Application of reducing agent in peroxidase catalytic reaction

Abstract

The invention discloses application of a reducing agent in heme peroxidase catalytic reaction, the reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid and pyrogallic acid, and the reducing agent has a remarkable promotion effect on oxidation reaction and hydroxylation reaction catalyzed by heme peroxidase. A new scheme is provided for improving the catalytic oxidation efficiency of the heme peroxidase, and the development and application potential is huge.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and more specifically, relates to the application of reducing agents in peroxidase-catalyzed reactions. Background Art

[0002] Heme-containing peroxidases (abbreviated as heme peroxidases) are widely distributed in various organisms such as animals, plants, fungi, and prokaryotes. These enzymes play important roles in the oxidative metabolism of a variety of exogenous and endogenous substrates. Among the heme-containing peroxidases, unspecific peroxidases (UPOs) have attracted great interest as "ideal biocatalysts" because they can use hydrogen peroxide (H2O2) as an oxidant to introduce oxygen into organic molecules containing inert C-H bonds ( Figure 1 A). In addition to UPOs, certain cytochrome P450s, such as CYP152 peroxidases (OleT JE , P450 SPα and P450 BSβ ) are also classified as H2O2-dependent peroxygenases. These enzymes can catalyze the hydroxylation or decarboxylation of fatty acids using H2O2 ( Figure 1 A).

[0003] It is well known that P450 monooxygenases require redox partners (ferredoxin and ferredoxin reductase) and the reducing agent NAD(P)H to activate molecular oxygen ( Figure 1 B), while both UPOs and P450 peroxidases exhibit a simpler catalytic structure, and they can directly generate iron-oxide-based heme (Compound I, Cpd I) as a catalytically active intermediate from H2O2 ( Figure 1 D), without a complex electron transfer chain. Although UPOs and P450 peroxidases have obvious advantages as selective oxidation functionalization catalysts, their peroxidative activity depends on H2O2 and there is irreversible oxidative inactivation, which greatly limits their industrial applications in selective oxidation functionalization chemistry. Summary of the Invention

[0004] Based on the known properties of heme peroxidases, we propose as Figure 1Regarding the idea of C, can a reducing agent be used to activate molecular oxygen to catalyze the reactions of UPO and P450 peroxidase, so as to get rid of the dependence on hydrogen peroxide and the irreversible inactivation of the enzyme. In our research on the catalytic oxidation reaction and hydroxylation reaction of heme peroxidase, it was found that adding reducing agents such as ascorbic acid had a significant promoting effect, and when this reducing agent participated in the catalytic reaction as a cosubstrate, oxygen was required, that is, this reaction was an oxygen-dependent reaction. Thus, a new O2 / reducing agent-dependent catalytic pathway of heme peroxidase was discovered. The discovery of this new pathway provides a new method for improving the catalytic oxidation efficiency of heme peroxidase. Specifically, the present invention includes the following technical solutions.

[0005] The first aspect of the present invention provides the use of a reducing agent in the peroxidase-catalyzed reaction, and the reducing agent is a reducing organic compound that can participate in the electron transfer process in the biological metabolic pathway (or biosynthetic pathway); the reaction refers to the oxidation reaction and / or hydroxylation reaction.

[0006] Preferably, the above-mentioned reducing agent is selected from ascorbic acid (AscA), dehydroascorbic acid (DHA), gallic acid (GA), and pyrogallic acid (PA).

[0007] Specifically, the above application is used to improve the catalytic efficiency of peroxidase in catalyzing the oxidation reaction and / or hydroxylation reaction of an organic compound containing an inert C-H bond.

[0008] Preferably, the oxygen source for the above oxidation reaction or hydroxylation reaction is oxygen or air, rather than hydrogen peroxide / H2O2.

[0009] In a preferred embodiment, the above peroxidase is selected from the following group: heme peroxidase, i.e., non-specific peroxidase (UPO), P450 peroxidase, chloroperoxidase (CPO), horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH), tryptophan hydroxylase (TrpH).

[0010] Specifically, the above non-specific peroxidase is selected from the following group: AaeUPO (NCBI accession number: B9W4V6.1) derived from Agrocybe aegerita, preferably the mutant PaDa I (PDB accession number: 2YOR) of AaeUPO, MroUPO (NCBI accession number: 5FUJ_A) derived from Marasmius rotula;

[0011] The above P450 peroxidase is selected from the following group: P450 derived from Sphingomonas paucimobilis SPα(NCBI accession number: WP_017980797.1), a P450 derived from Bacillus subtilis BSβ (NCBI accession number: WP_003246284.1), an OleT derived from Jeotgalicoccus sp. ATCC 8456 JE (NCBI accession number: WP_198687844.1);

[0012] The above-mentioned chloroperoxidase (CPO) is CfuCPO (NCBI accession number: P04963.3) derived from Caldariomyces fumago

[0013] The above-mentioned horseradish peroxidase (HRP) is HRP (NCBI accession number: P00433.2) derived from Armoracia rusticana

[0014] The above-mentioned tyrosine hydroxylase (TyrH) is TyrH (NCBI accession number: WP_051872337) derived from Streptomyces sclerotialus

[0015] The above-mentioned tryptophan hydroxylase (TrpH) is TrpH (NCBI accession number: WP_242375644.1) derived from Actinomadura luzonensis

[0016] When the above-mentioned peroxidase catalyzes the oxidation reaction and / or hydroxylation reaction of the reaction substrate, a reducing agent such as ascorbic acid (AscA), dehydroascorbic acid (DHA), gallic acid (GA) or pyrogallic acid (PA) is added to the reaction system as a co-substrate, and the addition amount of the reducing agent is linearly related to the amount of the reaction substrate used

[0017] The second aspect of the present invention is to provide a peroxidase catalytic system containing a reducing agent, wherein the reducing agent is selected from ascorbic acid (AscA), dehydroascorbic acid (DHA), gallic acid (GA) and pyrogallic acid (PA); the peroxidase is a heme-containing peroxidase (abbreviated as heme peroxidase), and is selected from the following group: unspecific peroxidase (UPO), P450 peroxidase, chloroperoxidase (CPO), horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH).

[0018] In the above peroxidase catalytic system, the peroxidase is selected from the following group: heme peroxidase, i.e., non-specific peroxidase (UPO), P450 peroxidase, chloroperoxidase (CPO), horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH), tryptophan hydroxylase (TrpH). Among them, the non-specific peroxidase can be selected from the following group: AaeUPO (NCBI accession number: B9W4V6.1) derived from Agrocybe aegerita, preferably the mutant PaDa I of AaeUPO (PDB accession number: 2YOR), MroUPO (NCBI accession number: 5FUJ_A) derived from Marasmius rotula;

[0019] The P450 peroxidase is selected from the following group: P450 derived from Sphingomonas paucimobilis SPα (NCBI accession number: WP_017980797.1), P450 derived from Bacillus subtilis BSβ (NCBI accession number: WP_003246284.1), OleT derived from Jeotgalicoccus sp. ATCC 8456 JE (NCBI accession number: WP_198687844.1);

[0020] The chloroperoxidase (CPO) is CfuCPO (NCBI accession number: P04963.3) derived from Caldariomyces fumago;

[0021] The horseradish peroxidase (HRP) is HRP (NCBI accession number: P00433.2) derived from Armoracia rusticana;

[0022] The tyrosine hydroxylase (TyrH) is TyrH (NCBI accession number: WP_051872337) derived from Streptomyces sclerotialus;

[0023] The tryptophan hydroxylase (TrpH) is TrpH (NCBI accession number: WP_242375644.1) derived from Actinomadura luzonensis.

[0024] The third aspect of the present invention lies in providing the use of the above peroxidase catalytic system in oxidation reactions and / or hydroxylation reactions.

[0025] Optionally, the peroxidase is a non-specific peroxidase AaeUPO (NCBI accession number: B9W4V6.1) derived from Agrocybe aegerita, preferably the AaeUPO mutant PaDa I (PDB accession number: 2YOR). The reaction substrate is ethylbenzene as shown in 1a, and the reaction products are (R)-1-phenylethanol as shown in 1b and acetophenone as shown in formula 1c:

[0026] Or

[0027] The peroxidase is a non-specific peroxidase MroUPO (NCBI accession number: 5FUJ_A) derived from Marasmius rotula. The reaction substrate is cyclohexane as shown in 2a, and the products are cyclohexanol as shown in 2b and cyclohexanone as shown in 2c:

[0028] Or

[0029] The peroxidase is a chloroperoxidase CfuCPO (NCBI accession number: P04963.3) derived from Caldariomyces fumago. The reaction substrate is benzyl methyl sulfide as shown in 3a, and the product is a sulfoxide compound as shown in 3b:

[0030] Or

[0031] The peroxidase is a P450 peroxygenase. The reaction substrate is lauric acid as shown in formula 4a, and the products are (2R)-hydroxylauric acid as shown in 4b and (3S)-hydroxylauric acid as shown in 4c:

[0032]

[0033] The peroxidase is tyrosine hydroxylase TyrH (NCBI accession number: WP_051872337). The reaction substrate is L-tyrosine as shown in 5a, and the product is dopa as shown in 5b:

[0034] Or

[0035] The peroxidase is tryptophan hydroxylase TrpH (NCBI accession number: WP_242375644.1). The reaction substrate is L-tryptophan as shown in 6a, and the product is 5-hydroxytryptophan as shown in 6b:

[0036] Or

[0037] The peroxidase is horseradish peroxidase HRP (NCBI accession number: P00433.2), the reaction substrate is 3,3',5,5'-tetramethylbenzidine (TMB) shown in 7a, and the products are the dimer shown in 7b and the quinoid conjugated monomer shown in 7c:

[0038]

[0039] Optionally, hydrogen peroxide may not be used as the oxygen source in the above oxidation reaction.

[0040] As an alternative embodiment, the above peroxidase is in the form of its expressing microorganism. The microorganism is, for example, Escherichia coli or yeast.

[0041] Optionally, other reducing agents such as NADPH (nicotinamide adenine dinucleotide phosphate, coenzyme II) and NADH (nicotinamide adenine dinucleotide, coenzyme I) may not be added to the reaction system catalyzed by P450 peroxidase, i.e., cytochrome P450 (abbreviated as P450).

[0042] The present invention discovers that several reducing agents, AscA, DHA, GA, and PA, can drive the catalytic reaction of heme-containing peroxidase under aerobic conditions and have much higher catalytic efficiency than hydrogen peroxide. Experiments prove that the oxygen in the product comes from oxygen and demonstrate the mutual influence between the enzyme and the reducing agent. When ascorbic acid is added to the reaction of AaeUPO catalyzing ethylbenzene, the TTN reaches 632100, which is the highest value reported so far. In addition, experiments confirm that these reducing agents can replace the traditional activator hydrogen peroxide for the color reaction of horseradish peroxidase (HRP) catalyzing the substrate tetramethylbenzidine (TMB), which also indicates the great potential of the present invention in industrial applications. Description of the Drawings

[0043] Figure 1 is a schematic diagram of the principle of the catalytic oxidation reaction of heme peroxidase. The figure shows different catalytic pathways for heme peroxidase to generate a ferryl oxygen intermediate (Compound I, Cpd I) as an oxidative species. Among them, A: Heme peroxidase uses hydrogen peroxide H2O2 to introduce oxygen into an organic molecule containing an inert C-H bond; B: P450 monooxygenase requires redox partners (ferredoxin and ferredoxin reductase) and a reducing agent NAD(P)H to activate molecular oxygen; D: Heme peroxidase catalyzes H2O2 to generate iron-oxide heme (Cpd I); C: The ideal concept to be achieved (a new catalytic pathway dependent on an oxidant).

[0044] Figure 2Summary of the catalytic benzyl hydroxylation of ethylbenzene (1a) by non-specific peroxidase AaeUPO via the O2 / reductant-dependent pathway. Among them, A: Reaction formula of AaeUPO-catalyzed ethylbenzene (1a); B: Comparative bar chart of the catalytic oxidation reaction efficiency (initial reaction rate) of 8 reductants assisting AaeUPO; C: HPLC result comparison chart of AaeUPO-catalyzed ethylbenzene reaction in different environments; D: Comparative bar chart of the initial reaction rate under aerobic and anaerobic conditions; E: Through 18 18O-labeling experiment to investigate that the oxygen in product 1b comes from air; F: Time-course curve of AaeUPO-catalyzed ethylbenzene reaction with AscA as a cosubstrate.

[0045] Figure 3 Shows the process of identifying the oxidation product dehydroascorbic acid (DHA) of ascorbic acid (AscA) as a cosubstrate for the AaeUPO-catalyzed oxidation reaction. Among them, A: Reaction formula of ascorbic acid AscA to DHA and subsequent DHAA and DKG; B: Comparative bar chart of catalytic activities (initial reaction rates) under the conditions of adding AscA, DHA, or DKG to the reaction system of AaeUPO-catalyzed ethylbenzene respectively; C: Comparative bar chart of catalytic activities (initial reaction rates) under anaerobic and aerobic conditions with DHA as a cosubstrate; D: Comparative bar chart of the consumption rates of AscA and DHA with and without AaeUPO.

[0046] Figure 4 Shows the identification of gallic acid (GA) and pyrogallic acid (PA) as cosubstrates for the AaeUPO-catalyzed benzyl hydroxylation of ethylbenzene 1a. Among them, A: Molecular structural formulas of gallic acid and pyrogallic acid; B: Comparative bar chart of enzyme catalytic activities (initial reaction rates) under aerobic and anaerobic conditions; C: Comparative bar chart of the consumption rates of GA and PA with and without AaeUPO.

[0047] Figure 5 Shows the catalytic oxidation reactions of different UPOs and P450 peroxidases using H2O2 and different reductants AscA, DHA, GA, and PA respectively. Among them, A: Comparative bar chart of the reaction of AaeUPO-catalyzed ethylbenzene 1a; B: Comparative bar chart of the reaction of MroUPO-catalyzed cyclohexane 2a; C: Comparative bar chart of the reaction of CfuCPO-catalyzed benzyl methyl sulfide 3a; D: P450 SPα Catalyzed lauric acid 4a reaction comparative bar chart; E: P450 BSβ Catalyzed lauric acid 4a reaction comparative bar chart; F: OleT JE Catalyzed lauric acid 4a reaction comparative bar chart.

[0048] Figure 6Shows the scale-up reaction of the biocatalytic system using a reducing agent as a raw material to prepare (R)-1-phenylethanol (1b) and α-OH lauric acid (4b). Among them, A: Reaction formula for the preparation of (R)-1-phenylethanol (1b) from ethylbenzene (1a) catalyzed by AaeUPO; B: Photo of the fermentation tank of AaeUPO cells and the reaction product; C: Product concentration progress curve of the reaction catalyzing ethylbenzene; D: P450 SPα Reaction formula for the preparation of α-OH lauric acid (4b) from lauric acid 4a catalyzed by P450; E: P450 SPα Photo of the fermentation tank of P450 cells and the reaction product; F: Product concentration progress curve of the reaction catalyzing lauric acid. Detailed implementation method

[0049] The discovery of the new O2 / reducing agent-dependent heme peroxidase-catalyzed pathway of the present invention has opened up a synthetic method for peroxidase-catalyzed oxidation reactions and / or hydroxylation reactions that avoids using hydrogen peroxide / H2O2 as a raw material, and only requires reaction in an air environment.

[0050] It was unexpectedly found in the experiment that the catalytic efficiency of heme peroxidase using oxygen driven by reducing agents AscA, DHA, GA, and PA is significantly improved compared to using hydrogen peroxide.

[0051] In this article, the above-mentioned terms "(catalytic efficiency) improvement" or "increase" mean an increase of at least 100% compared to the reference level, for example, at least 1 time, at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 20 times the increase compared to the reference level.

[0052] Furthermore, when the heme peroxidase is P450, the reaction system can even catalyze the oxidation of inert C-H bonds in the reaction substrate without adding the commonly used reducing agents NADPH and NADH, thus eliminating the need for the assistance of glucose dehydrogenase (GDH) / glucose or alcohol dehydrogenase (ADH) / isopropanol, which is economically beneficial.

[0053] In a specific application implementation method, the heme peroxidase may not be in the form of a pure enzyme, but in the form of the cells of its expressing microorganism or the crude enzyme of the fermentation broth in the reaction system. The microorganisms include bacteria and fungi. For example, the microorganism can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, or Saccharomyces cerevisiae.

[0054] When used as a biocatalyst for catalyzing the oxidation reaction and / or hydroxylation reaction of organic compounds containing inert C-H bonds, the peroxidase of the present invention can be in the form of an enzyme or a microbial cell. The form of the enzyme includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, enzyme immobilized on a carrier, etc.; the form of the microbial cell includes viable microbial cell, dead microbial cell, immobilized microbial cell, etc.

[0055] When microorganisms such as Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris no longer carry out fermentation and proliferation but are used for enzymatic catalysis reactions, they are themselves a natural immobilized enzyme, and without the need for disruption treatment, or even extraction and purification treatment, they can be used as an enzyme preparation for catalysis reactions.

[0056] The following further describes the present invention in detail with specific examples. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0057] Embodiment

[0058] In the examples herein, the addition amounts, contents and concentrations of various substances are involved, and the percentage content, unless otherwise specified, refers to the mass percentage content.

[0059] In the examples herein, if no specific description is made for the reaction temperature or operating temperature, the temperature generally refers to room temperature (10-40 °C).

[0060] Materials and Methods

[0061] Compounds such as ascorbic acid, dehydroascorbic acid, gallic acid, pyrogallic acid, ethylbenzene, cyclohexane, benzyl methyl sulfide, lauric acid, etc. used in the examples were all purchased from Sigma-Aldrich.

[0062] Mutants PaDa I (PDB accession number: 2YOR), MroUPO (NCBI accession number: 5FUJ_A), P450 peroxygenase P450 of non-specific peroxidase AaeUPO (NCBI accession number: B9W4V6.1) SPα (NCBI accession number: WP_017980797.1), P450 BSβ (NCBI accession number: WP_003246284.1), OleT JE(NCBI accession number: WP_198687844.1), chloroperoxidase CfuCPO (NCBI accession number: P04963.3), horseradish peroxidase HRP (NCBI accession number: P00433.2), tyrosine hydroxylase TyrH (NCBI accession number: WP_051872337), and tryptophan hydroxylase TrpH (NCBI accession number: WP_242375644.1) can all be purchased commercially or be recombinant enzyme proteins provided by the research group of Li Aitao from the College of Life Sciences, Hubei University.

[0063] The engineering bacteria expressing these enzymes were all constructed and preserved by the research group of Li Aitao from the College of Life Sciences, Hubei University. Any unit or individual can obtain this microorganism for verifying this invention, but it shall not be used for other purposes without the permission of Hubei University, including development and utilization, scientific research, and teaching.

[0064] The detection methods of high-performance liquid chromatography and gas chromatography are shown in the following table:

[0065]

[0066]

[0067] Example 1: Screening and Identification of Reducing Agents for Reconstituting UPO Activity

[0068] 1. Screening of Reducing Agents

[0069] Taking the reaction of AaeUPO catalyzing ethylbenzene ( Figure 2 A in it) as an example, eight reducing agents ( Figure 2 B in it), ascorbic acid (AscA), resorcinol (RC), hydroquinone (HQ), catechol (CC), CA, QA, FA, and L-cysteine (L-Cys), were screened to see if they could be used as cosubstrates.

[0070] The reaction system was 1 mL, including: 10 mM ethylbenzene, 20 mM reducing agent, 0.1 μM AaeUPO, 5% (m / v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 0.1 M potassium phosphate buffer at pH 8.0, and the reaction was carried out at 30 °C. The reaction results are shown in Figure 2 B in it. The comparison results showed that the group using ascorbic acid as the reducing agent and cosubstrate showed obvious catalytic activity.

[0071] The reaction conditions using ascorbic acid as the reducing agent and oxygen source were further investigated. The comparison chart of HPLC results is shown in Figure 2As shown in C. The comparison results show that the catalytic efficiency of ascorbic acid (rAaeUPO+1a+AscA, III) in promoting the enzymatic catalytic oxidation reaction in an air environment is significantly higher than that of hydrogen peroxide H2O2 as the oxygen source (rAaeUPO+1a+H2O2).

[0072] 2. The participation of the reductant as a cosubstrate in the catalytic reaction is an oxygen-dependent reaction

[0073] To investigate whether oxygen is required for the catalytic reaction, the reactions were designed to be carried out under aerobic and anaerobic conditions respectively. The reaction results are as Figure 2 shown in D. Under anaerobic conditions, trace amounts of product 1b were detected, and the catalytic reaction hardly proceeded; under aerobic conditions, a large amount of product 1b was detected, and the catalytic reaction proceeded smoothly. This indicates that when the reductant ascorbic acid acts as a cosubstrate in the catalytic reaction, oxygen is required, that is, this reaction is an oxygen-dependent reaction.

[0074] Next, as Figure 1 shown in E, we also proved by 18 18O labeling experiments that the oxygen in product 1b comes from the oxygen in the air. This further proves that the participation of the reductant AscA as a cosubstrate in the catalytic reaction is an oxygen-dependent reaction.

[0075] 3. Time course of the AaeUPO-catalyzed ethylbenzene reaction with AscA as the cosubstrate

[0076] To study the catalytic ability of AaeUPO when AscA is used as a cosubstrate, we added 100 mM of substrate 1a and 400 mM of AscA and carried out the catalytic reaction at pH 8.0. See Figure 2 F. After 72 h of reaction, the total products (1b + 1c) exceeded 60 mM, and the total turnover number reached 632,100 ± 37,073, which is the highest record reported so far for the free UPO-catalyzed reaction.

[0077] Example 2: Identification of the reconstitution of UPO activity by the oxidation product of AscA

[0078] It is known that AscA can be smoothly oxidized in aqueous solution to form dehydroascorbic acid (DHA), and then hydrolyzed to form 2,3-diketol-gulonic acid (DKG). In addition, DHA mainly exists in water in the form of DHA hydrate hemiacetal (DHAA) ( Figure 3 shown in A). Accordingly, we proposed that the further oxidation products (DHA, DHAA or DKG) may also be able to participate in the reaction.

[0079] To verify this hypothesis, the catalytic activity (initial reaction rate) of AaeUPO was compared by adding AscA, DHA, or DKG as reducing agents to the reaction system of AaeUPO-catalyzed ethylbenzene, respectively. See Figure 3 in B, the results showed that in the presence of DHA, the initial reaction rate reached 144 min -1 , which was 58% faster than the reaction rate with the addition of AscA. No activity was detected when using DKG. These findings demonstrated that DHA (or its hydrated hemiacetal DHA) could participate in the oxidation reaction catalyzed by UPO, while DKG could not.

[0080] Subsequently, experiments with DHA as a cosubstrate were carried out under anaerobic and aerobic conditions, respectively. Similar to the case when using AscA, high catalytic activity could only be achieved under aerobic conditions ( Figure 3 in C), indicating that both DHA and O2 are necessary for the enzyme-catalyzed reaction.

[0081] Next, to investigate whether the consumption of DHA depends on AaeUPO, we monitored the consumption rate of DHA in buffer solutions with and without AaeUPO. We observed that the presence of AaeUPO led to a 6-fold increase in the consumption rate of DHA and a 2-fold increase in the consumption of AscA compared to the reaction without the enzyme ( Figure 3 in D). These findings supported the conjecture that both AscA and DHA could serve as reducing agents to reconstruct UPO activity.

[0082] Example 3: Identification of gallic acid and pyrogallic acid as UPO cosubstrates

[0083] To investigate the potential of other reducing agents as cosubstrates for the UPO-catalyzed oxidation reaction, in addition to DHA and AscA found in fresh plant tissues, we hypothesized that polyphenolic compounds abundant in lignified tissues could also play a similar role. To verify this hypothesis, we detected polyphenolic compounds such as gallic acid (GA) and pyrogallic acid (PA) during the hydroxylation of compound 1a catalyzed by AaeUPO, respectively ( Figure 4 in A).

[0084] Referring to the experimental method in Example 2, the situation of AaeUPO-catalyzed 1a hydroxylation reaction was investigated with GA and PA as cosubstrates, respectively.

[0085] The study found that both GA and PA showed excellent catalytic activity as cosubstrates, with initial reaction rates of 265 min -1 and 141 min -1 ( Figure 4 in B), which was comparable to or even higher than the reaction rate when DHA was used as a cosubstrate.

[0086] In addition, consistent with previous findings, we found that GA and PA could only react under aerobic conditions ( Figure 4 in B).

[0087] The study also showed that the addition of UPO enzyme significantly accelerated the consumption rate of GA and PA ( Figure 4 in C). These findings all support the conjecture that GA and PA can also act as reducing agents in the UPO-catalyzed oxidation reaction.

[0088] Example 4: Universality of the O2 / reducing agent-dependent catalytic pathway in heme peroxidases

[0089] To verify whether the O2 / reducing agent-dependent pathway in heme peroxidases is universal, we focused on five representative peroxidases and four reducing agents, AscA, DHA, GA, and PA. These included the "short" MroUPO (from Marasmius rotula) involved in the hydroxylation of cyclohexane, CfuCPO (from Caldariomyces fumago) mediating the sulfoxidation of phenyl methyl sulfide, and three P450 peroxidases (P450 from Sphingomonas paucimobilis SPα , P450 from Bacillus subtilis BSβ and OleT from Jeotgalicoccus sp. ATCC 8456 JE ) catalyzing the hydroxylation or decarboxylation of lauric acid.

[0090] A comparative experiment was carried out with reference to the experimental method in Example 2, and the results are as Figure 5 shown. Compared with the H2O2-dependent reaction, the reducing agent-driven UPO showed significantly higher catalytic activity ( Figure 5 in A and B). For CfuCPO with high H2O2 tolerance, only DHA and GA showed better catalytic performance relative to the H2O2-dependent process ( Figure 5 in C). For the α- or β-hydroxylation of lauric acid catalyzed by P450 SPα and P450 BSβ , all the reactions catalyzed by reducing agents had higher activity than the H2O2-dependent reaction ( Figure 5 in D and E). It is worth noting that GA and PA achieved nearly 100% substrate conversion. In the decarboxylation reaction catalyzed by P450 OleT JE , most of the reactions catalyzed by reducing agents performed better than H2O2, although GA showed relatively poor activity (but still better than H2O2), which requires further study. Figure 5In (F). The above experimental results demonstrate the broad feasibility of the O2 / reductant-dependent catalytic pathway in heme-containing peroxidases and exhibit excellent catalytic performance. We believe that this O2 / reductant-dependent pathway may also be applicable to many other heme-containing enzymes.

[0091] Example 5: Scale-up reaction for product preparation

[0092] To demonstrate the industrial application potential of the O2 / reductant-dependent catalytic pathway, we attempted to scale up the production of high-value-added products using a biocatalytic system with a reductant raw material. The key points of investigation were two specific reactions: the phenyl hydroxylation of ethylbenzene 1a by AaeUPO to produce (R)-1-phenylethanol (1b) and the α-hydroxylation of lauric acid 4a by P450 SPα to produce α-OH lauric acid (4b). We chose AscA as the reductant because of its lower cost and higher availability compared to DHA. It is worth noting that both of these products have extensive applications in industries such as food, cosmetics, and pharmaceuticals.

[0093] The peroxidase AaeUPO-expressing microorganism is Pichia pastoris X33 (No. D4C-6, constructed and preserved by the research group of Li Aitao, School of Life Sciences, Hubei University), and the peroxidase P450 SPα expressing microbial cells are Escherichia coli engineering bacteria (No. P5B-2, constructed and preserved by the research group of Li Aitao, School of Life Sciences, Hubei University).

[0094] The fermentation conditions of Escherichia coli are as follows: Pick a single colony from the engineering bacteria LB plate into 5 mL of liquid LB medium containing Kan and culture overnight at 37 °C and 220 rpm; then transfer it to a shake flask containing 100 mL of liquid TB medium according to an inoculation amount of 5 v / v%, and culture at 37 °C and 220 rpm until the OD 600 reaches 0.6 - 0.8, and transfer it as the seed liquid to a 5 L fermenter (fermentation medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, 0.5 g / L antifoaming agent, pH 7.0 - 7.5, and the liquid loading volume of each tank of fermentation broth is 2 L. Feed medium: 50% glycerol.); After inoculation, culture at 400 - 800 rpm / min and 37 °C, and control the dissolved oxygen within the range of 25 - 30%; when the cell OD 600After reaching 20, IPTG was added to induce enzyme expression, and the final concentration of IPTG was 0.2 mM. The culture was continued at 25 °C for 20 - 24 h. During the fermentation process, ammonia water was used to control the pH value at 6.8 - 7.0, and the aeration rate was controlled within the range of 2.8 - 3.2. After the fermentation was completed, the cells were centrifuged at 4 °C and 10,000 rpm for 10 min, the cells were collected, resuspended with 0.1 M KP 8.0 buffer, the cells were centrifuged and collected, and stored at -80 °C for later use.

[0095] The fermentation conditions of Pichia pastoris are as follows: The strain of D4C-6 was activated on a YPD plate and incubated in an inverted position in a constant temperature incubator at 28 °C for 2 days; single colonies were picked from the YPD plate and inoculated into 100 mL of BMGY medium (in a 1 L Erlenmeyer flask), and 2 flasks were inoculated and cultured at 28 °C and 220 rpm; after 48 h, it was inoculated into a 5 L fermenter containing 2.5 L of BSM medium, and ammonia water was added to control the pH at 5.5; observing the dissolved oxygen in the fermenter, after about 26 h, the glycerol in BSM was consumed, and at this time, 50% glycerol containing 12 mL / L of PTM1 was fed into the fermenter, samples were taken at intervals for detection, and when OD 600 = about 200, the addition of glycerol was stopped, and carbon source starvation was carried out for more than half an hour; methanol was fed into the fermenter to induce the expression of the target protein, and the methanol feeding rate was controlled to keep the dissolved oxygen at 20% - 30%; after 4 - 5 d of induction, the cells were centrifuged at 4 °C and 10,000 rpm for 15 min, and the supernatant of the fermentation broth was collected and stored at 4 °C for later use.

[0096] The cells can be further subjected to cell disruption treatment, and separated and purified by a conventional enzyme extraction process to obtain crude enzyme or pure enzyme.

[0097] An exemplary enzyme extraction and purification process is briefly described as follows:

[0098] Escherichia coli: Take the freeze-thawed cells, resuspend the cells with a buffer (pH 8.0, 100 mM potassium phosphate buffer, 5% glycerol), and break the cells using a high-pressure homogenizer. The cell lysate was centrifuged at 12,000 rpm for 30 min, the supernatant was recovered, filtered through a 0.22 μm filter membrane to obtain a crude enzyme solution, and affinity purification was carried out at 4 °C.

[0099] Pichia pastoris: Take the fermentation broth stored at 4 °C, centrifuge at 12,000 rpm for 30 min, recover the supernatant, filter through a 0.22 μm filter membrane to obtain a crude enzyme solution, and carry out affinity purification at 4 °C.

[0100] Purification: Take a pre-packed column and install it on an AKTA, and the crude enzyme solution was passed through the column at a speed of 1 mL / min. The column was rinsed with a gradient of 10 - 250 mM imidazole, observing the change in the UV value, eluting the impurity proteins, and collecting the target protein. Finally, it was concentrated and desalted using an ultrafiltration tube and stored at -80 °C for later use.

[0101] Catalytic ethylbenzene reaction system 1 L: 200 mM compound 1a, 40 g AscA, 0.25 μM pure AaeUPO enzyme (or fermentation supernatant), 5% (m / v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 0.1 M phosphate buffer, pH 8.0, react at 30 °C, as Figure 6 shown in A - C.

[0102] After the reaction, enantiopure (R)-1-phenylethanol (1b) at 9.9 g / L and acetophenone (1c) at 1.8 g / L were obtained, corresponding to a catalytic turnover number of 395600. This result represents the highest product titer for the production of (R)-1-phenylethanol. In addition, the purified 1b was 7.72 g and 1c was 1.33 g, with a purity exceeding 98%.

[0103] Catalytic lauric acid reaction system 1 L: 10 g compound 4a (add an additional 2 g at 72 h), 30 g AscA, OD 600 ≈40 (enzyme concentration is about 1.5 μM) P450 SPα bacterial cells (or cell lysate supernatant), 0.1 M phosphate buffer, pH 8.0, 30% (m / v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 10% (v / v) DMSO, react at 30 °C, as Figure 6 shown in D - F.

[0104] After the reaction, the conversion rate of substrate 4a reached 99%, and the yield of α-OH lauric acid (4b) was 91%, which was 11.82 g. The catalytic conversion rate of the product titer of α-hydroxy lauric acid (TTN = 43,760) also set a new record, more than 6 times higher than the previously reported value. These results once again demonstrate the important synthetic potential of the combination technology of reducing agent raw materials and peroxygenase.

[0105] Example 6: Reactions of reducing agents in other heme peroxidases

[0106] Based on the good performance of the above reducing agents in the catalytic reactions of heme peroxidases, we also tried the reactions of horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase (TrpH).

[0107] TyrH reaction system: 6 μM pure TyrH enzyme, 0.1 M phosphate buffer (pH 8.0), 10 mM L-Tyr, 20 mM reducing agent, react at 30 °C;

[0108] TrpH reaction conditions: 20 μM pure TrpH enzyme, 0.1 M phosphate buffer (pH 8.0), 3 mM L-Tyr, 20 mM reducing agent, react at 30 °C;

[0109] HRP reaction conditions: 25 mU of pure HRP enzyme, 0.7 mM tetramethylbenzidine (TMB), 0.1 M phosphate buffer (pH 7.0), 1 mM reducing agent, reaction at 30 °C;

[0110] The reaction results showed that the reducing agents AscA, DHA, GA, and PA were all reflected in the reactions catalyzed by horseradish peroxidase (HRP) on the substrate tetramethylbenzidine (TMB), tyrosine hydroxylase (TyrH) on the substrate tyrosine, and tryptophan hydroxylase (TrpH) on the substrate tryptophan. See Table 1.

[0111] Table 1. Catalytic oxidation reactions of HRP, TyrH, and TrpH using different reducing agents AscA, DHA, GA, and PA

[0112]

[0113] These results once again demonstrated the universality of the O2 / reducing agent-dependent catalytic pathway in heme peroxidases and also reflected the application potential of ascorbic acid (AscA), dehydroascorbic acid (DHA), gallic acid (GA), and pyrogallic acid (PA) as alternatives to the traditional activator hydrogen peroxide.

[0114] Results and Discussion

[0115] It should be understood that the non-specific peroxidase (UPO) described in the solution of the present invention may include various UPOs, such as AaeUPO, MroUPO, CciUPO, CglUPO, GmaUPO, HspUPO, MthUPO, etc.; the reaction substrates that can be catalyzed include all substrates that UPO can catalyze, such as ethylbenzene, cyclohexane, vitamin D3, phenol, testosterone, quercetin, styrene, fatty acid compounds, etc. Similarly, other heme peroxidases should also include all peroxidases of the same family and type and all their catalytic reactions.

[0116] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. Application of a reducing agent in a peroxidase-catalyzed reaction, characterized in that, The reducing agent is a reducing organic compound participating in the electron transfer process in the biological metabolic pathway; the reaction refers to an oxidation reaction and / or a hydroxylation reaction.

2. The application according to claim 1, characterized in that, The reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid, and pyrogallic acid.

3. The application according to claim 3, wherein The oxygen source for the oxidation reaction or hydroxylation reaction is oxygen or air.

4. The application according to claim 1, characterized in that, The peroxidase is selected from the following group: heme peroxidase, i.e., non-specific peroxidase, P450 peroxidase, chloroperoxidase, horseradish peroxidase, tyrosine hydroxylase, tryptophan hydroxylase.

5. The application according to claim 4, wherein The non-specific peroxidase is selected from the following group: the mutant PaDa I (PDB accession number: 2YOR) of AaeUPO (NCBI accession number: B9W4V6.1) derived from Agrocybe aegerita; MroUPO (NCBI accession number: 5FUJ_A) derived from Marasmius rotula. The P450 peroxidase is selected from the group consisting of: P450 derived from Sphingomonas paucimobilis SPα (NCBI accession number: WP_017980797.1), P450 derived from Bacillus subtilis BSβ (NCBI accession number: WP_003246284.1), OleT derived from Jeotgalicoccus sp. ATCC 8456 JE (NCBI accession number: WP_198687844.1); The chloroperoxidase is CfuCPO (NCBI accession number: P04963.3) derived from Caldariomyces fumago. The horseradish peroxidase is HRP (NCBI accession number: P00433.2) derived from Armoracia rusticana. The tyrosine hydroxylase is TyrH (NCBI accession number: WP_051872337) derived from Streptomyces sclerotialus. The tryptophan hydroxylase is TrpH (NCBI accession number: WP_242375644.1) derived from Actinomadura luzonensis.

6. The application according to claim 1, characterized in that The peroxidase catalyzes the oxidation reaction and / or hydroxylation reaction of the reaction substrate, and ascorbic acid, dehydroascorbic acid, gallic acid, or pyrogallic acid is added as a co-substrate in the reaction system.

7. A peroxidase catalytic system containing a reducing agent, characterized in that, The reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid, and pyrogallic acid; the peroxidase is selected from the following group: heme-containing peroxidase, i.e., non-specific peroxidase, P450 peroxidase, chloroperoxidase, horseradish peroxidase, tyrosine hydroxylase, tryptophan hydroxylase.

8. The peroxidase-catalyzed system according to claim 7, wherein The non-specific peroxidase is selected from the following group: AaeUPO (NCBI accession number: B9W4V6.1) derived from Agrocybe aegerita, preferably the mutant PaDa I (PDB accession number: 2YOR) of AaeUPO, MroUPO (NCBI accession number: 5FUJ_A) derived from Marasmius rotula. The P450 peroxygenase is selected from the following group: P450 derived from Sphingomonas paucimobilis SPα (NCBI accession number: WP_017980797.1), P450 derived from Bacillus subtilis BSβ (NCBI accession number: WP_003246284.1), OleT derived from Jeotgalicoccus sp. ATCC 8456 JE (NCBI accession number: WP_198687844.1); The chloroperoxidase is CfuCPO (NCBI accession number: P04963.3) derived from Caldariomyces fumago. The horseradish peroxidase is HRP derived from Armoracia rusticana (NCBI accession number: P00433.2); The tyrosine hydroxylase is TyrH derived from Streptomyces sclerotialus (NCBI accession number: WP_051872337); The tryptophan hydroxylase is TrpH derived from Actinomadura luzonensis (NCBI accession number: WP_242375644.1).

9. Use of the peroxidase catalytic system according to claim 7 or 8 in an oxidation reaction and / or a hydroxylation reaction.

10. The use according to claim 9, characterized in that, Hydrogen peroxide is not used as an oxygen source in the oxidation reaction.

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