorobenzaldehyde can also be used as an intermediate for textile auxiliaries and photosensitive materials.

In recent years, with the continuous development of downstream products of p-chlorobenzaldehyde, its demand has increased year by year, while the production capacity of p-chlorobenzaldehyde in my country is relatively low, mainly due to the harsh production and oxidation process of aromatic hydrocarbons such as p-chlorobenzaldehyde. High cost of construction, strict environmental protection requirements, long-term dependence on imports and other reasons.

Therefore, the products after the oxidation of aromatic hydrocarbons and derivatives are far from meeting the broad market demand. Therefore, it is particularly important to develop a green and environmentally friendly oxidation method for aromatic hydrocarbons and derivatives.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an enzyme-catalyzed oxidation of aromatic hydrocarbons and derivatives to produce corresponding oxidation products. In the present invention, inert aromatic hydrocarbons or derivatives thereof are used as substrates, peroxidase is used as catalyst, and oxidation products are prepared in the presence of co-substrate hydrogen peroxide. In the method, the above-mentioned reaction components are added to a buffer with a certain pH at room temperature, and after a period of reaction, extraction and drying are performed to obtain the corresponding benzyl alcohol or benzaldehyde derivatives; or pure aromatic hydrocarbons are used as solvents and substrates, And the use of immobilized peroxidase and hydrogen peroxide to achieve oxidation reaction.

The preparation method of alcohol, aldehyde or ketone for synthesizing aromatic hydrocarbon provided by the present invention comprises the following steps:

所示含有取代基的苯为反应底物，于缓冲液反应体系或纯的苯反应体系中，在过氧化氢和过氧化酶存在下，得到式

或式III所示的醇或醛、酮产物；Equation

The benzene containing substituents is shown as the reaction substrate, in the buffer reaction system or the pure benzene reaction system, in the presence of hydrogen peroxide and peroxidase, the formula

Or the alcohol or aldehyde, ketone product shown in formula III;

中，R可为氢、甲基、丙基、异丙基、卤素（F、Cl、Br、I）、羰基、芳基、硝基、氨基中的一种或多种，存在于苯环的邻、间或对位；Mode

Among them, R can be one or more of hydrogen, methyl, propyl, isopropyl, halogen (F, Cl, Br, I), carbonyl, aryl, nitro, and amino, which are present in the benzene ring. Adjacent, intermittent or contraposition;

R ₁ can be H, methyl, propyl, isopropyl, carbonyl, aryl,

中R、R₁同式

中R、R₁；Mode

The same formula of R and R ₁

in R, R ₁ ;

中R、R₁同式

中R、R₁；Mode

The same formula of R and R ₁

in R, R ₁ ;

In the buffer reaction system, the concentration of halobenzene can be 1.0 mmol/L-100 mmol/L, specifically 2.5-10 mmol/L;

In order to improve the solubility of the substrate aromatic hydrocarbons, small molecular alcohol or acetonitrile, ethyl acetate, dimethyl sulfoxide, etc. can be added in the above reaction, wherein the small molecular alcohol can be one of methanol, ethanol, isopropanol or Several mixtures; small molecular alcohol accounts for 1-60% of the volume of the reaction system;

In the pure organic phase reaction system, the concentration of aromatic hydrocarbon substrate can be up to 7.0 mol/L;

The buffer solution can be any one of phosphate buffer, citrate buffer and tris(hydroxymethyl)aminomethane buffer, and its pH value is 3.5-11, specifically 4.5-9.5;

The peroxidase can be selected from at least one of the following (1)-(11). The peroxidase with heme activity (EC 1.11.2.1) does not need to provide cofactors to participate in catalysis, but directly utilizes the peroxidase. Oxide reacts with heme to generate reactive species (Compound I), which in turn catalyzes the selective transfer of single oxygen atoms from peroxide (H ₂ O ₂ , ROOH) to carbon-hydrogen bonds of different target molecules in the manner of monooxygenase above; it can be understood from the study of the examples that these enzymes are suitable for the present invention.

(1) Haloperoxidase Lfu CPO derived from Leptoxyphium fumago ; (SEQ ID NO.1);

(2) Haloperoxidase Cf CPO from Caldariomyces fumago (GenBank: GCA001660795.1);

(3) Peroxidase Aae UPO (SEQ ID NO. 2) derived from Agrocybe aegerita ;

(4) Peroxidase Cgl UPO from Chaetomium globosum (GenBank: XM 001219539.1);

(5) Peroxidase Mwe UPO (SEQ ID NO.3) derived from Marasmius wettsteinii ;

(6) Peroxidase Cci UPO (SEQ ID NO.4) derived from Coprinopsis cinerea ;

(7) Peroxidase Mth UPO (SEQ ID NO.5) derived from Myceliophthora thermophila ;

(8) Peroxidase Tte UPO (SEQ ID NO.6) derived from Thielavia terrestris ;

(9) Peroxidase Mro UPO (SEQ ID NO. 7) derived from Marasmius rotula ;

(10) Peroxidase Cra UPO from Coprinellus radians (GenBank: FM872459.1);

(11) Peroxidase Pab UPO (GenBank: MH880928) derived from Psathyrella aberdarensis.

The peroxidase or its mutant still having peroxidase function plays a catalytic role in the form of whole cells, crude enzyme powder, crude enzyme liquid, pure enzyme or immobilized enzyme;

In the reaction system, the concentration of peroxidase can be 5nmol/L-5μmol/L, specifically 200 nmol/L-1000 nmol/L, or 0.02-04 U/ml, where 1U refers to the The amount of enzyme that can convert 1 μmol of p-chlorotoluene in 1 minute).

The reaction time of the reaction may be 10-48 h, and the reaction temperature may be 20-50 °C.

After the reaction is completed, the reaction solution is extracted with an organic solvent, dried, filtered, separated and purified; when the substrate itself is used as a solvent in the reaction, the product can be directly distilled from it.

The present invention establishes an enzymatic catalysis method for oxidizing aromatic hydrocarbons or derivatives thereof under normal temperature and pressure, and can directly prepare halogenated benzyl alcohol, halogenated benzaldehyde, halogenated phenethyl alcohol, ( R )-1 through a one-pot method - High value-added compounds such as phenethyl alcohol, which can be used as intermediates of various pharmaceuticals or fine chemicals. In the preparation process, no other solvent is used except water or co-solvent. The method belongs to the green preparation method and has high atomic economical utilization. Therefore, the method of the present invention has the advantages of simple preparation process, high yield and high selectivity.

Description of drawings

1 is a gas chromatogram of 4-chlorobenzyl alcohol and 4-chlorobenzaldehyde reacted for 5 hours in Example 2.

2 is a gas chromatogram of 3-chlorobenzaldehyde reacted for 4 hours in Example 3.

3 is a gas chromatogram of 4-bromobenzaldehyde reacted for 4 hours in Example 4.

Figure 4 is a gas chromatogram of ( R )-1-(4-bromophenyl)ethanol or 4-bromoacetophenone prepared in Example 6.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Preparation of enzymes:

The UPO enzyme used in the following examples is Aae UPO, prepared by the following methods:

Recombinant protein expression: The Aae UPO DNA sequence (SEQ ID NO. 2) was subcloned into the pPICZαA vector with a His-tag at the C-terminus. The pPICZαA -Aae UPO recombinant plasmid was transformed into P. pastoris strain X-33, 100 μL of competent cells were added with linearized plasmid DNA for electroporation, and the cells were plated on YPD medium plates (containing 100 μg/mL bleomycin). Incubate at 30°C for 2 days. When a single colony grows on the YPD plate, pick a single colony, and then carry out PCR colony screening and verification. According to the positive result, transfer it into a conical flask containing 50 mL of BMGY medium, and cultivate it to OD ₆₀₀ = 1-1.5 at 30 °C and 200 rpm. ; 4000rpm, 5min to harvest bacteria; the pellet was resuspended with BMMY to OD ₆₀₀ =0.3 (about 100-200mL); transferred to a 500mL conical flask, 30 ℃, 200rpm began to induce expression for 3 days, adding 3% methanol every 24h.

Protein purification: Centrifuge the bacterial solution at 4°C and 4000 rpm for 20 minutes, and collect the supernatant (ie Aae UPO crude enzyme solution). The nickel column was pre-equilibrated with buffer A (20 mM Tris–HCl, pH=7.0), and the supernatant was passed through the HisTrapPHP prepacked column in the protein purifier at a flow rate of 1.5 mL/min. Adjust the ratio of elution buffer B (20 mM Tris–HCl, 2M NaCl pH=7.0,) from 0 to 15% for 5 min. Collect the eluted protein solution corresponding to the detected peak in separate tubes. Use an ultrafiltration tube to concentrate the target protein eluate and replace its buffer with size exclusion chromatography buffer.

Pass the protein solution through a molecular sieve Superdex® 200 Increase 10/300 GL at a flow rate of 0.5 mL/min. The protein solutions corresponding to the detected peaks were collected in separate tubes.

Among them, Aae UPO activity was determined by using the ABTS assay in NaPi buffer at pH 5.0.

Embodiment 1: the preparation of compound 4-chlorobenzyl alcohol of following formula

In a 2 mL reaction flask, 890 microliters of phosphate buffer solution (pH=7, 50 mmol/L) was added, 100 microliters of methanol solution of 4-chlorotoluene (50 mmol/L) was added, and 10 microliters of peroxidase UPO was added Enzyme (500 nmol/L), infused with H ₂ O ₂ at a rate of 5 mM/h using an infusion pump.

The above reaction system was stirred and reacted at 30 °C with a constant temperature mixer for 15 hours, the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, detected and analyzed by gas chromatography, and the yield was 85 %.

Embodiment 2: the preparation of compound 4-chlorobenzyl alcohol or 4-chlorobenzaldehyde of following formula

In a 1L reaction flask, add 890 mL of phosphate buffer solution (pH=7, 50 mmol/L), add 50 mL of methanol solution of 4-chlorotoluene (50 mmol/L), and add 5 mL of peroxidase UPO enzyme ( 500 nmol/L). H ₂ O ₂ was infused at a rate of 5 mmol/h using an infusion pump.

After the above reaction system was stirred at 30°C with a constant temperature oscillator for 5 hours, the reaction was terminated. The reaction solution was extracted twice with 500 ml of ethyl acetate, the extracts were combined, the organic phase was dried over anhydrous sodium sulfate, and the ethyl acetate was removed by rotary evaporation. After purification on a silica column, the overall yield was 71% (19% for 4-chlorobenzaldehyde and 52% for 4-chlorobenzaldehyde.

Fig. 1 is a gas chromatogram of 4-chlorobenzyl alcohol and 4-chlorobenzaldehyde under the reaction conditions for 5 hours.

Embodiment 3: the preparation of compound 3-chlorobenzyl alcohol or 3-chlorobenzaldehyde of following formula:

In a 2 mL reaction flask, 890 μl of phosphate buffer solution (pH=7, 50 mmol/L) was added, 100 μl of 3-chlorotoluene methanol solution (50 mmol/L) was added, and 10 μl of peroxidase was added UPO enzyme (500 nmol/L). H ₂ O ₂ was infused at a rate of 5 mmol/h using an infusion pump.

After the above reaction system was stirred at 30°C with a constant temperature mixer for 4 hours, the reaction was terminated, 100 microliters of the reaction solution was taken, 200 microliters of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, detected and analyzed by gas chromatography, and the yield was 81 % (ie the yield of 3-chlorobenzaldehyde). Figure 2 is a gas chromatogram of 3-chlorobenzaldehyde under the reaction conditions for 4 hours.

Embodiment 4: the preparation of compound 2-chlorobenzyl alcohol or 2-chlorobenzaldehyde of following formula:

In a 2 mL reaction flask, 890 microliters of phosphate buffer solution (pH=7, 50 mmol/L) was added, 100 microliters of methanol solution of 2-chlorotoluene (50 mmol/L) was added, and 10 microliters of peroxidase was added UPO enzyme (500 nmol/L). H ₂ O ₂ was infused at a rate of 5 mmol/h using an infusion pump.

After the above reaction system was stirred and reacted at 30°C with a constant temperature mixer for 23 hours, the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, detected and analyzed by gas chromatography, and the yield was 70%. % (that is, the yield of 2-chlorobenzaldehyde).

Embodiment 5: the preparation of compound 4-bromobenzyl alcohol or 4-bromobenzaldehyde of following formula:

In a 2 mL reaction flask, 890 microliters of phosphate buffer solution (pH=7, 50 mmol/L) was added, 100 microliters of methanol solution of 4-bromotoluene (50 mmol/L) was added, and 10 microliters of peroxidase was added UPO enzyme (500 nmol/L). H ₂ O ₂ was infused at a rate of 5 mmol/h using an infusion pump.

After the above reaction system was stirred at 30°C with a constant temperature mixer for 12 hours, the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, detected and analyzed by gas chromatography, and the yield was 68 % (that is, the yield of 4-bromobenzaldehyde). Figure 3 is a gas chromatogram of 4-bromobenzaldehyde under the reaction conditions for 4 hours.

Embodiment 6: the preparation of compound ( R )-1-(4-bromophenyl)ethanol or 4-bromoacetophenone of the following formula:

In a 2 mL reaction flask, 890 μl of phosphate buffer solution (pH=7, concentration of 50 mmol/L), add 100 μl of 4-bromoethylbenzene methanol solution (50 mmol/L), add 10 μl Peroxidase UPO enzyme (500 nmol/L). H ₂ O ₂ was infused at a rate of 5 mmol/h using an infusion pump.

After the above reaction system was stirred at 30 °C with a constant temperature mixer for 28 hours, the reaction was terminated, 100 microliters of the reaction solution was taken, 200 microliters of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, detected and analyzed by gas chromatography, and the yield was 75 % (i.e. the sum of the yields of (R)-1-(4-bromophenyl)ethanol and 4-bromoacetophenone). Figure 4 is a gas chromatogram of the prepared ( R )-1-(4-bromophenyl)ethanol or 4-bromoacetophenone.

Embodiment 7: the preparation of compound ( R )-1-(4-chlorophenyl)ethanol or 4-chloroacetophenone of the following formula:

In a 2 mL reaction flask, 890 μl of phosphate buffer solution (pH=7, concentration of 50 mmol/L), add 100 μl of 4-bromoethylbenzene methanol solution (50 mmol/L), add 10 μl Peroxidase UPO enzyme (500 nmol/L). H ₂ O ₂ was infused at a rate of 5 mmol/h using an infusion pump.

After the above reaction system was stirred and reacted at 30 °C with a constant temperature mixer for 26 hours, the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, detected and analyzed by gas chromatography, and the yield was 75 %.

Example 8: Immobilized peroxidase The compound 4-chlorobenzyl alcohol or 4-chlorobenzaldehyde of the following formula was prepared:

In a 2 mL reaction flask, add 1 mL of p-chlorotoluene, and add 200 mg of immobilized peroxidase UPO enzyme (400 nmol/L). Use an infusion pump to infuse tert-butanol peroxide at a rate of 3 mmol/h.

The above reaction system was stirred for 30 hours with a constant temperature mixer at 30°C, the reaction was terminated, and the distillation was carried out with a yield of 65% (that is, the total yield of the two substances).

The immobilization method of peroxidase UPO enzyme is as follows:

1. Raw materials:

(1) Amino carrier;

(2) Peroxidase (solid enzyme or liquid enzyme);

(3) Immobilized buffer: 0.01-0.05mol/L buffer that can ensure the activity and stability of the enzyme; here is a phosphate buffer solution (pH=7, concentration 50 mmol/L).

(4) Washing buffer (0.01-0.02mol/L buffer or deionized water for elution of enzymes that are not covalently bound); 25% glutaraldehyde solution;

2. Process

(1) Carrier balance: Immobilization buffer, wash repeatedly 2-4 times according to the ratio of amino carrier/buffer 1:5 (mass/volume ratio). (filter and drain after each cleaning)

(2) Prepare 2% glutaraldehyde solution with immobilization buffer;

(3) Carrier activation: 2% glutaraldehyde was added to the carrier, the ratio of carrier and glutaraldehyde buffer was 1:4 (mass/volume ratio), and stirred at 22°C for 60 min. After filtration, wash the support with immobilization buffer in a ratio of 1:4 (mass/volume ratio). The activated carrier should be used within 48h as much as possible. It is normal for the carrier to be orange to brown after activation.

(4) The recommended protein load is 50-100mg/g wet carrier, and the protein concentration is determined by the standard protein content determination method. The ratio of carrier and enzyme buffer is 1:3 (mass/volume ratio)

(5) The enzyme-containing buffer and carrier were added to the reactor, and the reaction was carried out at 800 rpm for 18 h. (Caution: Avoid using magnetic stirring, which may cause breakage of the carrier). The immobilization temperature can be selected at 25 °C, which mainly depends on the stability of the enzyme. At the same time, do not choose to immobilize at high temperature, which may cause side reactions of amino groups.

(6) After immobilization, the immobilized enzyme was collected by filtration, and the protein content in the serum was determined to calculate the immobilization rate and yield. Wash with wash buffer, repeat 2-4 times, and store at 2°C - 8°C.

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> A method for peroxidase catalytic oxidation of aromatic hydrocarbons and derivatives thereof

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 373

<212> PRT

<213> Haloperoxidase LfuCPO of Leptoxyphiumfumago

<400> 1

MFSKVLPFVGAVAALPHSVRQEPGSGIGYPYDNNTLPYVAPGPTDSRAPCPALNALANHGYIPHDGRAISRETLQNAFLNHMGIANSVIELALTNAFVVCEYVTGSDCGDSLVNLTLLAEPHAFEHDHSFSRKDYKQGVANSNDFIDNRNFDAETFQTSLDVVAGKTHFDYADMNEIRLQRESLSNELDFPGWFTESKPIQNVESGFIFALVSDFNLPDNDENPLVRIDWWKYWFTNESFPYHLGWHPPSPAREIEFVTSASSAVLAASVTSTPSSLPSGAIGPGAEAVPLSFASTMTPFLLATNAPYYAQDPTLGPNDKREAAPAATTSMAVFKNPYLEAIGTQDIKNQQAYVSSKAAAMASAMAANKARNL 373

<210> 2

<211> 330

<212> PRT

<213> Peroxidase AaeUPO of Agrocybeaegerita

<400> 2

AEPGLPPGPLENSSAKLVNDEAHPWKPLRPGDIRGPCPGLNTLASHGYLPRNGVATPAQIINAVQEGFNFDNQAAIFATYAAHLVDGNLITDLLSIGRKTRLTGPDPPPPASVGGLNEHGTFEGDASMTRGDAFFGNNHDFNETLFEQLVDYSNRFGGGKYNLTVAGELRFKRIQDSIATNPNFSFVDFRFFTAYGETTFPANLFVDGRRDDGQLDMDAARSFFQFSRMPDDFFRAPSPRSGTGVEVVVQAHPMQPGRNVGKINSYTVDPTSSDFSTPCLMYEKFVNITVKSLYPNPTVQLRKA LNTNLDFLFQGVAAGCTQVFPYGRDS 330

<210> 3

<211> 238

<212> PRT

<213> Peroxidase MweUPO of Marasmiuswettsteinii

<400> 3

ASAHPWKAPGPNDSRGPCPGLNTLANHGFLPRNGRNISVPMIVKAGFEGYNVQSDVLITAGKVGMLTSREADTISLEDLKLHGTIEHDASLSREDAAIGDNLHFNEAIFTTLANSNPGADVYNISSAAQVQHDRLADSLARNPNVTNTDVTATIRASESAFYLTVMSAGDPLRGEAPKKFVNVCFREERMPVKEGWKRSTTPINIPLLVPIIERIIELSDWKPTGDNCGAIVLSPDLS2

<210> 4

<211> 339

<212> PRT

<213> Peroxidase CciUPO of Coprinopsis cinerea

<400> 4

AFPPPPPEPIKDPWLKLVNDRAHPWRPLRRGDVRGPCPGLNTLASHGYLPRDGVATPAQIITAVQEGFNMEYGIATFVTYAAHLVDGNPLTNLISIGGKTRKTGPDPPPPAIVGGLNTHAVFEGDASMTRGDFHLGDNFNFNQTLWEQFKDYSNRYGGGRYNLTAAAELRWARIQQSMATNGQFDFTSPRYFTAYAESVFPINFFTDGRLFTSNTTAPGPDMDSALSFFRDHRYPKDFHRAPVPSGARGLDVVAAAYPIQPGYNADGKVNNYVLDPTSADFTKFCLLYENFVLKTVKGLYPNPKGFLRKALETNLEYFYQSFPGSGGCPQVFPWGKSDS 339

<210> 5

<211> 330

<212> PRT

<213> Peroxidase MthUPO of Myceliophthora thermophila

<400> 5

AGFDTWSPPGPTDVRAPCPMLNTLANHGFLPHDGKDITREQTENALFDALNINKTLASFLFDFALTTNPKNTSTFSLNDLGNHNILEHDASLSRADAYFGNVLQFNQTVFDETKTYWEGDTIDLRMAAKARLGRIKTSQATNPTYSMSELGDAFTYGESAAYVVVLGDLESRTVNRSWVEWFFEHEQLPQHLGWKRPAVSFEEEDLNRFMEEIEKYTKGLEGSNSTSGSQKHRLPHF

<210> 6

<211> 244

<212> PRT

<213> Peroxidase TteUPO of Thielaviaterrestris

<400> 6

AGFDSWHPPAPGDRRGPCPMLNTLANHGFLPHNGRNITKEITVNALNSALNVNKTLGELLFNFAVTTNPQPNATFFDLDHLSRHNILEHDASLSRADYYFGHDDHTFNQTVFDQTKSYWKTPIIDVQQAANARLARVLTSNATNPTFVLSQIGEAFSFGETAAYILALGEDRVSGTVPRQWVEYLFENERLPLELGWRRAKEVISNSDLDQLTNRVINATGALANITRKIKVR

<210> 7

<211> 238

<212> PRT

<213> Peroxidase MroUPO of Marasmius rotula

<400> 7

ASAHPWKAPGPNDSRGPCPGLNTLANHGFLPRNGRNISVPMIVKAGFEGYNVQSDILILAGKIGMLTSREADTISLEDLKLHGTIEHDASLSREDVAIGDNLHFNEAIFTTLANSNPGADVYNISSAAQVQHDRLADSLARNPNVTNTDLTATIRSSESAFFLTVMSAGDPLRGEAPKKFVNVFFREERMPIKEGWKRSTTPITIPLLGPIIERITELSDWKPTGDNCGAIVLSPELS

Claims (12)

1. a method for peroxidase catalyzed oxidation of aromatic hydrocarbons and derivatives thereof, comprising the steps: Taking benzene containing substituents shown in formula I as a reaction substrate, in a buffer reaction system or a pure substrate reaction system, in the presence of peroxide and a peroxidase with heme activity, formula II or formula is obtained. The alcohol, aldehyde or ketone product of III;

Wherein, R can be one or more of hydrogen, methyl, propyl, isopropyl, halogen (F, Cl, Br, I), carbonyl, aryl, nitro, and amino, and it is present in the benzene ring. Adjacent, intermittent or contraposition; R ₁ can be H, methyl, propyl, isopropyl, carbonyl, aryl. 2. method according to claim 1, is characterized in that: in buffer solution reaction system, substrate concentration is 1.0 mmol/L-100 mmol/L; In pure substrate reaction system, substrate concentration is 6.0 mmol/L L -8.0 mol/L. 3. The method according to any one of claims 1-2, characterized in that: small molecular alcohol, acetonitrile, ethyl acetate or dimethyl sulfoxide are also added in the reaction system. 4. method according to claim 3 is characterized in that: described small molecule alcohol is one or more mixtures in methanol, ethanol, isopropanol; Small molecule alcohol accounts for 1-60% of reaction system volume . 5. The method according to any one of claims 1-2, wherein the buffer is any one of phosphate buffer, citrate buffer and tris(hydroxymethyl)aminomethane buffer species with a pH of 3.5-11. 6. The method according to claim 5, wherein the pH value is 4.5-9.5. 7. The method according to any one of claims 1-2, wherein the peroxidase is selected from: (1) Haloperoxidase Lfu CPO derived from Leptoxyphium fumago ; (2) Haloperoxidase Cf CPO derived from Caldariomyces fumago ; (3) Peroxidase Aae UPO derived from Agrocybe aegerita ; (4) Peroxidase Cgl UPO derived from Chaetomium globosum ; (5) Peroxidase Mwe UPO derived from Marasmius wettsteinii ; (6) Peroxidase Cci UPO derived from Coprinopsis cinerea ; (7) Peroxidase Mth UPO derived from Myceliophthora thermophila ; (8) Peroxidase Tte UPO derived from Thielavia terrestris ; (9) Peroxidase Mro UPO derived from Marasmius rotula ; (10) Peroxidase Cra UPO derived from Coprinellus radians ; (11) Peroxidase Pab UPO from Psathyrella aberdarensis . 8 . The method according to claim 7 , wherein the peroxidase plays a catalytic role in the form of whole cells, crude enzyme powder, crude enzyme liquid, pure enzyme or immobilized enzyme. 9 . 9. according to the method described in any one of claim 1-2, it is characterized in that: in reaction system, the concentration of peroxidase is 5nmol/L-5 μmol/L, and its activity is 0.02～04 U/ml . 10. The method according to claim 9, wherein in the reaction system, the concentration of peroxidase is 200 nmol/L-1000 nmol/L. 11. The method according to any one of claims 1-2, wherein the reaction time is 10-48h, and the reaction temperature is 20-50°C. 12. The method according to any one of claims 1-2, characterized in that: further after the reaction ends, for the buffer reaction system, extract with an organic solvent, dry, filter, separate and purify; or for pure reaction bottom The reaction system of the product is directly distilled to obtain the product.

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