CN112442474B

CN112442474B - Preparation method of (-) gamma-lactam

Info

Publication number: CN112442474B
Application number: CN202011449726.5A
Authority: CN
Inventors: 倪晔; 周彤彤; 韩瑞枝; 许国超; 周婕妤; 董晋军
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2022-08-23
Anticipated expiration: 2040-12-09
Also published as: CN112442474A

Abstract

The invention discloses a preparation method of (-) gamma-lactam, belonging to the technical field of biological engineering. The (+) gamma-lactamase of the invention is derived from Fusarium oxysporum (Fusarium oxysporum) and can be used as a catalyst for preparing optically pure (-) gamma-lactams. The method has the advantages of good stability, strong stereoselectivity, mild applicable reaction conditions, environmental friendliness, avoidance of complicated steps such as crushing, centrifugation and the like due to whole-cell catalysis, simpler operation and good application and development prospects.

Description

Preparation method of (-) gamma-lactam

Technical Field

The invention relates to a preparation method of (-) gamma-lactam, belonging to the technical field of biological engineering and enzyme engineering.

Background

As an important chiral compound, the gamma-lactam can be used for synthesizing antiviral drugs of abacavir and peramivir, can also be used for synthesizing a ceramidase inhibitor, and draws wide attention in the application of the pharmaceutical chemistry field. The gamma-lactamase acts on amide bonds, not protein peptide bonds, and forms carboxylic acids. The gamma-lactamase with stereoselectivity can asymmetrically hydrolyze gamma-lactam to obtain optically pure gamma-lactam.

The initial production process of (-) gamma-lactam utilizes alkaline protease to selectively hydrolyze racemic gamma-lactam in mixed solvent of water/tetrahydrofuran, and the concentration of the substrate can reach 100 g/L. In 1996, Nakano hio et al, resolved racemic γ -lactams by lipase (Nakano hio et al tetrahedron: Asymmetry, 1996, 7 (8): 2381-. In 1999, Mahmoudian et al screened N-acetyl-L-phenylalanine as the sole carbon source to obtain (+) gamma-lactamase produced by Pseudomonas, which was able to resolve racemic gamma-lactam with high stereoselectivity, but had poor stability, easily lost activity, and failed to characterize the properties of the enzyme (Mahmoudian et al tetrahedron: Asymmetry, 1999, 10 (6): 1201-1206). In 2000, Wisdom et al reported that one strain of acid-feeding pseudomonas was screened, and the enzyme had higher stability than the previously identified enzyme and could perform a resolution reaction at a higher substrate concentration (microargnism, lactic enzyme immobilized thermum, and third use, 2000, United States patent, US006090616A), but wild bacteria had a complex genetic background, high fermentation cost, and a limited expression level of γ -lactamase, and a large number of cells were required to be added during the whole-cell catalytic wenstein-Barr reaction, limiting industrial applications.

In 2015, Zheng et al discovered (+) gamma-lactamase derived from thermophilic archaea, and the optimum reaction temperature was over 100 ℃ (Zheng G et al. appl Biochem Biotechnol, 2015, 176: 170-. In recent years, with the disclosure of massive genome data, the mining of new gamma-lactamase with superior performance from a database has become a hot research. Realizes the stable, efficient and soluble expression of the gamma-lactamase, is applied to the stereoselective resolution of racemic gamma-lactam, and is a potential method for industrially preparing optically pure (-) gamma-lactam.

The inventor of the present invention tried to express gamma-lactamase derived from Delftia acidovorans (Delftia acidovorans) in E.coli BL21 in the early stage of the project due to the advantage of the E.coli expression system as a production strain which is widely used in industry, but the gamma-lactamase has poor solubility and exists almost in the form of inclusion body, and the application in the reaction of resolving the Venus lactone is limited. Therefore, how to obtain a stable, efficient and soluble expressed gamma-lactamase to prepare optically pure (-) gamma-lactam by high-efficiency catalysis becomes a hotspot and difficulty of research.

Disclosure of Invention

The invention aims to solve the technical problems of low catalytic activity, poor thermal stability and the like of the reported (+) gamma-lactamase to a substrate Venus lactone, and provides a method for preparing optically pure (-) gamma-lactam by high-efficiency catalysis through the gamma-lactamase which is expressed stably, efficiently and solubly by genetic engineering bacteria.

The invention firstly provides a recombinant escherichia coli, which expresses (+) gamma-lactamase derived from Fusarium oxysporum (Fusarium oxysporum).

In one embodiment of the invention, the amino acid sequence of the (+) gamma-lactamase is shown as SEQ ID NO. 2.

In one embodiment of the invention, the nucleotide sequence of the (+) gamma-lactamase is shown as SEQ ID NO. 1.

In one embodiment of the invention, pET28a, pET32a, pET41a, pQE80L, pET20b, or pBAD are used as expression vectors.

In one embodiment of the invention, the recombinant Escherichia coli expresses (+) gamma-lactamase with a nucleotide sequence shown as SEQ ID NO.1 by taking BL21(DE3) as a host and pET28a as a vector.

The invention also provides a method for preparing the recombinant escherichia coli, which comprises the following steps:

1) cloning a gene FoLM for coding the (+) gamma-lactamase by adopting a chemical synthesis method or a PCR method;

2) carrying out double digestion on the FoLM gene obtained in the step 1) and a plasmid pET28a by using restriction endonucleases BamH I and Xho I at the same time, and then connecting by using T4 DNA ligase to obtain a recombinant expression vector pET28 a-FoLM;

3) transforming the recombinant expression vector pET28a-FoLM obtained in the step 2) into escherichia coli BL21(DE3) to obtain recombinant genetic engineering bacteria BL21(DE3)/pET28 a-FoLM.

The invention also provides a method for preparing (-) gamma-lactam by whole-cell transformation, which takes the recombinant escherichia coli as a cell catalyst and reacts in a reaction system containing a substrate Venus lactone.

In one embodiment of the invention, the cell concentration of the recombinant escherichia coli in the reaction system is 1-10 g/L.

In one embodiment of the present invention, the reaction conditions are: the temperature is 30-80 ℃, and the pH is 6-8.

In one embodiment of the invention, the concentration of the substrate vinelactone is 5-500 mmol/L; .

The invention also provides a method for preparing (-) gamma-lactam by an enzyme method, which applies the recombinant Escherichia coli to produce (+) gamma-lactamase, and uses the produced (+) gamma-lactamase for enzyme catalytic reaction of the Venus lactone.

In one embodiment of the invention, the method for producing (+) gamma-lactamase by using the recombinant Escherichia coli comprises the following steps: inoculating the recombinant Escherichia coli into a culture medium and culturing to OD ₆₀₀ And (3) reaching 0.5-0.7, adding IPTG, carrying out induced culture at 16-30 ℃ for 8-24 h, then centrifugally collecting thalli, and crushing cells to obtain (+) gamma-lactamase crude enzyme solution.

In one embodiment of the invention, the fermentation medium is LB liquid medium.

In one embodiment of the present invention, the LB liquid medium contains: 10-20 g/L of peptone, 5-10 g/L of yeast extract, 10g/L of NaCl and 7.0 of pH.

In one embodiment of the present invention, the method is to inoculate the recombinant Escherichia coli into LB culture medium containing kanamycin to culture to OD ₆₀₀ Reaching 0.5-0.7, carrying out induction culture at 16-30 ℃ for 8-24 h under the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.1-1.0 mmol/L, and then efficiently expressing the recombinant (+) gamma-lactamase, centrifugally collecting thalli and crushing cells to obtain (+) gamma-lactamase crude enzyme solution.

In one embodiment of the invention, the concentration of the wenstein lactone is 5 to 500 mmol/L.

In one embodiment of the invention, the dosage of the (+) gamma-lactamase is 1 to 10 g/L.

In one embodiment of the present invention, the reaction conditions are: the pH value is 6-8, and the temperature is 30-80 ℃.

Advantageous effects

(1) The invention realizes the soluble expression of (+) gamma-lactamase derived from Fusarium oxysporum (Fusarium oxysporum) in escherichia coli.

(2) The recombinant escherichia coli provided by the invention can express (+) gamma-lactamase with good solubility, low Km value, high catalytic efficiency (the conversion rate is more than or equal to 50%), strong stereoselectivity (e.e. > 99.9%), mild applicable reaction conditions and environmental friendliness.

Drawings

FIG. 1: PCR amplification electropherogram of gene FoLM; m, Marker; 1, gene FoLM.

FIG. 2: pET28a-FoLM recombinant plasmid physical map.

FIG. 3: protein electrophoretogram of recombinant (+) gamma-lactamase; m, Marker; lane 1 and Lane 2 are the supernatant and the precipitate of the recombinant genetically engineered bacterium BL21(DE3)/pET28a-FoLM after induction, respectively.

FIG. 4 is a schematic view of: protein electrophoretogram of recombinant (+) gamma-lactamase; m, Marker; lane 1 is the pure enzyme FoLM.

FIG. 5: selective analytical liquid phase detection of (+) gamma-lactamase.

FIG. 6: thermal stability profile of (+) -gamma-lactamase FoLM.

Detailed Description

The media involved in the following examples are as follows:

LB solid medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl, 20g/L of agar and 7.0 of pH.

LB liquid medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 7.0 of pH.

The detection methods referred to in the following examples are as follows:

detection of enzyme activity of (+) gamma-lactamase:

the measuring system is as follows: appropriate amount of enzyme solution, 10 mmol. L ^-1 Placing the wenscolide in a reaction vessel, and carrying out oscillation reaction at 30 ℃ for 30 min; and after the reaction is finished, sampling and carrying out liquid phase detection. Liquid phase detection conditions: the column was Diamonsil Plus C18(25 cm. times.4.6 mm,5 μm), and the mobile phase was methanol: water (20: 80) at a flow rate of 0.2 to 1mL/min ^-1 The detection wavelength is 225 nm.

Definition of enzyme activity unit (U): the amount of enzyme required for the (+) -gamma-lactamase to catalyze the hydrolysis of 1. mu. mol of gamma-lactam at 30 ℃ is defined as one enzyme activity unit (U).

Example 1: construction and culture of recombinant Escherichia coli BL21(DE3)/pET28a-FoLM

(1) The nucleotide sequence of the chemical synthesis is shown as (+) gamma-lactamase gene FoLM in SEQ ID NO. 1.

(2) Construction of recombinant E.coli:

plasmids pET28a and FoLM were double digested with restriction enzymes BamH I and Xho I in a 37 ℃ water bath overnight, purified by agarose gel electrophoresis the next day and the fragments of interest recovered using agarose recovery kit (as shown in FIG. 1). At 37 ℃, the gene FoLM is connected with an enzyme-cut plasmid pET28a by using T4 DNA ligase to obtain a recombinant expression vector pET28a-FoLM (shown in figure 2), the constructed recombinant expression vector pET28a-FoLM is transformed into an escherichia coli BL21(DE3) competence through heat, the competent escherichia coli BL21 is coated in an LB solid culture medium containing kanamycin resistance, and colony PCR verification is carried out after overnight culture, and a positive clone is the recombinant escherichia coli BL21(DE3)/pET28 a-FoLM.

(3) Culturing the recombinant Escherichia coli:

selecting positive clone, culturing in LB liquid culture medium for 12 hr to obtain seed liquid, transferring the seed liquid into fresh LB liquid culture medium according to 1% (v/v) transfer amount, and culturing to OD ₆₀₀ When the concentration reaches 0.6-0.8, 0.2 mmol/L is added ^-1 IPTG, induced culture at 16 ℃ for 24 hours, and then centrifugation at 8000r/min for 10min at 4 ℃ to collect thalli.

The collected cells were suspended in a sodium phosphate buffer (100 mmol. multidot.L) ^-1 pH 7.0), and protein expression was analyzed by SDS-PAGE (shown in figure 3).

As can be seen from FIG. 3, the major portion of the target protein was found in the supernatant, indicating that the recombinant enzyme could be expressed in E.coli in a soluble manner.

Example 2: separation and purification of recombinant Escherichia coli enzyme and (+) gamma-lactamase

(1) The recombinant Escherichia coli BL21(DE3)/pET28a-FoLM obtained in example 1 was suspended in solution A (20 mmol. L) ^-1 Sodium phosphate, 500 mmol. L ^-1 NaCl，20mmol·L ^-1 Imidazole, pH 7.4), and obtaining a crude enzyme solution after ultrasonic crushing and centrifugation.

(2) Purification of (+) -gamma-lactamase:

the column used for purification was an affinity column, HisTrap FF column, which was affinity-bound using a histidine tag on the recombinant protein. Firstly, using solution A to balance nickel column, loading crude enzyme solution obtained in step (1), continuously using solution A to elute penetration peak, after balancing, using solution B (20 mmol. L) ^-1 Sodium phosphate, 500 mmol. L ^-1 NaCl，1000mmol·L ^-1 Imidazole, pH 7.4), eluting the recombinant protein bound on the nickel column to obtain the recombinant (+) gamma-lactamase.

The purified protein was subjected to enzyme activity assay (visfate as substrate) and SDS-PAGE analysis (as shown in FIG. 4). As can be seen from FIG. 4, nickel column purificationAnd then, a single band is shown at about 45kDa, and the impurity protein is less, which indicates that the nickel column has better purification effect. Then, purified (+) gamma-lactamase was replaced with PBS (100 mmol. L.) using a HiTrap desaling Desalting column (GE Healthcare) ^-1 pH 7.0) buffer.

Example 3: properties of (+) gamma-lactamase

(1) At the temperature of 30 ℃ and under the condition of pH 7.0, according to the method for detecting the enzyme activity of (+) gamma-lactamase, after reacting for 30min, the enzyme activity of (+) gamma-lactamase to substrate vinelactone is measured, and the enzyme activity is as follows: 0.8 U.mg ^–1 。

(2) Preparation of 100 mmol. L ^-1 Buffers at different pH: Tris-HCl (8.0-9.0), sodium phosphate buffer (pH 6.0-8.0), and citric acid-sodium citrate buffer (pH 5.0-6.0). Then, racemic γ -lactam was used as a substrate to determine the relative enzyme activities of FoLM in buffers with different pH, as shown in table 1. The optimum reaction pH of the FoLM is 7.0-8.0, and the enzyme activity is 0.7-0.8 U.mg ^–1 。

TABLE 1 relative enzyme Activities of FoLM in buffers of different pH

(3) The enzyme activities of the FoLM at different temperatures (20-80 ℃) are respectively measured by taking racemic gamma-lactam as a substrate, the maximum enzyme activity is defined as 100%, and the enzyme activities measured at other temperatures are calculated according to the percentage relative to the maximum enzyme activity, and are specifically shown in table 2. The results show that the optimum reaction temperature of the FoLM is 60-70 ℃ and 2.7-2.8 U.mg ^–1 。

TABLE 2 relative enzyme Activity of the FoLM at different temperatures

(4) And respectively measuring the enzyme activities of the FoLM after heat preservation for different times at different temperatures (50, 70 and 90 ℃) by using racemic gamma-lactam as a substrate, wherein the highest enzyme activity measured by 0 hour of heat preservation at the same temperature is defined as 100%, and the enzyme activities measured at other times are calculated according to the percentage relative to the highest enzyme activity. The results show that the half-lives of the FoLM at 50, 70 and 90 ℃ are 32, 17 and 4h respectively. As shown in fig. 6, the residual activity of FoLM after incubation at 50 ℃ for 60h was close to 30%.

(5) Kinetic parameters of the FoLM on the substrate racemic γ -lactam were determined. The enzyme activity assay system is listed as follows: sodium phosphate buffer solution (100 mmol. L) ^-1 pH 7.0), racemic γ -lactam (0 to 100 mmol. multidot.L) ^-1 ). The reaction rate was characterized by calculating the specific enzyme activity, and thus the kinetic parameters were calculated. The kinetic parameters of the FOLM on the substrate racemic gamma-lactam were determined to be K _m 37.6 mmol. multidot.L ^-1 ，V _max Is 4.4. mu. mol/min ^–1 ·mg ^–1 。

(6) Substrate profiling

The enzyme activity of different substrates catalyzed by (+) gamma-lactamase (FoLM) is measured, the enzyme activity measured by taking visolactone as the substrate is taken as a 100 percent contrast, and the enzyme activity measured by other substrates is calculated by the percentage of the two. The measurement results are shown in Table 3.

TABLE 3 substrate spectra of FoLM

Table 3 shows that FoLM is able to catalyze caprylolactam with a relative activity of only 7% of that of γ -lactam, with very low activity on the substrates heptanolactam, caprolactam, lauryllactam, butyramide, valeramide.

(7) Stereoselective analysis

The selectivity of the FoLM catalytic substrate for racemic γ -lactam was determined. The reaction system (10mL) was: purified enzyme solution of appropriate amount, 10 mmol. multidot.L ^-1 Racemic γ -lactam. The reaction was shaken at 30 ℃ for 24 h. After the reaction is finished, sampling and carrying out liquid phase detection. Detection conditions are as follows: daicel Chiralpack AS-H column (25 cm. times.4.6 mm,5 μm), detection wavelength 230nm, mobile phase acetonitrile: isopropanol (80: 20) at a flow rate of 0.2-1 mL/min. As can be seen from FIG. 5, the enzyme has very good stereoselectivity, preferablyThe value of e.e. can reach 99.9% when (+) gamma-lactam is hydrolyzed.

Example 4: application of (+) gamma-lactamase in asymmetric resolution of Venus lactone to prepare (-) gamma-lactam

Taking 5-10 g of the obtained expression recombinant (+) gamma-lactamase cells in a phosphate buffer (pH 6-8, 100 mmol. L) ^-1 ) Adding 100-500 mmol.L ^-1 Wenslactone (Table 4), and the total volume of the reaction solution was 10 mL. Placing the reaction at 50-70 ℃, sampling, detecting and converting, wherein the conditions are as follows: daicel Chiralpack AS-H column (25 cm. times.4.6 mm,5 μm), detection wavelength 230nm, mobile phase acetonitrile: isopropanol (80: 20) at a flow rate of 0.2-1 mL/min.

TABLE 4 FoLM Whole cell catalysis for preparation of (-) gamma-lactams by asymmetric separation of vesselnolide

The results show that the reaction cycle: 14h, amount of catalytic conversion substrate per mass of catalyst: 5.4 g/g.

10g/L of the whole FoLM cells catalyse the completion of the resolution of 500mmol/L of wenscolide in 14h with a substrate to catalyst ratio (S/C) of 5.4, higher than the reported E.coli RutB (S/C,0.872) (see Zhu et al Bioorganic & Medicinal Chemistry Letters,2014,24(20):4899-4902.), and recombinant E.coli rosetta (DE3)/pDxcc-est-gla (S/C,2.5) (see Wang et al applied Microbiology and Biotechnology,2014,98(16): 6991-7001.).

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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Claims

1. A method for preparing (-) gamma-lactam by whole cell transformation is characterized in that recombinant Escherichia coli is used as a cell catalyst and reacts in a reaction system containing a substrate wenstein, and the recombinant Escherichia coli expresses a gene derived from fusarium oxysporum (F.oxysporum) (R) (), (R) () gamma-lactamFusarium oxysporum) The amino acid sequence of the (+) gamma-lactamase is shown in SEQ ID NO. 2.

2. The method of claim 1, wherein the recombinant E.coli has pET28a, pET32a, pET41a, pQE80L, pET20b, or pBAD as an expression vector.

3. The method of claim 1, wherein the nucleotide sequence encoding the (+) gamma-lactamase is set forth in SEQ ID No. 1.

4. The method of claim 3, wherein the cell concentration of the recombinant Escherichia coli in the reaction system is 1-10 g/L.

5. The method of claim 4, wherein the concentration of the wenskolide is 5 to 500 mmol/L.

6. The method of claim 5, wherein the reaction conditions are: the temperature is 30-80 ℃, and the pH is 6-8.

7. A method for preparing (-) gamma-lactam by an enzyme method is characterized in that recombinant Escherichia coli is used for producing (+) gamma-lactamase, and the produced (+) gamma-lactamase is used for enzyme catalytic reaction of Venus lactone, wherein the recombinant Escherichia coli expresses (+) gamma-lactamase derived from fusarium oxysporum, and the amino acid sequence of the (+) gamma-lactamase is shown in SEQ ID NO. 2.

8. The method of claim 7, wherein the recombinant E.coli is inoculated into a culture medium and cultured to OD ₆₀₀ And (3) reaching 0.5-0.7, adding IPTG, carrying out induction culture at 16-30 ℃ for 8-24 h, centrifuging and collecting thalli, and crushing cells to obtain (+) gamma-lactamase crude enzyme solution.

9. The method of claim 7 or 8, wherein the concentration of vinelactone is 5-500 mmol/L.

10. The method of claim 9, wherein the (+) gamma-lactamase is present in an amount of 1 to 10 g/L.

11. The method of claim 10, wherein the reaction conditions are: the pH value is 6-8, and the temperature is 30-80 ℃.