CN112961791B

CN112961791B - Recombinant strain of non-specific peroxygenase and construction method and application thereof

Info

Publication number: CN112961791B
Application number: CN202110205876.XA
Authority: CN
Inventors: 蓝东明; 乜晓爽; 王永华; 马云建; 王方华; 杨博
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2022-06-14
Anticipated expiration: 2041-02-24
Also published as: CN112961791A

Abstract

The invention discloses a recombinant strain of nonspecific peroxygenase, a construction method and application thereof, belonging to the field of enzyme engineering application. The recombinant strain takes pPICZ alpha A plasmid as a starting vector and takes pichia pastoris as a host bacterium to express nonspecific peroxygenase GmaUPO; the amino acid sequence of the nonspecific peroxygenase GmaUPO is shown as SEQ ID NO: 1 is shown. According to the invention, through a genetic engineering technology, GmaUPO is successfully heterologously expressed in pichia pastoris, the fermentation condition is optimized, and preliminary research on catalytic application of recombinase is carried out, so that the resource library of UPO biological enzyme is enlarged, the existing chemical method is replaced to carry out efficient and clean production of aromatic hydrocarbon, and a certain research basis is provided for the catalytic application of aromatic hydrocarbon.

Description

Recombinant strain of non-specific peroxygenase and construction method and application thereof

Technical Field

The invention belongs to the field of enzyme engineering application, and particularly relates to a non-specific peroxygenase recombinant strain as well as a construction method and application thereof.

Background

Non-specific peroxygenases (UPO, EC 1.11.2.1) are one type of available H ₂O₂Heme oxidases that achieve catalytic oxidative functionalization of C-H bonds have been the subject of research in recent years due to their multifunctional oxidative catalytic activity, and catalyze various types of reactions such as hydroxylation of C-H bonds, epoxidation of C ═ C bonds, and sulfonation of thioethersThe regioselectivity and stereoselectivity of (a) are considered to be a new generation of biocatalysts for the oxidative functionalization of organic compounds. The catalytic process of the P450 monooxygenase with the same function needs the nicotinyl coenzyme factor NAD (P) H as an electron donor, and is accompanied with a cofactor regeneration system, and the complex electron transfer chain of the monooxygenase makes the reaction difficult to be widely applied. And UPO can directly utilize H₂O₂As an oxygen donor and an electron acceptor, thereby having higher practical application value.

The achievement of heterologous high-efficiency expression of fungal heme oxygenases has been a bottleneck limiting the above studies. The selection of a suitable expression system for recombinant expression of the enzyme gene is critical. Expression systems reported to be capable of heterologously expressing UPO include Pichia pastoris, Saccharomyces cerevisiae, Aspergillus oryzae, and Escherichia coli. Although the escherichia coli expression system expresses inclusion bodies which are easy to form, and renaturation can be carried out in an in vitro activation mode, no report is provided for a UPO inclusion body renaturation scheme. In addition, because the endocrine of the heme-containing enzyme in a heterologous host cell is insufficient, the synthesis efficiency is too low, the combination of heme and a new recombinant protein is directly influenced, trace expression of a recombinase is often caused, and the application of the enzyme in the biotransformation industry is severely restricted.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a recombinant strain of non-specific peroxygenase.

Another object of the present invention is to provide a method for constructing the recombinant strain of the above nonspecific peroxygenase.

It is still another object of the present invention to provide use of the recombinant strain of non-specific peroxygenase.

The purpose of the invention is realized by the following technical scheme:

a recombinant strain of nonspecific peroxygenase, the recombinant strain takes pPICZ alpha A plasmid as an initial vector, takes pichia pastoris as a host bacterium, and expresses the nonspecific peroxygenase; the amino acid sequence of the non-specific peroxygenase (GmaUPO) is shown as SEQ ID NO: 1 is shown. The nucleotide sequence of the non-specific peroxygenase (GmaUPO) is shown as SEQ ID NO: 2, respectively.

The target gene of the nonspecific peroxygenase and the original signal peptide fragment are fused into a fusion gene which is connected between EcoR I and SalI sites of the pPICZ alpha A plasmid, and then the fusion gene is electrically transformed into pichia pastoris for expression.

Preferably, the original Signal Peptide (SP)_Gma) The amino acid sequence of (a) is as shown in SEQ ID NO: 3, original Signal Peptide (SP)_Gma) The nucleotide sequence of (a) is shown as SEQ ID NO: 4, respectively.

Preferably, the pichia pastoris is pichia pastoris X-33.

The construction method of the non-specific peroxygenase recombinant strain comprises the following steps:

1) taking pPICZ alpha A as a template, taking an upstream primer F1 and a downstream primer R1 as primers, and amplifying to obtain a plasmid pPICZ alpha A containing EcoRI and SalI restriction enzyme sites;

the upstream primer F1: 5'-ATCCCAACACTCGAGGCCCGAGTCGACCATCATCATCATCATCATTGAGTT-3', respectively;

the downstream primer R1: 5'-GAACAGGGGAAAATATTTCATGAATTCCTCGTTTCGAATAAT-3', respectively;

2) then, a target gene of GmaUPO is taken as a template, an upstream primer F2 and a downstream primer R2 are taken as primers, and a target gene fragment is obtained through amplification; taking the original signal peptide as a template and taking the primers F3-F6 as primer fragments, and amplifying to obtain an original signal peptide fragment; fusing a target gene and an original signal peptide by using a PCR amplification technology, purifying a PCR product of the fused fragment and a plasmid pPICZ alpha A containing EcoRI and SalI restriction enzyme sites, and connecting to obtain a positive plasmid;

the upstream primer F2: 5'-ATTATTCGAAACGAGGAATTCTTTCCAGCTTACGGATCACTA-3', respectively;

the downstream primer R2: 5'-AACTCAATGATGATGATGATGATGGTCGACTTTGCCATAGGGAAAGACTTG-3', respectively;

primer F3: 5'-ATTATTCGAAACGAGGAATTCATGA-3', respectively;

Primer F4: 5'-AAAGCAATCAAAACAGCAAATCTAGCAGCACCTCTCATGAATTCCTCGTTTCGAATAAT-3', respectively;

primer F5: 5'-CTAGATTTGCTGTTTTGATTGCTTTGTTTACTCATGCCGCTATCGCTTTTCCAGCTTAC-3', respectively;

primer F6: 5'-TAGTGATCCGTAAGCTGGAAAAGCGATAGC-3', respectively;

3) transforming the obtained positive plasmid into DH5 alpha colibacillus competent cells, coating the competent cells on an LB solid plate containing bleomycin for culture, picking a single colony into an LB liquid culture medium, carrying out shake culture, carrying out whole-gene sequencing on the recombinant plasmid to obtain the positive plasmid, and marking the positive plasmid as the recombinant plasmid pPICZ alpha A-SP_Gma-GmaUPO(SP_GmaRepresenting the original signal peptide);

4) the recombinant plasmid pPICZ alpha A-SP with correct sequencing is subjected to_GmaLinearization is carried out on GmaUPO, the strain is transformed into a pichia host strain competent cell through an electric shock penetration method, and the strain is coated on a YPD solid plate containing bleomycin and cultured; subsequently, the recombinant single colony is picked into a YPD liquid culture medium, is subjected to shaking culture, and is screened for positive recombinant bacteria by a PCR method.

Preferably, in the step 3), the final concentration of the bleomycin in the LB solid plate containing the bleomycin is 25 μ g/mL;

preferably, in the step 3), the culture condition is that the culture is carried out at 37 +/-0.5 ℃ for 12-16 h;

Preferably, in the step 3), the shaking culture condition is to culture for 12-16 h at 37 +/-0.5 ℃ and 200-220 rpm; further culturing for 12-16 h at 37 +/-0.5 ℃ and 200 rpm;

preferably, in step 4), the final concentration of bleomycin in the YPD solid plate containing bleomycin is 100. mu.g/mL.

Preferably, in the step 4), the culture condition is to culture at 29 +/-1 ℃ for 36-48 h;

preferably, in the step 4), the shaking culture condition is to culture for 16-18 h at 29 +/-1 ℃ and 220-280 rpm; further culturing for 16-18 h at 29 +/-1 ℃ and 250 rpm;

the application of the non-specific peroxygenase recombinant strain in preparing the recombinant non-specific peroxygenase by fermentation.

The fermentation preparation method of the recombinant non-specific peroxygenase comprises the following steps:

inoculating the non-specific peroxygenase recombinant strain into a BMGY liquid culture medium, and culturing until the OD of the strain is 0.8-1.0; inoculating the BMMY liquid culture medium with an inoculation amount of 5-10% (v/v), and adding aminolevulinic acid with a final concentration of 5-85 mu M; performing shake culture, and adding methanol every 24h for induction; after fermentation, the supernatant is centrifuged and contains recombinant non-specific peroxygenase.

Preferably, the culture is carried out until the thallus OD is 0.8-1.0 under the conditions of 29 +/-1 ℃ and 240-250 rpm until the thallus OD is 0.8-1.0; further, the cells were cultured at 29. + -. 1 ℃ and 250rpm until the OD of the cells was 0.8 to 1.0.

Preferably, during the induction period, the shaking culture condition is to culture for 96 hours at the temperature of 22-28 ℃ and the rotating speed of 240-250 rpm; further cultured for 96h at 22 ℃ and 250 rpm.

Preferably, the final concentration of the aminolevulinic acid is 25-65 mu M; further 25-45 μ M; further 45. mu.M.

Preferably, the final concentration of methanol is 1. + -. 0.1% (v/v).

The recombinant non-specific peroxygenase can be used as a catalyst for synthesizing aromatic hydrocarbons.

Compared with the prior art, the invention has the following advantages and effects:

the invention discovers a new non-specific peroxygenase and constructs a recombinant strain thereof through a genetic engineering technology, and realizes heterologous expression through optimization of fermentation parameters; and provides a method for efficiently and cleanly producing aromatic hydrocarbon instead of the existing chemical method.

Drawings

FIG. 1 shows the expression of GS115/pPIC9K-GmaUPO recombinant engineering bacteria tested by Western blot.

FIG. 2 is X-33/pPICZ α A-SP _GmaFermentation optimization of the GmaUPO recombinant engineering bacteria.

FIG. 3 shows Western blot analysis of X-33/pPICZ. alpha. A-SP_GmaExpression of GmaUPO recombinant engineering bacteria, theoretical molecular weight 38.3 kDa.

FIG. 4 shows the detection of the enzyme activity of GmaUPO using NBD as a substrate; wherein A is a standard curve for enzyme activity calculation; b is 4-nitrocatechol color development reaction, and 1, NBD is used as a substrate to detect the enzyme activity of the empty carrier fermentation liquid; 2, detecting the enzyme activity of the GmaUPO fermentation liquor by taking NBD as a substrate; 3, NBD is used as a substrate to detect GmaUPO fermentation liquid inactivation control.

FIG. 5 is the effect of pH on GmaUPO enzyme activity and stability; wherein A is the influence of pH on the activity of GmaUPO enzyme; and B is the influence of pH on the activity stability of the GmaUPO enzyme.

FIG. 6 is the effect of temperature on the activity and heat resistance of GmaUPO enzyme; wherein A is the influence of temperature on the activity of GmaUPO enzyme; and B is the influence of temperature on the thermal stability of the activity of the GmaUPO enzyme.

FIG. 7 shows that the GmaUPO enzyme cascade catalytic reaction is detected by GC-MS with ethylbenzene as a substrate.

FIG. 8 shows the detection of GmaUPO enzyme cascade catalysis reaction by GC-MS with methyl phenyl sulfide as a substrate.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention successfully expresses a novel non-specific peroxygenase (GmaUPO) in pichia pastoris in a heterologous way, optimizes the fermentation condition, carries out preliminary research on the catalytic application of the recombinase, aims to expand the resource library of the UPO biological enzyme and provides a certain research basis for the catalytic application of the UPO biological enzyme.

In order to successfully heterologously express a novel non-specific peroxygenase (GmaUPO) and successfully apply the non-specific peroxygenase to the catalytic conversion reaction of a non-activated carbon-hydrogen bond, the invention selects pPICZ alpha A as a vector and constructs pPICZ alpha A-SP _Gma-a recombinant vector of GmaUPO; pichia pastoris X-33 is selected as a heterologous expression host, and is applied to the catalytic conversion of non-activated carbon-hydrogen bonds through fermentation optimization to construct a new enzyme cascade catalytic reaction system for catalytically converting ethylbenzene into alcohols, so that the aim of expanding a resource library of UPO biological enzymes is fulfilled, and a certain research basis is provided for the catalytic application of the UPO biological enzymes.

Firstly, according to the characteristics of related gene segments, utilizing seamless cloning and enzyme digestion enzyme-linked technology to construct engineering bacteria to obtain a transformation recombinant vector pPICZ alpha A-SP_Gma-recombinant engineering bacteria of GmaUPO and optimization of fermentation condition related factors; finally, the method is applied to organic matter catalytic conversion to identify the successful expression and application of a novel nonspecific peroxygenase (GmaUPO).

Materials and reagents used in the following examples: the pichia pastoris expression strain X-33 and the plasmid pPICZ alpha A are conventional commercial materials; e.coli DH 5. alpha. competent cells, Biotech Ltd only; restriction enzymes EcoRI and SalI, TaKaRa Dajieshan Biotech Co., Ltd; seamless cloning kit, zhongmeitai and biotechnology (beijing) ltd; plasmid extraction kit, bio-engineering (Shanghai) Co., Ltd; the primary antibody of Western blot was a mouse anti-His-tag monoclonal antibody, and the secondary antibody was a goat anti-mouse IgG (H & L) secondary antibody (HRP-labeled), both from Jinruis Biotech. PCR apparatus (T100) from Burle Bio-rad, USA; an Infine M NANO multifunctional microplate reader from Dirkon (Shanghai) trade company Limited; an electrophoresis apparatus (model DYY-8C) from six instruments, Beijing; SW-CJ-1F superclean bench; HVE-50 autoclave of Hirayama, Japan; constant temperature incubator of Shanghai Shenxian constant temperature Co., Ltd.

The amino acid sequence of the GmaUPO is shown as KDR72024.1, and is specifically shown as SEQ ID NO: 1 is shown.

The nucleotide sequence of the GmaUPO is shown as SEQ ID NO: 2, respectively.

The SP_GmaThe amino acid sequence of (a) is as shown in SEQ ID NO: 3, the SP_GmaThe nucleotide sequence of (a) is shown as SEQ ID NO: 4, respectively.

Comparative example 1

According to the literature, "Ponengwei, Pombo and the like", the expression and biochemical characterization of agaricus bisporus-derived non-specific peroxygenase in Pichia pastoris and modern food technology 2021,37(01) ", a BamHI-digested pPIC9K vector and an amplified GmaUPO target gene are ligated, transferred into DH5 alpha Escherichia coli competent cells and plated on solid LB plates containing 100. mu.g/mL ampicillin. The next day, the recombinant plasmid was extracted and sent to Shanghai Biotech for sequencing. And (3) electrically transforming the successfully sequenced recombinant plasmid into pichia pastoris GS115 for culture, identifying, marking the identified recombinant bacterium as a positive recombinant bacterium GS115/pPIC9K-GmaUPO, and detecting by Western blot to have no expression, as shown in figure 1. Subsequently, the vector is replaced for recombinant expression again.

According to the sequence characteristics of a target gene, an original signal peptide and an expression vector pPICZ alpha A, an amplification primer is designed.

Example 1: transformation recombinant vector pPICZ alpha A-SP _GmaConstruction of engineering bacteria of GmaUPO

2) then, the gene of interest of GmaUPO (SEQ ID NO: 2) taking an upstream primer F2 and a downstream primer R2 as primers as a template, and amplifying to obtain a target gene fragment; using original signal peptide gene (SEQ ID NO: 4) as template, using primer F3-F6 as primer fragment, amplifying to obtain original signal peptide fragment; fusing a target gene and an original signal peptide by a PCR amplification technology, purifying a PCR product of the fused fragment and a plasmid pPICZ alpha A containing EcoRI and SalI restriction enzyme sites, and connecting to obtain a positive plasmid;

primer F3: 5'-ATTATTCGAAACGAGGAATTCATGA-3';

primer F6: 5'-TAGTGATCCGTAAGCTGGAAAAGCGATAGC-3', respectively;

3) transforming the obtained positive plasmid into DH5 alpha escherichia coli competent cells, coating the cells on an LB solid plate containing bleomycin with the final concentration of 25 mu g/mL, culturing for 12-16 h at 37 ℃, picking single colonies into 5mL of LB liquid culture medium, culturing for 12-16 h at 37 ℃ and 200rpm, extracting the plasmid, performing whole-gene sequencing on the recombinant plasmid, and recording the recombinant plasmid with correct sequencing as pPICZ alpha A-SP_Gma-GmaUPO。

4) The recombinant plasmid pPICZ alpha A-SP with correct sequencing is subjected to_GmaAnd (4) linearizing GmaUPO, transforming the linearized GmaUPO into pichia pastoris X-33 competent cells by an electric shock penetrating method, coating the competent cells on a YPD solid plate containing 100 mu g/mL bleomycin at the final concentration, and culturing the competent cells at the temperature of 30 ℃ for 36-48 h. Then, picking the recombinant single colony into 5mL YPD liquid culture medium, culturing for 16-18 h under the conditions of 30 ℃ and 250rpm, and screening positive recombinant bacteria by using a PCR method, wherein the positive recombinant bacteria are marked as X-33/pPICZ alpha A-SP_Gma-GmaUPO。

Example 2: X-33/pPICZ alpha A-SP_Gma-fermentation of a GmaUPO recombinant protein engineering bacterium comprising the steps of:

1) positive monoclonal X-33/pPICZ alpha A-SP _Gma-GmaUPO is inoculated into 50mL BMGY liquid medium and cultured under the conditions of 30 ℃ and 250rpm until the OD of the thalli is 0.8-1.0.

2) The cells were inoculated into 400mL BMMY liquid medium at 10% (v/v) inoculation amount, while delta-aminolevulinic acid (ALA) was added to the medium at a final concentration of 45. mu.M, and the cells were induced at 22 ℃ and 250rpm for 96 hours, and methanol (1%) was added every 24 hours for induction, while Pichia pastoris strains transformed with no target gene empty vector pPICZ. alpha.A were used as a control.

3) After fermentation, the fermentation broth was centrifuged at 12,000rpm at 4 ℃ for 10min in a refrigerated centrifuge, and the supernatant was collected and assayed.

Example 3: X-33/pPICZ alpha A-SP_GmaFermentation optimization of GmaUPO recombinant engineering bacteria

(1) Optimizing ALA concentration: 5 ALA concentrations were selected for fermentation optimization of the engineered bacteria (5. mu.M, 25. mu.M, 45. mu.M, 65. mu.M, 85. mu.M) as shown in example 2, with the other conditions unchanged.

(2) Optimizing the induction temperature: 3 induction temperatures were selected for fermentation optimization of the engineering bacteria (22 ℃, 25 ℃, 28 ℃), the ALA concentration was tested according to the optimal ALA concentration obtained in step (1) of example 3, and the rest steps were as shown in example 2, with the other conditions being unchanged.

(3) And (3) taking the fermentation supernatant obtained at the optimal ALA concentration and the optimal induction temperature as a sample, and detecting the expression condition of the recombinant protein by using Western Blot, wherein the detected antibody is a specific His tag antibody.

The fermentation process was optimized for relevant fermentation conditions including induction temperature (28 ℃, 25 ℃, 22 ℃) and ALA concentration (5. mu.M, 25. mu.M, 45. mu.M, 65. mu.M, 85. mu.M), and the results are shown in FIG. 2. The results show that: when the induction temperature is 22 ℃; when the final concentration of ALA is 45 mu M, the enzyme activity is highest.

As a result of detecting the expression of the recombinant protein by Western Blot, the antibody to be detected was a specific His-tag antibody, as shown in FIG. 3. The results show that there is a clear protein expression band in the fermentation supernatant sample with a molecular weight of about 40kDa, consistent with the predicted size of GmaUPO (theoretical molecular weight of 38.3 kDa).

Example 4: enzyme activity detection of GmaUPO recombinant protein

Enzyme activity of UPO (UPO) detected by using 5-nitro-1, 3-benzodioxazole (NBD) as substrateAnd the fermentation supernatant obtained from the optimal ALA concentration and the optimal induction temperature in example 3 was used as an enzyme solution sample. The method comprises the following steps: to a 96-well plate, 100. mu.L of phosphate buffer (PBS, 100mM, pH 6.0), 30. mu.L of NBD (6.6mM), 20. mu.L of deionized water, 30. mu.L of enzyme solution, 20. mu. L H were added₂O₂(100mM), and the absorbance value was measured at 425 nm. All experiments were performed in triplicate, with the high temperature inactivated supernatant used as a control.

Calculating a standard curve of enzyme activity: mu.L of 4-nitrophthalol (product of NBD catalyzed by GmaUPO enzyme), 20. mu.L of hydrogen peroxide, 20. mu.L of water, 130. mu.L of PBS were added to a 96-well plate, and the absorbance value was measured at 425 nm. All experiments are carried out in parallel by three groups, namely standard curves, and the enzyme activity is calculated by using the standard curves according to OD values measured by an NBD method.

The unit of enzyme activity is defined as: the amount of enzyme required to hydrolyze the substrate to 1. mu. mol of 4-nitrophthalol per minute at 35 ℃ and pH 9 was taken as 1 activity unit (U). (4-Nitrocellulose is a color indicator that turns red under alkaline conditions).

NBD is used as a substrate, the GmaUPO enzyme activity phenomenon and a product label graph are detected, and the GmaUPO enzyme activity is quantitatively calculated, and the result is shown in figure 4. The result shows that the enzyme activity of the GmaUPO is 6U/L according to the definition of the enzyme activity unit and the calculation of a standard curve.

Example 5: biochemical characterization of GmaUPO recombinant proteins

1) NBD is used as a detection substrate, and 7 buffer solutions with pH ranges of 4.0-10.0 are selected to influence the activity change of GmaUPO (citrate buffer solution with pH values of 4.0 and 5.0 being 100 mM; phosphate buffer at 100mM at 6.0, 7.0, 8.0; Tri-HCl buffer at 100mM 9.0, 10.0). The highest enzyme activity was defined as 100%, and the remainder were expressed as relative enzyme activity. Further researching the influence of pH on enzyme stability, namely incubating GmaUPO in buffer solutions with different pH values at 4 ℃ for 2h, determining the residual value of enzyme activity, and detecting the tolerance of the pH. The enzyme activity before incubation was defined as 100%, and the rest was expressed as relative enzyme activity. The results are shown in fig. 5A, the optimal reaction pH of the GmaUPO enzyme solution is 9.0, the enzyme activity can be maintained at about 60% of the highest activity when the solution pH is 8.0 and 10.0, and the enzyme activity in other pH buffers is inhibited. Subsequently, the stability of the GmaUPO incubated for 2h in different pH buffers is studied, as shown in FIG. 5B, the residual enzyme activity of the GmaUPO incubated in the buffer solution with the pH value of below 7.0 is lower than 70%, and the residual enzyme activity of the GmaUPO incubated in the buffer solution with the pH value of 7.0-10.0 is higher than 70%, which indicates that the catalytic activity of the GmaUPO in an alkaline environment is better.

2) Under the optimal pH value, NBD is used as a detection substrate, different temperatures (20-45 ℃) are selected to study the activity change of the GmaUPO enzyme liquid, the highest enzyme activity is defined as 100%, and the rest is expressed by relative activity. Further researching the influence of temperature on enzyme stability, a proper amount of GmaUPO is placed at different temperatures, and samples are taken at regular intervals to detect the enzyme activity so as to determine the temperature tolerance of the GmaUPO. The enzyme activity before heat treatment was defined as 100%, and the remainder was expressed as relative enzyme activity. The result is shown in FIG. 6A, the optimum temperature of NBD catalyzed by the GmaUPO enzyme liquid is 35 ℃, and the enzyme activity is still maintained to be more than 60% at other temperatures (20-40 ℃). The results of the thermal stability experiments are shown in fig. 6B, where GmaUPO maintained 80% of the initial activity within 1h and less than 30% after 3h at 25 ℃ and 30 ℃. After incubation for 3h at 40 ℃, the activity of the GmaUPO enzyme can not be detected basically.

Example 6: hydroxylation and sulfonation products generated by catalyzing conversion of ethylbenzene and methyl phenyl sulfide by utilizing GmaUPO recombinant protein

On-line production of H by choline oxidase catalytic choline chloride₂O₂The method is characterized in that a cascade catalytic reaction system is researched, the GmaUPO catalyzes and oxidizes ethylbenzene and methyl phenyl sulfide to serve as substrates, and fermentation supernatant obtained in the embodiment 3 at the optimal ALA concentration and the optimal induction temperature is used as an enzyme solution sample. And detecting and analyzing the reaction product by using a triple series quadrupole gas chromatograph-mass spectrometer GC-MS. The specific catalytic reaction steps are as follows: mu.L of phosphate buffer (50mM, pH 7.0) and 100. mu.L of choline chloride solution (1M) were added to a reaction glass vial, after oxygenation, 50. mu.L of choline oxidase (100. mu.M, AnChox) enzyme solution, 400. mu.L of GmaUPO enzyme solution (supernatant) and 5mM substrate (ethylbenzene or methylphenylsulfide) were added, and finally the reaction volume was made up to 1mL with phosphate buffer (50mM, pH 7.0). Putting the reaction system into a constant-temperature oil bath kettle for feeding The reaction is carried out at the reaction temperature of 30 ℃ and the rotation speed of 500 r/min. After the reaction, the mixture was extracted with 1mL of ethyl acetate, centrifuged, and the upper organic layer was collected and examined.

The GC-MS detection conditions of the triple series quadrupole gas chromatograph-mass spectrometer are as follows: heating the initial 50 deg.C to 190 deg.C at a heating rate of 10 deg.C/min, maintaining for 3min, heating to 230 deg.C at a heating rate of 5 deg.C/min, and maintaining for 6 min.

Calculation of product concentration: accurately weighing beta-phenethyl alcohol (or methyl phenyl sulfoxide) to prepare beta-phenethyl alcohol (or methyl phenyl sulfoxide) solutions with the concentrations of 1 mM, 2 mM, 3 mM, 4 mM and 5mM, performing GC-MS detection on the standard substance, and drawing a concentration standard curve of the beta-phenethyl alcohol (or methyl phenyl sulfoxide) according to an absorption peak of a gas chromatogram. Substituting the peak area in the extract and the absorption peak corresponding to the product into a standard curve to obtain the product concentration.

The hydroxylated product is generated by conversion with ethylbenzene as a substrate, and analyzed by a triple tandem quadrupole gas chromatograph-mass spectrometer GC-MS, and the results are shown in FIG. 7, wherein the retention time of ethylbenzene and beta-phenylethyl alcohol is 5.071min and 9.431min respectively.

The methyl phenyl sulfide is used as a substrate to be converted into a sulfonated product, and the sulfonated product is analyzed by a triple tandem quadrupole gas chromatograph-mass spectrometer GC-MS, so that the retention time of the methyl phenyl sulfide and the retention time of the methyl phenyl sulfoxide are 9.574min and 14.451min respectively as shown in figure 8.

Table 1 shows that the conversion of ethylbenzene and methyl phenyl sulfide as substrates to produce hydroxylated and sulfonated products (beta-phenethyl alcohol and methyl phenyl sulfoxide) reaches 3.4% and 22.86% within 4 h.

TABLE 1 GmaUPO enzyme cascade catalytic reaction product yield

Product of	Beta-phenylethyl alcohol	Methyl phenyl sulfoxide
			Initial concentration (mM)	0	0
Reaction 4h concentration (mM)	0.17	1.143
			Conversion (%)	3.4	22.86

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of southern China's science

<120> a non-specific peroxygenase recombinant strain, its construction method and application

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<210> 2

<211> 1059

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of non-specific peroxygenase

<400> 2

tttccagctt acggatcact agctggtcta actagggaac aactagacga aatcctgcct 60

acacttgaaa tccgagagcc tggaaagcct cctggaccac ttaaggatac ttctgctaaa 120

cttgttaatg ataaggcaca tccttggaaa cctgtagcac ctgcagatat aagaggtcca 180

tgtccaggtt tgaacacatt agcatcacat ggatggttac ctagaaacgg tatcgcttca 240

ccttcagaaa tcattacggc agtacaggaa gggtttaaca tggacaacgg acttgcaata 300

tttgtcacct acgctgctca cttagtagac ggaaacattc tgacggataa actgtctatc 360

ggtgggaaga ctggtttgac aggtccaaat cctccagctc cagctattgt tggtggattg 420

aatacacatg cagtgtttga aggagataca tctatgacga gaggagattt cttctttggt 480

aacaaccacg acttcaacga gaccttgttt gacgaatttg tggactttag taaccgtttc 540

ggtgctggca aatacaacct gaccgttgct ggtgagttta gatggcaaag aatacaggac 600

tctattgcca ccaacccaga gtttagtttc gttagtccaa gattctttac cgcctacgcc 660

gaatctacat ttccaattaa tttcttcatt gacggtaggc aaaccgatgg tcaactggat 720

ttgaccgtgg ctaggggatt ctttcagaac tcccgaatgc cagatgactt tcatcgtgct 780

aacggtacta gaggaactga aggtattgat ttggtggctg aggcccaccc aattgagccc 840

ggttctaatg ttggaggggt taataattat gttgttgatc ccacttccgc cgatttctcc 900

actttctgtt tgttatatga gaatttcgtc aataagactg tcaagggctt gtatcccaat 960

cccactgggg ccttacgtaa agcccttaat actaatttgg gcttcttctt tagtggtatt 1020

tccgattccg gctgcactca agtctttccc tatggcaaa 1059

<210> 3

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of original signal peptide

<400> 3

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

1 5 10 15

Ala Ala Ile Ala

20

<210> 4

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of original signal peptide

<400> 4

atgagaggtg ctgctagatt tgctgttttg attgctttgt ttactcatgc cgctatcgct 60

<210> 5

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer F1

<400> 5

atcccaacac tcgaggcccg agtcgaccat catcatcatc atcattgagt t 51

<210> 6

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer R1

<400> 6

gaacagggga aaatatttca tgaattcctc gtttcgaata at 42

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer F2

<400> 7

attattcgaa acgaggaatt ctttccagct tacggatcac ta 42

<210> 8

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer R2

<400> 8

aactcaatga tgatgatgat gatggtcgac tttgccatag ggaaagactt g 51

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer F3

<400> 9

attattcgaa acgaggaatt catga 25

<210> 10

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer F4

<400> 10

aaagcaatca aaacagcaaa tctagcagca cctctcatga attcctcgtt tcgaataat 59

<210> 11

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer F5

<400> 11

ctagatttgc tgttttgatt gctttgttta ctcatgccgc tatcgctttt ccagcttac 59

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer F6

<400> 12

tagtgatccg taagctggaa aagcgatagc 30

Claims

1. The fermentation preparation method of the recombinant nonspecific peroxygenase is characterized in that: the method comprises the following steps:

inoculating the non-specific peroxygenase recombinant strain into a BMGY liquid culture medium, and culturing until the OD of the strain is 0.8-1.0; inoculating the BMMY liquid culture medium with an inoculation amount of 5-10% v/v, and adding aminolevulinic acid with a final concentration of 5-85 mu M; performing shake culture, and adding methanol every 24h for induction; after fermentation is finished, centrifuging to obtain supernatant, wherein the fermented supernatant contains recombinant non-specific peroxygenase;

The nonspecific peroxygenase recombinant strain takes pPICZ alpha A plasmid as a starting vector and pichia pastoris as a host bacterium to express nonspecific peroxygenase GmaUPO; the amino acid sequence of the nonspecific peroxygenase GmaUPO is shown as SEQ ID NO: 1 is shown in the specification;

non-specific peroxygenase target gene and primary signal peptide SP_GmaThe fragments are fused into a fusion gene which is connected to pPICZ alpha A plasmid, and then the fusion gene is electrically transformed into pichia pastoris for expression;

original signal peptide SP_GmaThe amino acid sequence of (a) is as shown in SEQ ID NO: 3 is shown in the specification;

the pichia is pichia X-33.

2. The fermentative preparation method according to claim 1, wherein:

the culture is carried out until the thallus OD is 0.8-1.0 under the conditions of 29 +/-1 ℃ and 240-250 rpm until the thallus OD is 0.8-1.0;

the shaking culture condition is that the culture is carried out for 96 hours at the temperature of 22-28 ℃ and the rotating speed of 240-250 rpm;

the final concentration of the aminolevulinic acid is 25-65 mu M;

the final concentration of methanol was 1. + -. 0.1% v/v.

3. The fermentative production method according to claim 1, wherein:

non-specific peroxygenase target gene and primary signal peptide SP_GmaThe fragment was fused into a fusion gene, ligated between EcoRI and SalI sites of pPICZ. alpha.A plasmid, and then electrically transformed into Pichia pastoris for expression.

4. The fermentative production method according to claim 1, wherein:

the nucleotide sequence of the nonspecific peroxygenase GmaUPO is shown as SEQ ID NO: 2 is shown in the specification;

original signal peptide SP_GmaThe nucleotide sequence of (a) is shown as SEQ ID NO: 4, respectively.

5. The fermentative preparation method according to any one of claims 1 to 4, wherein:

the upstream primer F1: 5'-ATCCCAACACTCGAGGCCCGAGTCGACCATCATCATC ATCATCATTGAGTT-3', respectively;

2) then, a target gene of GmaUPO is taken as a template, an upstream primer F2 and a downstream primer R2 are taken as primers, and a target gene fragment is obtained through amplification; with the original signal peptide SP_GmaUsing the gene as a template and using primers F3-F6 as primer fragments to amplify to obtain an original signal peptide fragment; fusing a target gene and an original signal peptide by using a PCR amplification technology, purifying a PCR product of the fused fragment and a plasmid pPICZ alpha A containing EcoRI and SalI restriction enzyme sites, and connecting to obtain a positive plasmid;

primer F3: 5'-ATTATTCGAAACGAGGAATTCATGA-3', respectively;

primer F6: 5'-TAGTGATCCGTAAGCTGGAAAAGCGATAGC-3', respectively;

3) transforming the obtained positive plasmid into DH5 alpha colibacillus competent cells, coating the competent cells on an LB solid plate containing bleomycin for culture, picking a single colony into an LB liquid culture medium, carrying out shake culture, carrying out whole-gene sequencing on the recombinant plasmid to obtain the positive plasmid, and marking the positive plasmid as the recombinant plasmid pPICZ alpha A-SP_Gma-GmaUPO；

6. The fermentative preparation method according to claim 5, wherein:

In the step 3), the culture condition is that the culture is carried out for 12-16 h at 37 +/-0.5 ℃;

in the step 3), the shaking culture condition is to culture for 12-16 h at 37 +/-0.5 ℃ and 200-220 rpm;

in the step 4), the culture condition is that the culture is carried out for 36-48 h at 29 +/-1 ℃;

in the step 4), the shaking culture condition is to culture for 16-18 h at 29 +/-1 ℃ and 220-280 rpm.

7. The use of a non-specific peroxygenase as a catalyst in the synthesis of aromatic hydrocarbons, characterized in that: the amino acid sequence of the non-specific peroxygenase is shown as SEQ ID NO: 1 is shown.

8. Use according to claim 7, characterized in that: the nucleotide sequence of the non-specific peroxygenase is shown as SEQ ID NO: 2, respectively.