Application of reducing agent in peroxidase catalytic reaction

Abstract

Disclosed in the present invention is an application of a reducing agent in a heme peroxidase catalytic reaction. The reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid and pyrogallic acid. The reducing agent has a significant promotion effect on oxidation reaction and hydroxylation reaction catalyzed by heme peroxidase.

Description

Application of reducing agents in peroxidase-catalyzed reactions Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to the application of a reducing agent in a peroxidase catalytic reaction. 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 an important role in the oxidative metabolism of a variety of exogenous and endogenous substrates. Among heme-containing peroxidases, nonspecific 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 CH bonds (Figure 1A). In addition to UPOs, certain cytochrome P450s, such as CYP152 peroxidase (OleT JE ,P450 SPα and P450 BSβ ) are also classified as H2O2-dependent peroxygenases. These enzymes can utilize H2O2 to catalyze the hydroxylation or decarboxylation of fatty acids (Figure 1A).

[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 1B), while UPO and P450 peroxidases both exhibit simpler catalytic structures and can directly generate oxidized iron-based heme (compound I, Cpd I) as a catalytically active intermediate from H2O2 (Figure 1D) without the need for a complex electron transport chain. Although UPO and P450 peroxidases have obvious advantages as selective oxidative functionalization catalysts, their peroxidative activity depends on H2O2 and they are subject to irreversible oxidative deactivation, which greatly limits their industrial application in selective oxidative functionalization chemistry. Summary of the Invention

[0004] Based on the known properties of heme peroxidase, we proposed the idea shown in Figure 1C: whether it is possible to use a reducing agent to activate molecular oxygen to cause UPO and P450 peroxidase to catalyze the reaction, thereby getting rid of the dependence on hydrogen peroxide and the irreversible inactivation of the enzyme. In our research on the oxidation and hydroxylation reactions catalyzed by heme peroxidase, we found that the addition of reducing agents such as ascorbic acid has a significant promoting effect, and when the reducing agent participates in the catalytic reaction as a co-substrate, oxygen is required to participate, that is, the reaction is an oxygen-dependent reaction. This has led to the discovery of a new O2 / reducing agent-dependent heme peroxidase catalytic pathway. 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 a peroxidase-catalyzed reaction, wherein the reducing agent is a reducing organic compound capable of participating in the electron migration process in a biological metabolic pathway (or biosynthetic pathway); the reaction is an oxidation reaction and / or a hydroxylation reaction.

[0006] Preferably, the 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 organic compounds containing inert CH bonds.

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

[0009] In a preferred embodiment, the peroxidase is selected from the group consisting of heme peroxidase, i.e., nonspecific peroxidase (UPO), P450 peroxidase, chloroperoxidase (CPO), horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase (TrpH).

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

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

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

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

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

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

[0016] When the peroxidase catalyzes the oxidation and / or hydroxylation of the reaction substrate, a reducing agent, ascorbic acid (AscA), dehydroascorbic acid (DHA), gallic acid (GA), or pyrogallic acid (PA), is added to the reaction system as a co-substrate. The amount of the reducing agent added is linearly correlated with the amount of the reaction substrate.

[0017] The second aspect of the present invention is to provide a peroxidase catalytic system comprising 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 (heme peroxidase for short), selected from the following group: nonspecific 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., nonspecific peroxidase (UPO), P450 peroxidase, chloroperoxidase (CPO), horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase (TrpH). The nonspecific peroxidase can be selected from the following group: AaeUPO (NCBI accession number: B9W4V6.1) from Agrocybe aegerita, preferably a mutant of AaeUPO, PaDa I (PDB accession number: 2YOR), and MroUPO (NCBI accession number: 5FUJ_A) from Marasmius rotula.

[0019] The P450 peroxidase is selected from the group consisting of: P450 from Sphingomonas paucimobilis; SPα (NCBI accession number: WP_017980797.1), P450 from Bacillus subtilis BSβ (NCBI accession number: WP_003246284.1), OleT 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 derived from the horseradish Armoracia rusticana (NCBI accession number: P00433.2);

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

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

[0024] The third aspect of the present invention is to provide use of the above-mentioned 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) from Agrocybe aegerita, preferably an AaeUPO mutant PaDa I (PDB accession number: 2YOR), the reaction substrate is ethylbenzene shown in formula 1a, and the reaction products are (R)-1-phenylethanol shown in formula 1b and acetophenone 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 shown in 2a, and the products are cyclohexanol shown in 2b and cyclohexanone shown in 2c:

[0028] or

[0029] The peroxidase is chloroperoxidase CfuCPO (NCBI accession number: P04963.3) derived from Caldariomyces fumago. The reaction substrate is thioanisole shown in 3a, and the product is a sulfoxide compound 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 formula 4b and (3S)-hydroxylauric acid as shown in formula 4c:

[0032] The peroxidase is tyrosine hydroxylase TyrH (NCBI accession number: WP_051872337), the reaction substrate is L-tyrosine shown in 5a, and the product is DOPA shown in 5b:

[0033] or

[0034] The peroxidase is tryptophan hydroxylase TrpH (NCBI accession number: WP_242375644.1), the reaction substrate is L-tryptophan shown in 6a, and the product is 5-hydroxytryptophan shown in 6b:

[0035] or

[0036] 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 quinone-type conjugated monomer shown in 7c:

[0037] Optionally, hydrogen peroxide may not be used as an oxygen source in the above oxidation reaction.

[0038] As an optional embodiment, the peroxidase is in the form of a microorganism expressing the peroxidase, such as Escherichia coli or yeast.

[0039] Alternatively, other reducing agents such as NADPH (nicotinamide adenine dinucleoside 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).

[0040] The present invention discovered that several reducing agents, including AscA, DHA, GA, and PA, can drive the catalytic reaction of heme-containing peroxidases under aerobic conditions, with significantly higher catalytic efficiency than hydrogen peroxide. Experiments confirmed that the oxygen in the product originates from oxygen and demonstrated the interaction between the enzyme and the reducing agent. Adding ascorbic acid to the AaeUPO-catalyzed reaction of ethylbenzene resulted in a TTN of 632,100, the highest value reported to date. Furthermore, experiments confirmed that these reducing agents can replace the traditional activator hydrogen peroxide in the color development reaction of horseradish peroxidase (HRP) catalyzed by the substrate tetramethylbenzyldiamine (TMB), indicating the significant potential of the present invention for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram of the principles of heme peroxidase-catalyzed oxidation reactions. The figure shows different catalytic pathways by which heme peroxidases generate a ferric oxide intermediate (Compound I, Cpd I) as an oxidizing species. A: Heme peroxidases use hydrogen peroxide (H2O2) to introduce oxygen into organic molecules containing inert C-H bonds; B: P450 monooxygenases require redox partners (ferredoxin and ferredoxin reductase) and the reducing agent NAD(P)H to activate molecular oxygen; D: Heme peroxidases catalyze H2O2 to generate ferric oxide heme (Cpd I); C: The desired concept (a new oxidant-dependent catalytic pathway).

[0042] Figure 2 is a summary of the benzyl hydroxylation of ethylbenzene (1a) catalyzed by the nonspecific peroxidase AaeUPO through an O2 / reducing agent-dependent pathway. A: The reaction equation for ethylbenzene (1a) catalyzed by AaeUPO; B: A bar graph comparing the efficiency (initial reaction rate) of the catalytic oxidation reaction of 8 reducing agents assisted by AaeUPO; C: A comparison of the HPLC results of ethylbenzene catalyzed by AaeUPO in different environments; D: A bar graph comparing the initial reaction rates under aerobic and anaerobic conditions; E: 18 O labeling experiment investigated whether the oxygen in product 1b originated from air; F: Time course curve of the ethylbenzene reaction catalyzed by AaeUPO with AscA as cosubstrate.

[0043] Figure 3 shows the identification of dehydroascorbic acid (DHA), an oxidation product of ascorbic acid (AscA), as a co-substrate in the AaeUPO-catalyzed oxidation reaction. A: The reaction equation for the conversion of ascorbic acid AscA to DHA and subsequently to DHAA and DKG; B: A bar graph comparing the catalytic activity (initial reaction rate) in the AaeUPO-catalyzed ethylbenzene reaction system with the addition of AscA, DHA, or DKG; C: A bar graph comparing the catalytic activity (initial reaction rate) with the addition of DHA as a co-substrate under anaerobic and aerobic conditions; D: A bar graph comparing the consumption rates of AscA and DHA in the presence and absence of AaeUPO.

[0044] Figure 4 shows the identification of gallic acid (GA) and pyrogallic acid (PA) as cosubstrates in the benzylic hydroxylation of ethylbenzene 1a catalyzed by AaeUPO. A: Molecular structures of gallic acid and pyrogallic acid; B: Bar graph comparing enzyme catalytic activity (initial reaction rate) under aerobic and anaerobic conditions; C: Bar graph comparing GA and PA consumption rates in the presence and absence of AaeUPO.

[0045] Figure 5 shows the oxidation reactions catalyzed by different UPOs and P450 peroxidases using H2O2 and different reducing agents AscA, DHA, GA, and PA. A: Comparative bar graph of the reaction of ethylbenzene 1a catalyzed by AaeUPO; B: Comparative bar graph of the reaction of cyclohexane 2a catalyzed by MroUPO; C: Comparative bar graph of the reaction of anisole 3a catalyzed by CfuCPO; D: Comparative bar graph of the reaction of P450 SPα Comparative bar chart of the reaction of lauric acid 4a; E: P450 BSβ Comparative bar chart of the reaction of lauric acid 4a catalyzed by OleT JE Comparative bar chart of the reactions catalyzed by lauric acid 4a.

[0046] Figure 6 shows the scaled-up reaction of the biocatalytic system using a reducing agent as the raw material to prepare (R)-1-phenylethanol (1b) and α-OH lauric acid (4b). A: The reaction equation for the preparation of (R)-1-phenylethanol (1b) from ethylbenzene (1a) catalyzed by AaeUPO; B: A photo of the fermentation of AaeUPO cells in a fermenter and the reaction products; C: The product concentration curve of the catalytic ethylbenzene reaction; D: The P450 SPα Reaction formula for the preparation of α-OH lauric acid (4b) from lauric acid 4a; E: P450 SPα Photos of the fermentation and reaction products in the bacterial fermentation tank; F: Product concentration progression curve of the catalytic lauric acid reaction. DETAILED DESCRIPTION

[0047] The discovery of the new O2 / reducing agent-dependent heme peroxidase catalytic pathway of the present invention has opened up a synthetic method for peroxidase-catalyzed oxidation and / or hydroxylation reactions that avoids the use of hydrogen peroxide / H2O2 as raw materials and only requires the reaction in an air environment.

[0048] The experiment unexpectedly found that the catalytic efficiency of heme peroxidase driven by reducing agents AscA, DHA, GA and PA in utilizing oxygen was significantly improved compared to when using hydrogen peroxide.

[0049] As used herein, the term "(catalytic efficiency) improvement" or "increase" as used above means an increase of at least 100% compared to a reference level, for example, an increase of at least 1-fold, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold compared to a reference level.

[0050] Furthermore, when the heme peroxidase is a P450, the oxidation of inert C-H bonds in the reaction substrate can be catalyzed in the reaction system even without the addition of the commonly used reducing agents NADPH and NADH, thereby eliminating the need for the assistance of glucose dehydrogenase (GDH) / glucose or alcohol dehydrogenase (ADH) / isopropanol, which is economically advantageous.

[0051] In a specific application embodiment, the heme peroxidase may not be in the form of a pure enzyme, but rather in the form of a bacterial cell of an expressing microorganism or a crude enzyme in a fermentation broth in a reaction system. The microorganism includes bacteria and fungi, for example, the microorganism may be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, or Saccharomyces cerevisiae.

[0052] When used as a biocatalyst to catalyze the oxidation and / or hydroxylation 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 enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, and enzymes immobilized on carriers; the microbial cells include living cells, dead cells, and immobilized cells.

[0053] When microorganisms such as Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris are no longer used for fermentation and proliferation but are used for enzyme-catalyzed reactions, they themselves become natural immobilized enzymes and do not require crushing or even extraction and purification. They can be used as enzyme preparations for catalytic reactions.

[0054] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0055] Example

[0056] The examples herein involve the addition amounts, contents and concentrations of various substances, wherein the percentages mentioned, unless otherwise specified, are by mass percentages.

[0057] In the examples herein, if no specific description is given for the reaction temperature or the operating temperature, the temperature generally refers to room temperature (10-40° C.).

[0058] Materials and methods

[0059] Ascorbic acid, dehydroascorbic acid, gallic acid, pyrogallic acid, ethylbenzene, cyclohexane, thioanisole, lauric acid and other compounds used in the examples were purchased from Sigma-Aldrich.

[0060] The mutants of nonspecific peroxidase AaeUPO (NCBI accession number: B9W4V6.1), PaDa I (PDB accession number: 2YOR), MroUPO (NCBI accession number: 5FUJ_A), P450 peroxygenase P450 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 as recombinant enzyme proteins provided by the research group of Li Aitao, School of Life Sciences, Hubei University.

[0061] The engineered bacteria expressing these enzymes were constructed and preserved by Li Aitao's research group at the School of Life Sciences of Hubei University. Any organization or individual can obtain these microorganisms for verification of the present invention, but they may not be used for other purposes, including development, utilization, scientific research, and teaching, without the permission of Hubei University.

[0062] The HPLC and GC detection methods are shown in the following table:

[0063] Example 1: Screening and identification of reducing agents that reconstitute UPO activity

[0064] 1. Screening of reducing agents

[0065] Taking the reaction of ethylbenzene catalyzed by AaeUPO (Figure 2A) as an example, eight reducing agents (Figure 2B) including ascorbic acid (AscA), resorcinol (RC), hydroquinone (HQ), catechol (CC), CA, QA, FA, and L-cysteine ​​(L-Cys) were screened for their potential as cosubstrates.

[0066] The reaction system consisted of 1 mL of 10 mM ethylbenzene, 20 mM reducing agent, 0.1 μM AaeUPO, 5% (m / v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), and 0.1 M potassium phosphate buffer (pH 8.0) at 30°C. The results are shown in Figure 2B. Comparative results show that the group using the reducing agent ascorbic acid as a cosubstrate exhibited significant catalytic activity.

[0067] Further investigations were conducted using ascorbic acid as a reducing agent and an oxygen source. A comparison of the HPLC results is shown in Figure 2, C. These results demonstrate that the catalytic efficiency of the enzyme-catalyzed oxidation reaction promoted by ascorbic acid in air (rAaeUPO + 1a + AscA, III) is significantly higher than that achieved using hydrogen peroxide (H2O2) as the oxygen source (rAaeUPO + 1a + H2O2).

[0068] 2. The reducing agent participates in the catalytic reaction as a co-substrate, which is an oxygen-dependent reaction.

[0069] To investigate whether the catalytic reaction requires oxygen, the reaction was conducted under both aerobic and anaerobic conditions. The results are shown in Figure 2(D). Under anaerobic conditions, trace amounts of product 1b were detected, indicating little catalytic reaction. Under aerobic conditions, significant amounts of product 1b were detected, indicating smooth catalytic reaction. This suggests that when the reducing agent ascorbic acid acts as a co-substrate in the catalytic reaction, oxygen is required, indicating that the reaction is oxygen-dependent.

[0070] Next, as shown in Figure 1E, we also pass 18 O labeling experiments demonstrated that the oxygen in product 1b originated from oxygen in the air, further demonstrating that the catalytic reaction catalyzed by the reducing agent AscA as a cosubstrate is an oxygen-dependent reaction.

[0071] 3. Time course of the reaction of ethylbenzene catalyzed by AaeUPO using AscA as a co-substrate

[0072] To investigate the catalytic activity of AaeUPO with AscA as a cosubstrate, we used 100 mM substrate 1a and 400 mM AscA at pH 8.0. As shown in Figure 2F, after 72 hours of reaction, the total product (1b + 1c) exceeded 60 mM, and the total turnover number reached 632,100 ± 37,073, which is the highest reported for a free UPO-catalyzed reaction to date.

[0073] Example 2: Identification of UPO activity reconstituted by AscA oxidation products

[0074] AscA is known to be readily oxidized in aqueous solution to form dehydroascorbic acid (DHA), which is then hydrolyzed to 2,3-diketo-l-gulonic acid (DKG). Furthermore, DHA exists primarily in water as hydrated DHA hemiacetal (DHAA) (Figure 3A). Based on this, we proposed that further oxidation products (DHA, DHAA, or DKG) may also participate in the reaction.

[0075] To verify this hypothesis, the catalytic activity (initial reaction rate) of AaeUPO was compared by adding AscA, DHA, or DKG as a reducing agent to the reaction system of ethylbenzene catalyzed by AaeUPO. As shown in Figure 3B, the results show that in the presence of DHA, the initial reaction rate reached 144 min -1 , 58% faster than the reaction rate with AscA. No activity was detected when DKG was used. These findings demonstrate that DHA (or its hydrated hemiacetal DHA) can participate in the UPO-catalyzed oxidation reaction, while DKG cannot.

[0076] Subsequently, experiments were conducted using DHA as a co-substrate under both anaerobic and aerobic conditions. Similar to the situation with AscA, high catalytic activity was achieved only under aerobic conditions (Figure 3, C), indicating that both DHA and O2 are required for the enzyme-catalyzed reaction.

[0077] Next, to investigate whether DHA consumption is dependent on AaeUPO, we monitored the rate of DHA consumption in buffer solutions with and without AaeUPO. We observed that the presence of AaeUPO resulted in a six-fold increase in DHA consumption and a two-fold increase in AscA consumption compared to the reaction without enzyme (Figure 3D). These findings support the hypothesis that both AscA and DHA can act as reducing agents to reconstitute UPO activity.

[0078] Example 3: Identification of Gallic Acid and Pyrogallic Acid as UPO Co-substrates

[0079] To investigate the potential of other reducing agents as co-substrates for UPO-catalyzed oxidation reactions, in addition to DHA and AscA found in fresh plant tissues, we hypothesized that polyphenolic compounds abundant in ligninized tissues could play a similar role. To test this hypothesis, we examined polyphenolic compounds, such as gallic acid (GA) and pyrogallic acid (PA), during the hydroxylation of compound 1a catalyzed by AaeUPO (Figure 4A).

[0080] Referring to the experimental method in Example 2, GA and PA were used as co-substrates to investigate the hydroxylation reaction of 1a catalyzed by AaeUPO.

[0081] The study found that GA and PA as co-substrates both showed excellent catalytic activity, with initial reaction rates of 265 min -1 and 141 minutes -1 (B in FIG4 ), the reaction rate is comparable to or even higher than that when DHA is used as a co-substrate.

[0082] Furthermore, consistent with previous findings, we found that GA and PA could only react under aerobic conditions (Fig. 4B).

[0083] The study also showed that the addition of UPO enzyme significantly accelerated the consumption of GA and PA (Figure 4C). These findings support the hypothesis that GA and PA can also act as reducing agents in the UPO-catalyzed oxidation reaction.

[0084] Example 4: Universality of O2 / reductant-dependent catalytic pathways in heme peroxidases

[0085] To verify whether the O2 / reductant-dependent pathway is universal in heme-containing peroxygenases, we focused on five representative peroxygenases and four reductants, AscA, DHA, GA, and PA. These included the "short" form MroUPO (from Marasmius rotula) involved in the hydroxylation of cyclohexane, CfuCPO (from Caldariomyces fumago) mediating the sulfoxidation of phenylmethyl sulfide, and three P450 peroxygenases (P450 from Sphingomonas paucimobilis). SPα , P450 from Bacillus subtilis BSβ and OleT from Jeotgalicoccus sp. ATCC 8456 JE ) catalyzes the hydroxylation or decarboxylation of lauric acid.

[0086] Comparative tests were conducted with reference to the experimental method in Example 2. The results are shown in Figure 5. Compared with the reaction dependent on H2O2, the reductant-driven UPO showed significantly higher catalytic activity (Figure 5 A and B). For CfuCPO with higher H2O2 tolerance, only DHA and GA showed better catalytic performance relative to the H2O2-dependent process (Figure 5 C). SPα and P450 BSβ For the α- or β-hydroxylation of lauric acid catalyzed by P450 OleT, all reductant-catalyzed reactions showed higher activity than the H2O2-dependent reactions (Figure 5D and E). Notably, GA and PA achieved nearly 100% substrate conversion. JE In the decarboxylation reactions catalyzed by most reductants, the performance was better than that of H2O2, although GA exhibited relatively poor activity (still superior to H2O2), which warrants further investigation (Figure 5F). These experimental results demonstrate the widespread feasibility of O2 / reductant-dependent catalytic pathways in heme-containing peroxygenases and demonstrate excellent catalytic performance. We believe that this O2 / reductant-dependent pathway may also be applicable to many other heme-containing enzymes.

[0087] Example 5: Amplification reaction of product preparation

[0088] To demonstrate the industrial application potential of the O2 / reductant-dependent catalytic pathway, we conducted a large-scale production attempt of high value-added products using a biocatalytic system with a reductant feedstock. The focus of the investigation was on two specific reactions: the phenyl hydroxylation of ethylbenzene 1a catalyzed by AaeUPO to produce (R)-1-phenylethanol (1b) and the P450 SPαAscA catalyzes the α-hydroxylation of lauric acid 4a to produce α-OH lauric acid (4b). We chose AscA as the reducing agent due to its lower cost and higher availability compared to DHA. Notably, both products have a wide range of applications in the food, cosmetics, and pharmaceutical industries.

[0089] The peroxidase AaeUPO expression microorganism is Pichia pastoris X33 (number D4C-6, constructed and preserved by Li Aitao's research group at the School of Life Sciences, Hubei University), and the peroxidase P450 SPα The expression microorganism is an engineered Escherichia coli (number P5B-2, constructed and preserved by the research group of Li Aitao, College of Life Sciences, Hubei University).

[0090] The fermentation conditions for E. coli were as follows: a single colony was picked from the engineered bacteria LB plate and transferred to 5 mL of liquid LB medium containing Kan, and cultured overnight at 37°C and 220 rpm; then, the inoculum was transferred to a shake flask containing 100 mL of liquid TB medium at a volume concentration of 5 v / v%, and cultured at 37°C and 220 rpm until the OD 600 When the pH reaches 0.6-0.8, the solution is transferred as seed liquid to a 5L fermenter (fermentation medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4.3H2O, 2.31g / L KH2PO4, 5g / L glycerol, 0.5g / L defoamer, pH 7.0-7.5, the fermentation liquid volume of each tank is 2L. Feed medium: 50% glycerol.); after inoculation, the solution is cultured at 400-800rpm / min and 37°C, and the dissolved oxygen is controlled within the range of 25-30%. When the bacterial OD 600 After reaching 20, IPTG was added to induce enzyme expression at a final concentration of 0.2 mM. Culture was continued at 25°C for 20-24 hours. Ammonia was used to control the pH of the fermentation process to 6.8-7.0, and the aeration rate was controlled within the range of 2.8-3.2. After fermentation, the cells were harvested by centrifugation at 10,000 rpm for 10 minutes at 4°C. The cells were resuspended in 0.1 M KP 8.0 buffer, collected by centrifugation, and refrigerated at -80°C until further use.

[0091] The fermentation conditions of Pichia pastoris were as follows: D4C-6 was activated on a YPD plate and incubated in an inverted incubator at 28°C for 2 days; a single colony was picked from the YPD plate and placed in 100 mL of BMGY medium (1 L triangular flask), inoculated into two flasks, and incubated at 28°C and 220 rpm; after 48 hours, the colony was inoculated into a 5 L fermentor containing 2.5 L of BSM medium, and ammonia was added to control the pH at 5.5; the dissolved oxygen content in the fermentor was observed. After approximately 26 hours, the glycerol in the BSM was completely consumed. At this time, 50% glycerol containing 12 mL / L of PTM1 was added to the fermentor, and samples were taken at intervals for testing.600 =200, stop adding glycerol and starve the fermenter of carbon source for more than half an hour; add methanol to the fermenter to induce the expression of target protein, control the methanol flow acceleration to keep the dissolved oxygen at 20% to 30%; after 4-5 days of induction, centrifuge at 4°C and 10,000 rpm for 15 minutes, collect the supernatant of the fermentation liquid, and refrigerate at 4°C for later use.

[0092] The bacteria can be further subjected to cell lysis treatment, and the crude enzyme or pure enzyme can be separated and purified from the bacteria through conventional enzyme extraction technology.

[0093] An exemplary enzyme extraction and purification process is briefly described as follows:

[0094] Escherichia coli: Resuspend frozen-thawed cells in buffer (pH 8.0, 100 mM potassium phosphate buffer, 5% glycerol) and disrupt the cells using a high-pressure homogenizer. Centrifuge the cell lysate at 12,000 rpm for 30 minutes, recover the supernatant, and filter through a 0.22 μm filter to obtain the crude enzyme solution. Affinity purification was performed at 4°C.

[0095] Pichia pastoris: Take the fermentation broth stored at 4°C and centrifuge it at 12,000 rpm for 30 min. Recover the supernatant and filter it through a 0.22 μm filter membrane to obtain the crude enzyme solution. Affinity purification operation is performed at 4°C.

[0096] Purification: Load the prepacked column onto an AKTA column and pass the crude enzyme solution through the column at 1 mL / min. Elute the column with a gradient of 10-250 mM imidazole, monitor the UV light readings, elute impurities, and collect the target protein. Finally, concentrate and desalt the solution using an ultrafiltration tube and store at -80°C until ready for use.

[0097] The catalytic ethylbenzene reaction system (1 L) includes: 200 mM compound 1a, 40 g AscA, 0.25 μM AaeUPO pure enzyme (or fermentation supernatant), 5% (m / v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 0.1 M phosphate buffer, pH 8.0, and the reaction is carried out at 30° C., as shown in FIG6A to C.

[0098] After the reaction, 9.9 g / L of enantiomerically pure (R)-1-phenylethanol (1b) and 1.8 g / L of acetophenone (1c) were obtained, corresponding to a catalytic turnover of 395,600. This result represents the highest product titer ever achieved for (R)-1-phenylethanol production. Furthermore, the purified yields of 1b were 7.72 g and 1c were 1.33 g, with purities exceeding 98%.

[0099] Catalytic lauric acid reaction system 1L: 10g compound 4a (additional 2g at 72h), 30g AscA, OD 600 ≈40 (enzyme concentration is about 1.5μM) P450SPα Bacteria (or supernatant of disrupted bacteria), 0.1 M phosphate buffer, pH 8.0, 30% (m / v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 10% (v / v) DMSO were reacted at 30°C, as shown in Figure 6 DF.

[0100] After the reaction, substrate 4a conversion reached 99%, and the yield of α-OH lauric acid (4b) was 91%, or 11.82 g. The catalytic conversion of α-hydroxylauric acid (TTN = 43,760) also set a new record, exceeding the previously reported value by more than six times. These results further demonstrate the significant synthetic potential of the combined reducing agent and peroxygenase technology.

[0101] Example 6: Reduction of other heme peroxidases

[0102] Based on the good performance of the above reducing agents in heme peroxidase-catalyzed reactions, we also tried reactions with horseradish peroxidase (HRP), tyrosine hydroxylase (TyrH), and tryptophan hydroxylase (TrpH).

[0103] TyrH reaction system: 6 μM pure TyrH enzyme, 0.1 M phosphate buffer (pH 8.0), 10 mM L-Tyr, 20 mM reducing agent, reaction at 30°C;

[0104] TrpH reaction conditions: 20 μM TrpH pure enzyme, 0.1 M phosphate buffer (pH 8.0), 3 mM L-Tyr, 20 mM reducing agent, reaction at 30°C;

[0105] HRP reaction conditions: 25 mU pure HRP enzyme, 0.7 mM tetramethylbenzidine (TMB), 0.1 M phosphate buffer (pH 7.0), 1 mM reducing agent, reaction at 30°C;

[0106] The reaction results showed that the reducing agents AscA, DHA, GA and PA were all catalyzed by horseradish peroxidase (HRP) for the reaction of substrate tetramethylbenzyldiamine (TMB), by tyrosine hydroxylase (TyrH) for the reaction of substrate tyrosine, and by tryptophan hydroxylase (TrpH) for the reaction of substrate tryptophan, as shown in Table 1.

[0107] Table 1. Oxidation reactions catalyzed by HRP, TyrH, and TrpH using different reducing agents, AscA, DHA, GA, and PA.

[0108] These results once again demonstrate the universality of the O2 / reductant-dependent catalytic pathway in heme peroxidases and also reflect 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.

[0109] Results and Discussion

[0110] It should be understood that the nonspecific peroxidase (UPO) described in the present invention may include various UPOs, such as AaeUPO, MroUPO, CciUPO, CglUPO, GmaUPO, HspUPO, MthUPO, etc.; the catalyzed reaction substrates include all substrates that can be catalyzed by UPO, 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 of their catalytic reactions.

[0111] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. Use 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, wherein The peroxidase is selected from the following group: heme peroxidase, i.e., non-specific peroxidase, P450 peroxidase, chloroperoxidase, horseradish peroxidase, tyrosine hydroxylase, and tryptophan hydroxylase.

5. The application according to claim 4, characterized in that, 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, wherein 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, and tryptophan hydroxylase.

8. The peroxidase catalytic system according to claim 7, characterized in that, 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, and 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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