CN110272856B - A kind of recombinant bacteria expressing D-threonine aldolase and its construction method and application - Google Patents

Abstract

The invention discloses a recombinant bacterium for expressing D-threonine aldolase and a construction method and application thereof, belonging to the technical field of enzyme engineering. The recombinant strain expresses D-threonine aldolase with an amino acid sequence shown as SEQ ID NO. 2. The invention provides a D-threonine aldolase which can be used as a catalyst for synthesizing chiral beta-hydroxy-alpha-amino acid, has high catalytic efficiency (the conversion rate is more than 65%), strong stereoselectivity (e.e. > 99%, d.e. > 95%), mild reaction conditions and environmental friendliness. The D-threonine aldolase has good catalytic effect, wide substrate applicability and good application and development prospects.

Description

A kind of recombinant bacteria expressing D-threonine aldolase and its construction method and application

technical field

The invention relates to a recombinant bacterium expressing D-threonine aldolase, a construction method and application thereof, and belongs to the technical field of enzyme engineering.

Background technique

Chiral β-hydroxy-α-amino acids are a very important class of compounds, which are widely used in the manufacture of fine chemicals such as medicines and materials because of their two functional groups, chiral hydroxyl and amino acids. The chemical synthesis of chiral β-hydroxy-α-amino acids has the disadvantages of expensive catalysts, heavy metal pollution, long synthetic routes, and the stereoselectivity of products can only be improved under harsher conditions, which is not conducive to the scale-up of industrial production. Compared with chemical methods, enzymatic methods do not require any protective groups, have better stereoselectivity, and the reaction can be completed in one step. Therefore, the enzymatic synthesis of β-hydroxy-α-amino acids has more potential for application development.

Threonine aldolase is a kind of pyridoxal phosphate-dependent aldolase, which is a powerful tool for carbon-carbon bond formation in organic synthesis. It can catalyze the specific hydroxylation of aldehydes and glycines with different substituents. Aldehyde condensation produces highly valuable β-hydroxy-α-amino acids with high selectivity at the α-carbon of the two chiral centers formed, while the β-carbon is less stereoselective. Therefore, the development of high-efficiency, high-stereoselective threonine aldolase, for the technical transformation of chiral intermediates (l-syn-p-methylsulfonylphenylserine) for the synthesis of thiamphenicol, florfenicol and other drugs Upgrading is important.

At present, most of the enzymatic production of l-syn-p-methylsulfonylphenylserine is obtained by enzymatic separation of dl-syn-p-methylsulfonylphenylserine. Among them, in a two-phase system (dichloromethane, dichloroethane, cyclohexanone), D-threonine aldolase derived from Delftiasp.RIT313 can completely resolve 300 mmol·L ^-1 dl-syn-pair Methylsulfonylphenylserine, the highest substrate concentration at present (Catalysis Science & Technology, 2017, 7, 5964-5973). However, there are few reports on the enzymatic synthesis of l-syn-p-methylsulfonylphenylserine. The l-threonine aldolase from P. putida catalyzes the synthesis of p-methylsulfonylbenzaldehyde and glycine to l-syn-p-methylsulfonylphenylserine with a yield of 68% and a de value of only 53% (Tetrahedron, 2007 , 63, 918-926). Low enzymatic activity and stereoselectivity have always been the bottleneck restricting the application of threonine aldolase. It is urgent to develop a new type of threonine aldolase with high activity and high stereoselectivity to meet the requirements of industrial application.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a recombinant bacterium expressing D-threonine aldolase, and the produced D-threonine aldolase can efficiently catalyze the synthesis of l-syn- For p-methylsulfonyl phenylserine, the yield is over 65%, and the d.e. value is over 95%. This process has the advantages of mild conditions and simple operation.

The first object of the present invention is to provide a recombinant bacterium expressing D-threonine aldolase, the recombinant bacterium expressing D-threonine aldolase whose amino acid sequence is shown in SEQ ID NO.2.

Further, the recombinant bacteria use bacteria, fungi, plants, insects or animal cells as host cells.

Further, the recombinant bacteria take Escherichia coli as the host bacteria. E. coli BL21(DE3) is preferred.

Further, the expression vector of the recombinant bacteria is bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or mammalian cell virus.

Further, the expression vectors of the recombinant bacteria are pET series expression vectors. PET28a is preferred.

The second object of the present invention is to provide the construction method of described recombinant bacteria, comprises the steps:

(1) Construction of recombinant plasmid pET28a-ApDTA: The D-threonine aldolase gene ApDTA is connected with the digested plasmid pET28a to obtain a recombinant expression vector pET28a-ApDTA;

(2) Construction of recombinant strain E.coli BL21(DE3)/pET28a-ApDTA: The constructed recombinant expression vector pET28a-ApDTA was thermally transferred into E. coli BL21(DE3) competent, and the recombinant strain E.coli BL21 was obtained by culture and screening. (DE3)/pET28a-ApDTA.

The third object of the present invention is to provide the application of the recombinant bacteria in the synthesis of chiral β-hydroxy-α-amino acids.

Further, the application is to use the D-threonine aldolase produced by fermentation of the recombinant bacteria as a catalyst to synthesize chiral β-hydroxy-α-amino acids.

Further, the application is specifically to catalyze the synthesis of chiral β-hydroxy-α-aryl amino acids from aldehyde and glycine.

Further, the catalysis reacts for 10 to 20 hours under the conditions that the reaction temperature is 5-15° C. and the pH is 5.5-6.5.

The beneficial effects of the present invention are:

The invention provides a D-threonine aldolase which can be used as a catalyst for the synthesis of chiral β-hydroxy-α-amino acids, and has high catalytic efficiency (conversion rate>65%) and strong stereoselectivity (e.e.> 99%, d.e.>95%), the applicable reaction conditions are mild and environmentally friendly. The D-threonine aldolase of the invention has good catalytic effect, wide substrate applicability and good application and development prospect.

Description of drawings

Fig. 1 is the electrophoresis map of PCR amplification of gene ApDTA; M, Marker; 1, gene ApDTA;

Fig. 2 is the physical map of pET28a-ApDTA recombinant plasmid;

Figure 3 is the protein electrophoresis image of recombinant d-threonine aldolase; M, Marker; lanes 1, 2, and 3 are the supernatant, precipitation and purification of recombinant bacteria E.coli BL21(DE3)/pET28a-ApDTA after induction, respectively post-enzyme;

Fig. 4 is the reaction formula that d-threonine aldolase catalyzes the condensation of p-methylsulfonylbenzaldehyde and glycine into p-methylsulfonylphenylserine;

Fig. 5 HPLC spectrum of reaction liquid product l-syn-p-methylsulfonyl phenylserine.

Detailed ways

The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

The HPLC analysis conditions of the reaction products catalyzed by D-threonine aldolase are: chromatographic column Diamonsil Plus C18 (25cm×4.6mm, 5μm), mobile phase is V(CH ₃ CN):V(50mmol·L ^-1 dihydrogen phosphate Potassium solution) = 15:85, the flow rate is 0.2-1 mL·min ^-1 , the detection wavelength is 338 nm, the column temperature is 40 °C, and the OPA-NAC pre-column derivatization liquid phase measurement is performed. At the same time, the l-syn-p-methylsulfonyl phenylserine standard was used as a control to determine the peak time and sequence of the product, and based on this, the enzyme activity was measured. Determination of D-threonine aldolase activity: The enzyme activity assay system of D-threonine aldolase to substrate p-methylsulfonylbenzaldehyde is: an appropriate amount of enzyme solution, 5mmol·L ^-1 p-methylsulfonylbenzaldehyde , 50mmol·L ^-1 glycine, 50μmol·L ^-1 pyridoxal phosphate (PLP), 50μmol·L ^-1 Mn ²⁺ were shaken at 30℃ for 10min. After the reaction, samples were taken for liquid phase detection. One unit of enzyme activity (Unit) is defined as the amount of biocatalyst required to catalyze the production of 1 μmol of l-syn-p-methylsulfonylphenylserine from p-methylsulfonylbenzaldehyde per minute. Using bovine serum albumin as a standard, the protein concentration was determined by the Bradford method.

Example 1: Screening of D-threonine aldolase by gene mining technology based on probe enzyme sequence

According to the reported gene sequence of D-threonine aldolase (Alcaligenes xylosoxydans) with aldehyde condensation to p-methylsulfonyl phenylserine, this sequence was used as a probe to search in the NCBI database and BLAST comparison analysis, to find the same Candidate enzyme genes whose probe sequences have 40% to 70% homology, select enzyme genes with higher sequence consistency, and ensure that the source strains of these enzyme genes are different from the probes, and then design primers according to the retrieved gene sequences , using PCR amplification to obtain DNA encoding these enzymes, clone and express them, and finally screen the target substrate (p-methylsulfonylbenzaldehyde) for activity and stereoselectivity, that is, to obtain high activity and high selection Sexual D-threonine aldolase.

Example 2: Cloning of D-threonine aldolase gene

The recombinant plasmid pET28a-ApDTA was constructed by "one-step cloning method" (homologous recombination).

(1) First, use a nutrient gravy agar medium (10.0 g of peptone, 3.0 g of beef extract, 5.0 g of NaCl, 15.0 g of agar, 1.0 L of distilled water, pH 7.0) to activate and rejuvenate the above-mentioned Microbacillus depigmentosa at 25°C . Then, after the colony has grown, the single colony is inserted into the liquid medium for cultivation. After the cells were obtained by centrifugation, the genomic DNA of Achromobacter pectoralis was extracted using a bacterial genomic DNA kit.

(2) Design primers according to the reported D-threonine aldolase gene (upstream primer: CAGCAAATGGGTCGC GG ATCC ATGTCCCAGGAAGTCATACGCG, downstream primer: TGCGGCCGCAAGCTT GTCGAC TCAGCGCGAGAAGCCGCG, where GGATCC is the BamHI restriction site and GTCGAC is the Sal I restriction site ), using Achromobacter pyogenes genomic DNA as template to carry out PCR, the system is as follows (μL): MgSO ₄ 0.6, dNTP 1.0, upstream primer 0.4, downstream primer 0.4, KOD Buffer 1.0, KOD enzyme 0.2, template 0.4, ddH ₂ O 6.0. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 10 min, denaturation at 98°C for 20s, annealing at 65°C for 20s, extension at 68°C for 50s, repeated 30 cycles, and extension at 68°C for 10 min. The PCR product was identified by agarose gel electrophoresis, and a band in the range of 800-1200 bp was recovered by agarose gel DNA recovery kit (Fig. 1), namely the D-threonine aldolase gene. The obtained D-threonine aldolase gene was named ApDTA, the nucleotide sequence is shown in SEQ ID No. 1: the full length is 1140 bp, the start codon is ATG, and the stop codon is TGA. There is no intron in the sequence, the coding sequence starts from the first nucleotide to 1140 nucleotides, and the encoded protein sequence is shown in SEQ ID No.2. The sequence has been submitted to the NCBI database with the GenBank accession number KNY11228.1.

Example 3: Construction and culture of recombinant Escherichia coli BL21(DE3)/pET28a-ApDTA

The plasmid pET28a was double digested with restriction enzymes BamH I and Sal I in a water bath at 37°C for 4 hours, identified by agarose gel electrophoresis, and purified and recovered by agarose DNA recovery kit. At 37°C, the gene ApDTA was ligated with the digested plasmid pET28a using the recombinase in the recombination kit to obtain the recombinant expression vector pET28a-ApDTA (Fig. 2). The constructed recombinant expression vector pET28a-ApDTA was thermally transferred into E. coli BL21(DE3) competent, coated with LB solid plate containing kanamycin resistance, and the colony PCR was carried out after overnight culture, and the positive clone was the gene Recombinant engineering bacteria E.coli BL21(DE3)/pET28a-ApDTA. Pick positive clones and culture them in LB medium overnight, transfer to fresh LB medium at 2% inoculum the next day, and culture until the OD ₆₀₀ reaches 0.6-0.8, add 0.2 mmol·L ^-1 IPTG, 25°C After induction and culture for 10 h, the cells were collected by centrifugation at 4°C, 8000 r·min ^-1 for 5 min. The collected bacterial cells were suspended in HEPES buffer (100 ^{mmol·L -1} , pH 8.0), sonicated, and the protein expression was analyzed by SDS-PAGE ( FIG. 3 ). It can be seen from Figure 3 that the target protein is all in the supernatant (lane 1), and there is basically no band in the precipitate (lane 2), indicating that the recombinase is highly soluble and expressed in E. coli.

Example 4: Separation and purification of D-threonine aldolase ApDTA

The induced recombinant cells were harvested and suspended in buffer A (20mmol·L ^-1 sodium phosphate, 500mmol·L ^-1 NaCl, 20mmol·L ^-1 imidazole, pH 7.4), sonicated (300W, working for 1 second, paused) 3 seconds) for 10 min, and centrifuged at 4°C for 20 min at 8000 r·min ^-1 to remove cell debris to obtain the supernatant. The column used for purification is HisTrap FF crude, an affinity column for preparing and purifying histidine-tagged recombinant proteins. First, use buffer A to equilibrate the nickel column, pass the above supernatant through the nickel column, and continue to use buffer A to elute the proteins that are not bound to the nickel column. (20mmol·L ^-1 sodium phosphate, 500mmol·L ^-1 NaCl, 1000mmol·L ^-1 imidazole, pH 7.4) carry out gradient elution, and the recombinant protein bound to the nickel column is eluted to obtain recombinant D-threonine acid aldolase. The purified protein was assayed for enzyme activity (with p-methylsulfonylbenzaldehyde as the substrate) and analyzed by SDS-PAGE (Fig. 3). It can be seen from Figure 3 that after purification by the nickel column, a single band is displayed at about 40 kDa, and there are fewer impurity proteins, indicating that the purification effect of the nickel column is better (lane 3). Afterwards, the purified d-threonine aldolase was substituted into HEPES (100 ^{mmol·L -1} , pH 8.0) buffer using a HiTrapDesalting column, and the enzymatic properties were analyzed.

Example 5: Substrate profiling of recombinant ApDTA

The enzymatic activity of D-threonine aldolase ApDTA catalyzing different aldehyde substrates was determined. The enzyme activity measured with p-methylsulfonylbenzaldehyde as the substrate is the 100% control, and the enzyme activity measured with other substrates is calculated as the percentage of the two. The measurement results are shown in Table 1.

Table 1 Substrate spectrum of ApDTA

It can be seen from Table 1 that ApDTA exhibits a broad substrate spectrum, exhibits good activity on aliphatic, aromatic and heterocyclic aldehyde substrates, and has good application prospects.

Example 6: Optimum pH for recombinant ApDTA

Prepare 100 ^{mmol·L -1} buffers with different pH: MES buffer (pH 5.0-6.5); HEPES buffer (pH 7.0-8.5); CHES buffer (pH 9.0-10.0). Using p-methylsulfone benzaldehyde as the substrate, the condensation reaction activity of ApDTA in different pH buffers was determined. The optimum enzyme activity pH of ApDTA was 8.0, and the specific activity was 10.0-12.0 U·mg ^-1 . In the CHES-NaOH buffer with pH 9.0～10.0, the enzyme activity decreased rapidly. Using d-threonine as the substrate, the relative cleavage activity of ApDTA in different pH buffers was determined. Under the condition of acidic buffer (pH 5.0-6.5), the cleavage activity of ApDTA was low, and when the pH increased to 8.0, the cleavage activity reached the maximum. Likewise, viability decreases gradually with increasing buffer alkalinity.

Example 7: Optimum temperature for recombinant ApDTA

Using p-methylsulfonylbenzaldehyde as the substrate, the enzyme activity of ApDTA at different temperatures (20-45°C) was determined. The highest enzyme activity measured was set as 100%. The enzyme activity measured at other temperatures was relative to Percentage calculation of maximum vitality. The results showed that different temperatures had a certain effect on the condensation activity of ApDTA, and the reaction temperature showed a skewed normal distribution. The optimum temperature is 30℃, and the enzyme activity is relatively low below or above 30℃.

Example 8: Kinetic parameter determination

The specific activity of ApDTA at different concentrations of p-methylsulfonylbenzaldehyde was determined, and the kinetic parameters were calculated according to the double-reciprocal curve of the specific activity and the reciprocal of the substrate concentration. The determined kinetic parameters of ApDTA for p-methylsulfonylbenzaldehyde were K _m of 30.0 ^{mmol·L -1} and V _max of 40.3 μmol·min ^-1 ·mg ^-1 , respectively.

Example 9: Effect of metal ions on enzyme activity

The effects of metal ions and metal ion chelators such as Mn ²⁺ , Fe ²⁺ , Mg ²⁺ , Ca ²⁺ , Al ³⁺ , Cu ²⁺ , Co ²⁺ , Ni ²⁺ and EDTA on the enzymatic activity of condensation reaction were determined. The metal ions in the form of chloride salts or sulfate salts with a final concentration of ^{0.1mmol·L -1} were added to the activity measurement system. Methylsulfonylbenzaldehyde was used as the substrate to determine its enzymatic activity. Under the same conditions, the enzyme activity measured without adding any metal ions was set as 100% control, and the enzyme activity measured by adding metal ions was calculated as the percentage of control. The results showed that metal ions could activate the enzyme activity to a certain extent. The enzyme activity was the highest when Mn ²⁺ was added, indicating that Mn ²⁺ could promote the catalytic active center or correct conformation of the enzyme. D-threonine aldolase from A. xylosoxidans, X. oryzae, S. pomeroyi has been reported to exhibit similar activation; however, D-threonine aldolase from S. variicoloris and Pseudomonas sp. has also been reported Threonine aldolase is a non-metal ion dependent type.

Example 10: Influence of reaction temperature on the aldol reaction of p-methylsulfonylbenzaldehyde catalyzed by recombinant threonine aldolase ApDTA

At 10℃ and 30℃, respectively, in MES buffer (100mmol·L ^-1 , pH 6.0), add the final concentration of 50mmol·L ^-1 glycine, 5mmol·L ^-1 p-methylsulfonylbenzaldehyde, 50μmol· L ^-1 PLP, 50 μmol·L ^-1 Mn ²⁺ , the total volume of the reaction solution was 10 mL, and the reaction was carried out at 200 rpm for 12 h. Conversion and de values were analyzed by liquid chromatography. As shown in table 2.

Table 2 Effects of different reaction temperatures on aldehyde condensation catalysis for p-methylsulfonylbenzaldehyde

As can be seen from Table 2, the reaction temperature has a certain influence on the aldehyde condensation catalysis p-methylsulfonylbenzaldehyde. The higher conversion and d.e. value were obtained at 10°C than at 30°C, and the low temperature was beneficial to the stability and improvement of the d.e. value of the product. The improvement of the conversion rate indicated that the direction of the aldehyde condensation reaction was an exothermic reaction. There are different preferences for the reaction direction at different temperatures. At low temperature, the reaction proceeds toward the direction of low activation energy and high speed, and the reaction is controlled by kinetics. Therefore, the reaction temperature was chosen to be 10°C.

Example 11: The effect of pH on the catalysis of recombinant threonine aldolase ApDTA on the aldol catalysis of methylsulfonylbenzaldehyde

In Example 6, different pH buffers (100mmol·L ^-1 , pH 6.0, 7.0, 8.0) were added with final concentrations of 50mmol·L ^-1 glycine, 5mmol·L ^-1 p-methylsulfonylbenzaldehyde, 50μmol· L ^-1 PLP, 50 μmol·L ^-1 Mn ²⁺ , the total volume of the reaction solution was 10 mL, and the reaction was carried out at 10° C. and 200 rpm for 12 h. Conversion and de values were analyzed by liquid chromatography. The results are shown in Table 3.

Table 3 The effect of buffer pH on aldehyde condensation catalysis for p-methylsulfonylbenzaldehyde

buffer pH Conversion rate% d.e. value 5.0 4% 83% 6.0 65% 95% 7.0 50% 75% 8.0 50% 80% 9.0 50% 94% 10.0 29% 90%

It can be seen from Table 3 that the pH of the reaction system has a certain influence on the catalysis of ApDTA aldehyde condensation on methylsulfonylbenzaldehyde. In the buffer solution of pH 6.0, the conversion rate and d.e. value reach the maximum, and the d.e. value in the whole reaction process. relatively stable. At pH 6.0, the lyase activity of ApDTA was low, and the reverse cleavage direction was inhibited.

Example 12: Recombinant threonine aldolase ApDTA for the preparation of l-syn-p-methylsulfonylphenylserine

The reaction system was enlarged to 500mL, including MES buffer (100mmol·L ^-1 , pH 6.0), 1mol·L ^-1 glycine, 100mmol·L ^-1 p-methylsulfonylbenzaldehyde, 50μmol·L ^-1 PLP, 50μmol· L ^-1 Mn ²⁺ and 25kU·L ^-1 ApDTA were reacted at 10°C and 200rpm for 12h. The reaction process is shown in Figure 4, and the liquid phase analysis chromatogram of the reaction product is shown in Figure 5. After the reaction, 4 times the volume of methanol was directly added to the reacted mixture, stored at 4° C. overnight, the precipitate was collected by filtration, and washed with methanol to recover glycine. The filtrate was concentrated in vacuo and passed through alkali treated Dowex-1 anion exchange resin and washed with water. The product (containing glycine) was eluted with 20% acetic acid and the residue was collected and concentrated in vacuo and purified on an ODS column to give the desired product. The purity of the product is over 99%, and the yield is over 75%.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

sequence listing

<110> Jiangnan University

<120> A recombinant bacterium expressing D-threonine aldolase and its construction method and application

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Gly Gln Leu Ala Arg Val Ala Lys Met Ser Val Cys Val Asp Asn Ala

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His Asn Leu Ala Gln Leu Ser Gln Ala Met Thr Gln Ala Gly Ala Gln

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Ile Asp Val Leu Val Glu Val Asp Val Gly Gln Gly Arg Cys Gly Val

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Ser Asp Asp Ala Leu Val Leu Ala Leu Ala Gln Gln Ala Arg Asp Leu

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Claims (5)

1. The application of a recombinant bacterium for expressing D-threonine aldolase in the synthesis of l-syn-p-methylsulfonylphenylserine is characterized in that the recombinant bacterium is prepared fromE. coli BL21(DE3) is used as a host, pET28a is used as an expression vector, and D-threonine aldolase with an amino acid sequence shown as SEQ ID NO.2 is expressed.

2. The application of claim 1, wherein the construction method of the recombinant bacterium comprises the following steps:

(1) construction of the recombinant plasmid pET28a-ApDTA: the D-threonine aldolase geneApThe DTA is connected with the digested plasmid pET28a to obtain a recombinant expression vector pET28a-ApDTA；

(2) Construction of recombinant bacteriaE. coli BL21(DE3)/pET28a-ApDTA: the constructed recombinant expression vector pET28a-ApDTA heat transfer into escherichia coli BL21(DE3) competence, and recombinant bacteria are obtained by culture and screeningE. coli BL21(DE3)/pET28a-ApDTA。

3. Use according to claim 1, characterized in that: the application is to synthesize l-syn-p-methylsulfonylphenylserine by using the D-threonine aldolase produced by fermentation of the recombinant bacteria as a catalyst.

4. Use according to claim 3, characterized in that: the application is particularly to catalyzing aldehyde and glycine to synthesize l-syn-p-methylsulfonyl phenyl serine.

5. Use according to claim 3, characterized in that: the application is that the reaction temperature is 5-15 DEG C^oC, reacting for 10-20 hours under the condition that the pH value is 5.5-6.5.

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