阅读说明：本技术 一种细胞色素p450环氧酶及其应用 (Cytochrome P450 epoxidase and application thereof ) 是由 刘立明 燕宇 宋伟 陈修来 刘佳 高聪 于 2020-11-24 设计创作，主要内容包括：本发明公开了一种细胞色素P450环氧酶及其应用,属于生物工程技术领域。本发明提供了一种细胞色素P450环氧酶CytP,通过在大肠杆菌中表达所述环氧酶CytP,利用全细胞转化法,将降冰片烯转化为环氧降冰片烷。并从反应的pH、温度、相比和诱导时间等因素对CytP酶的两相催化体系进行优化,克服了之前P450单加氧酶环氧化活性低且催化特异性差的局限,可使得环氧降冰片烷产量达到36.81g/L,催化特性达到99％,环氧化反应摩尔产率达31.46％。从而大大降低了之前的底物成本和环境污染等问题,为环氧降冰片烷的工业化生产和绿色生产奠定了基础。(The invention discloses cytochrome P450 epoxidase and application thereof, and belongs to the technical field of bioengineering. The invention provides cytochrome P450 epoxidase CytP, which is expressed in escherichia coli, and norbornene is converted into epoxy norbornane by a whole-cell conversion method. The two-phase catalytic system of the CytP enzyme is optimized according to factors such as pH, temperature, phase ratio and induction time of the reaction, the limitation that the prior P450 monooxygenase has low epoxidation activity and poor catalytic specificity is overcome, the yield of epoxy norbornane can reach 36.81g/L, the catalytic property reaches 99%, and the molar yield of the epoxidation reaction reaches 31.46%. Thereby greatly reducing the problems of substrate cost, environmental pollution and the like, and laying a foundation for the industrial production and the green production of the epoxy norbornane.)

1. A method for synthesizing norbornane oxide, characterized in that norbornane oxide is produced using a strain expressing an epoxidase derived from Pseudomonas putida, using norbornene as a substrate.

2. The method according to claim 1, wherein the amino acid sequence of the epoxidase is as shown in SEQ ID No. 1.

3. The method as claimed in claim 1, wherein the strain is Escherichia coli BL21(DE 3).

4. The method according to claim 3, characterized in that the epoxidase is expressed in Escherichia coli BL21(DE3) using pET28a as expression vector.

5. The method according to claim 1, wherein the reaction system comprises an organic phase and an aqueous phase, and the volume ratio of the organic phase to the aqueous phase is (2-6: 1).

6. The method of claim 1, wherein the reaction pH is 8.0 to 8.5.

7. The process according to claim 1, wherein the reaction temperature is 30-37 ℃.

8. The method of claim 1, wherein the strain is induced for 12-13 hours or 16-17 hours, and the cells are collected after the induction and added to the reaction system for reaction.

9. The application of the epoxidase with an amino acid sequence shown as SEQ ID NO.1 in preparing epoxy norbornane.

10. The method according to claim 9, wherein the epoxygenase or a genetically engineered bacterium expressing the epoxygenase is used as a catalyst to catalyze norbornene to produce norbornane oxide.

Technical Field

The invention relates to cytochrome P450 epoxidase and application thereof, and belongs to the technical field of bioengineering.

Background

Epoxy norbornane, also known as 3-epoxypropyl [3,2,1,0 ]^2,4]Octane, one of the derivatives of norbornane. As a saturated bridged ring compound of formula C₇H₁₀O, melting point 126 ℃. It is formed by that the carbon skeleton of cyclohexane is used to bridge a methylene group at the 1, 4 position, and at the same time the 2, 3 position and an oxygen atom are formed into epoxy structure.

At present, the main production method of epoxy norbornane is a chemical synthesis method, mainly a hydrogen peroxide method, but the method has high yield and large energy consumption, can generate toxic action on the environment, and does not meet the requirements of green production, safe production and sustainable development. The preparation of the epoxy norbornane by the biological method has the characteristics of stable and safe product quality, mild process conditions, high efficiency, environmental protection and the like, and can reduce the environmental and resource pressure, so that an effective biological method for efficiently preparing the epoxy norbornane is urgently needed.

At present, the production of epoxy norbornane by a microbiological method relates to a key enzyme Cytochrome P450 epoxidase (CytP), which has wide substrate heterouniversality and can catalyze norbornene to epoxidize a C ═ C double bond to form epoxy norbornane. The preparation of epoxy norbornane by a microbiological method is mainly an enzymatic conversion method. At present, the enzymatic conversion method has the advantages of favorable environment, mild reaction, simple and convenient operation and the like, thereby having industrial application value. However, the large-scale preparation of epoxynorbornane by enzymatic conversion is limited by the following limitations: (1) norbornene as a substrate is insoluble in water, and CytP is inhibited by adding an organic solvent in a reaction system; (2) hydrogen peroxide is used as one of reactants to provide oxygen atoms for products, so that irreversible loss is further caused to enzyme activity; (3) along with the catalytic reaction, CytP generates byproducts due to strong catalytic capability and substrate heterozygosity, so that the catalytic specificity is reduced, the yield and the later separation and purification of epoxy norbornane are greatly limited, and the method is a key problem in the current research. Furthermore, as reported by the mechanism of olefin Epoxidation by the P450 enzyme studied by Shengxijin, Thomas M, Makris et al, the P450 enzyme acts as an oxygenase and favors the catalytic hydroxylation reaction when a C ═ C double bond is present as a substrate, whereas the Epoxidation reaction has been an undesirable side reaction with low conversion (disclosed in Epoxidation of Olefins by Hydroperoxo-ferroelectric Cytocoromes P450, 2003). P450 enzymes often require mutational engineering of specific key residues to achieve epoxidation, and specifically catalyze epoxidation is a challenge.

Disclosure of Invention

In the current research, most enzymes in the P450 epoxidase can not realize single catalytic epoxidation on a substrate, so that the catalytic efficiency is low and the yield is extremely low.

In recent years, the intensive research on the catalytic path of cytochrome P450 enzyme provides a new idea for catalyzing single epoxidation reaction

-a Coupling II shunt pathway. Unlike the classical hydroxylated monooxygenation pathway of the P450 enzyme, the couplinii shunt pathway requires neither additional redox chaperones nor nad (P) H cofactors, with hydrogen peroxide as the sole source of oxygen in the pathway, ultimately producing a single epoxidation product. The Coupling II shunt approach breaks through the barrier of low catalytic specificity for the preparation of epoxy norbornane by the P450 enzymatic conversion method.

However, the present inventors have found in previous studies that not all of the enzymes of the bulky P450 enzyme system catalyze the formation of epoxynorbornane from norbornene.

Therefore, the invention provides cytochrome P450 epoxidase CytP, and the epoxidase is used for catalyzing norbornene to prepare epoxy norbornane. The epoxidase CytP is used for constructing recombinant bacteria, and the epoxy norbornane is produced by transformation of a whole cell method, so that the epoxidase CytP has the advantages of low environmental damage, high catalytic specificity, reduction of byproducts in the transformation and the like, and the industrial production efficiency is greatly improved.

The corresponding parent amino acid sequence of the cytochrome P450 epoxidase CytP provided by the invention is shown as SEQ ID NO.1, and the nucleotide sequence thereof is shown as SEQ ID NO. 2.

The invention provides a method for synthesizing epoxy norbornane, which is characterized in that a strain expressing epoxidase CytP derived from Pseudomonas putida KT2440 is used for producing the epoxy norbornane by taking norbornene as a substrate.

In one embodiment of the invention, the amino acid sequence of the epoxidase CytP is shown in SEQ ID NO. 1.

In one embodiment of the invention, the strain is Escherichia coli BL21(DE3) as a starting strain.

In one embodiment of the invention, the CytP enzyme is expressed in Escherichia coli BL21(DE3) using pET28a as the expression vector.

In one embodiment of the present invention, the reaction system comprises an organic phase and an aqueous phase, and the volume ratio of the organic phase to the aqueous phase is (2-6: 1).

In one embodiment of the invention, the organic phase is ethyl acetate and the aqueous phase is phosphate buffer.

In one embodiment of the invention, the reaction pH is from 8.0 to 8.5.

In one embodiment of the invention, the reaction temperature is from 30 to 37 ℃.

In one embodiment of the invention, after the strain is induced by IPTG for 12-13h or 16-17h, the cells are collected and added into a reaction system for reaction.

The invention provides application of epoxidase CytP with an amino acid sequence shown as SEQ ID NO.1 in preparation of epoxy norbornane.

In one embodiment of the invention, the cyclooxygenase CytP or the genetically engineered bacterium expressing the cyclooxygenase CytP is used as a catalyst to catalyze norbornene to generate norbornane epoxide.

In one embodiment of the invention, the amino acid sequence of the epoxidase CytP is shown in SEQ ID NO. 1.

In one embodiment of the invention, the genetically engineered bacterium is started from Escherichia coli BL21(DE 3).

The invention has the beneficial effects that: the invention provides cytochrome P450 epoxidase CytP which is used for catalyzing and producing epoxy norbornane. The amino acid sequence of the epoxidase CytP is shown in SEQ ID NO. 1. The epoxidase greatly improves the epoxidation activity of P450 enzyme, has high catalytic specificity to a substrate, improves the production capacity of a unit catalyst, and effectively reduces the production cost. When the cyclooxygenase provided by the invention takes norbornene as a substrate, the yield of epoxy norbornane can reach 36.81g/L, the molar yield can reach 31.46%, the yield of hydroxylation by-products is less than 0.01g/L, and the molar yield is less than 0.0084%, thus accelerating the industrial process of producing epoxy norbornane by an enzymatic conversion method.

Drawings

FIG. 1 is a SDS-PAGE pattern of cytochrome P450 epoxidase-induced expression according to the invention; lanes 1-3 are the sizes of the target protein bands in whole cells, supernatants and pellets after induction expression at 25 ℃ with 0.2mM IPTG, respectively.

FIG. 2 is a graph showing the verification of the enzyme activity, in which the components in the conversion system are subjected to deletion control, and the formation of the epoxidized product and the hydroxylated by-product are compared.

FIG. 3 is a graph showing the relationship between the pH of the conversion buffer and the amount of production of norbornane oxide.

FIG. 4 is a graph showing the relationship between the conversion temperature and the amount of norbornane oxide produced.

FIG. 5 is a graph showing the relationship between the ratio of two phases and the amount of norbornane oxide produced.

FIG. 6 is a graph showing the relationship between the induction time of the recombinant bacterium and the amount of norbornane epoxide produced.

Detailed Description

Example 1: expression and purification of CytP enzyme

Construction of genetically engineered bacteria and expression of proteins:

the nucleotide sequence (shown in SEQ ID NO. 2) of a target protein coding gene in Pseudomonas putida KT2440 is taken as a template, F1 and R1 are taken as primers (the restriction sites of BamH I and EcoR I are underlined respectively) for PCR amplification, and the amplification conditions are as follows:

95 ℃ for 5min, 29 cycles (98 ℃ for 10s, 55 ℃ for 15s, 72 ℃ for 1.5min), 72 ℃ for 5 min.

F1：caaatgggtcgcggatccATGGAGATCC(SEQ ID NO.3)；

R1：tcgacggagctcgaattcTTACCAAATCAC(SEQ ID NO.4)。

Obtaining a CytP gene coding region cDNA sequence, obtaining a recombinant expression plasmid pET-28a (+) -p28p by homologous recombination and connection with a pET-28a (+) plasmid vector which is subjected to the same double enzyme digestion after recovering a PCR product, transforming the recombinant plasmid pET-28a (+) -p28p into E.coli BL21(DE3), and obtaining a positive engineering bacterium named as E.coli BL21/pET-28a (+) -p28p through PCR identification.

Inoculating engineering bacteria E.coli BL21/pET-28a (+) -p28p into an LB culture medium, culturing for 12h to obtain an activation solution, inoculating the activation solution into a fresh TB culture medium, culturing for 2h, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.2mM, culturing for 14h at 25 ℃, and carrying out induced expression on the recombinant target protein. Collecting thallus by taking 150mL of induced fermentation broth through 6000r/min centrifugation

The results are shown in FIG. 2: lanes 1 to 3 are the sizes of the bands of the proteins contained in the whole cells, the supernatant and the pellet, respectively, and it can be seen that the target protein is expressed in all the cells, the supernatant and the pellet, and the bands are the same in size.

Example 2: CytP enzyme activity verification

The strain preserved in a glycerol tube is coated on an LB solid culture medium, the strain is cultured at the constant temperature of 37 ℃ until a monoclonal grows out, the monoclonal is picked up to be put into a fresh LB liquid culture medium, the constant temperature culture is carried out at 200rpm and 37 ℃ for 12h to obtain an activating solution, the activating solution is inoculated into a fresh TB culture medium in the amount of 1mL/100mL, the I PTG with the final concentration of 0.2mM is added after the culture is carried out for 2h, the induction culture is carried out at 25 ℃ for 14h, and the cells are collected after the completion.

0.2g of whole cells expressing CytP protein after induction culture and 1g of norbornene (C) were added to 100mL Erlenmeyer flasks, respectively₇H₁₀O, NBE), 320. mu.L of 30% hydrogen peroxide, 2.18mL of phosphate buffer (100mmol/L KCL, pH7.4) and 7.5mL of ethyl acetate, reacted at 25 ℃ for 48 hours, centrifuged at 5000r/min for 10min, the upper organic phase was extracted, dried over anhydrous magnesium sulfate, passed through a 0.22 μm organic membrane, and analyzed by gas chromatography.

The specific gas chromatographic analysis method comprises the following steps: the sample analysis adopts a gas chromatograph Agilent GC-7890B and a chiral gas chromatographic column DB-5; the temperature of a sample inlet is 250 ℃; the initial column temperature is 45 ℃ for 2min, and the temperature is increased to 250 ℃ at the speed of 10 ℃/min; the carrier gas is helium, the flow rate is 1.0mL/min, and the split ratio is 10: 1. Under the detection conditions, the retention time of ethyl acetate, norbornene and epoxy norbornane (EPO-NBE) is 2.255min, 3.315min and 6.195min respectively.

EPO-NBE molar yield ═ P/S₀)×100％；

Wherein: p represents the final molar concentration of EPO-NBE, S₀Represents the initial molar concentration of NBE.

The specific results are shown in FIG. 2, and it is understood from FIG. 2 that the enzymatic effect of the whole cells was 8.21% molar yield. From the results, it can be seen that the CytP enzyme not only has obvious epoxidation activity, but also has good catalytic specificity. In contrast, the blank control and the e.coli control group without the target gene recombinant vector both have no corresponding enzymolysis effect, and the molar yield is significantly reduced in a reaction system without organic solvent ethyl acetate and without hydrogen peroxide.

Example 3: whole cell optimum reaction pH

Referring to example 2, 0.2g of whole cells were transformed for 48 hours in 10mL of a two-phase transformation system (organic phase: aqueous phase: 3: 1) composed of phosphate buffers of ph6.0, ph6.5, ph7.0, ph7.5, ph8.0, ph8.5 and ph9.0, respectively, and the yield of epoxynorbornane was measured according to the above-described detection method to calculate the molar yield of epoxidation. The results show that the epoxidation activity of the CytP enzyme increases with increasing pH at pH6.0-pH8.5, reaches 10.67g/L at pH8.0, peaks around pH8.5, and has a yield of 11.17g/L of epoxynorbornane of 9.54% molar yield, and subsequently the epoxidation activity decreases with further increasing pH. This indicates that higher pH (alkaline environment) is more favorable for the catalytic epoxidation reaction of CytP enzyme, and the whole cell has better epoxidation activity at pH 8.0-8.5.

Example 4: optimum reaction temperature of whole cells

See example 1 for a difference in that the yield of epoxynorbornane converted by CytP at various temperatures (16, 20, 25, 30, 37 ℃) for 48h was determined at a buffer pH of 8.5 and the molar yield of epoxidation was calculated. The result shows that the epoxidation activity of CytP enzyme is reduced along with the rise of temperature in the range of 16-25 ℃, the epoxidation activity is increased along with the rise of temperature in the range of 25-30 ℃, the peak can be reached at 30 ℃, the yield of the epoxy norbornane is 17.32g/L, the molar yield is 14.8%, and the yield can also reach 17.18g/L and 14.68% at 37 ℃. Thus, the catalytic epoxidation reaction of CytP enzymes is more favored at a conversion temperature of 30-37 ℃, and CytP enzymes have better epoxidation activity at that temperature.

Example 5: optimal phase ratio for whole cell transformation

See example 1 for a difference in that the yield of epoxynorbornane converted by CytP under two different volume ratio conditions (organic phase: aqueous phase: 1: 6-6: 1) for 48h was determined at 30 ℃ and buffer pH8.5, and the molar yield of epoxidation was calculated.

The organic phase was ethyl acetate and the aqueous phase was phosphate buffer (containing 320. mu.L of 30% hydrogen peroxide).

The results show that the epoxidation activity of the CytP enzyme increases with increasing phase ratio in the range of 1:6 to 2:1 in the two-phase ratio and peaks at 2:1 in the range of 25.17g/L of epoxynorbornane with a molar yield of 21.51%, and that the epoxidation activity subsequently decreases with further increase in the phase ratio. This shows that the substrate dissolution and the inhibition of the organic solvent to the enzyme activity can be better counteracted when the ratio is 2:1, which is more beneficial to the catalytic epoxidation reaction of CytP enzyme.

Example 6: whole cell transformation Induction time analysis

See example 2 for a difference in that the time to induce the strain with IPTG was changed to 12-17 h.

The yield of epoxy norbornane converted by CytP for 48h under different induction time conditions (12-17h) is measured under the conditions of 30 ℃, the pH of a buffer solution and a two-phase ratio of 2:1, and the epoxidation molar yield is calculated.

As shown in FIG. 6, the epoxidation activity of CytP enzyme is reduced along with the increase of the induction time within the induction time range of 12-15h, the short-time induction can promote the transformation, the yield of epoxy norbornane is 35.42g/L and the molar yield is 30.27% at the induction time of 12h, and the yield of epoxy norbornane is 34.45g/L and the molar yield is 29.44% at the induction time of 13 h; while the epoxidation activity increased with the increase of the induction time within the range of 15-16h and reached a peak value around 16h, the yield of the epoxy norbornane was 36.81g/L and the molar yield was 31.46%, and at the induction time of 17h, the yield of the epoxy norbornane was 35.98g/L and the molar yield was 30.75%. Therefore, the cells have better epoxidation activity when being induced for 12-13 and 16-17 hours, and are more beneficial to catalyzing epoxidation reaction.

Comparative example 1

Referring to example 2, the difference is that whole cells of recombinant bacteria BL21-P28BU7 expressing CytBU7 enzyme (derived from Bacillus megaterium) in P450 enzyme system are added into the reaction system, after the reaction is finished, the content of epoxy norbornane is measured, and the result shows that the yield of epoxy norbornane is 0.044g/L, the molar yield is only 0.037%, and is far lower than that of CytP enzyme.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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