Novel dipeptide synthetase and application thereof

Abstract

The invention discloses a dipeptide synthetase and application thereof, and relates to the technical field of biology, and the nucleotide sequence of the dipeptide synthetase is as shown in SEQ ID NO: 1; the method comprises the following steps: cloning a dipeptide synthetase gene from streptomyces albogriseolus 1-36 by virtue of a PCR (Polymerase Chain Reaction) technology; the recombinant plasmid is connected to NcoI and XhoI restriction enzyme cutting sites of a high-efficiency expression vector pET-28a (+), and is transformed into competent cells of escherichia coli DH5alpha, and plasmids are extracted; and transforming to an expression host strain BL21 (DE3) to obtain a genetic engineering strain for efficiently expressing the dipeptide synthetase. The dipeptide synthetase is applied to synthesis of a dipeptide compound or synthesis of a compound taking the dipeptide compound as a precursor. The dipeptide synthetase obtained by the invention can identify carboxyl donors and amino donors from non-natural amino acids to generate dipeptide compounds, and has the advantages of mild enzyme catalysis conditions and high catalysis efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a novel dipeptide synthetase and application thereof. Background Art

[0002] An amide bond is a bond formed by the dehydration condensation of a carboxyl group (-COOH) and an amine group (-NH), with the chemical formula -CO-NH-. It is a widely existing structure in natural products, which greatly enriches the diversity of product structures and participates in many different physiological processes in primary and secondary metabolism. According to statistics, the synthesis of more than 60% of candidate drugs involves the synthesis of amide bonds, such as Atorvastatin, Acetaminophen and Gleevec, which are commonly used in clinical practice. Peptide compounds formed by amide bond connection also show many important biological activities, such as antibiotics and pheromones, and are widely used in pharmaceuticals, food, cosmetics, agriculture and industry. Among them, dipeptide compounds not only provide the premise for the synthesis of complex peptide compounds, but also have other physiological and pharmacological activities. For example, the widely used sweetener aspartame (L-aspartic acid-L-phenylalanine), the dipeptide compound (tyrosine-leucine) with activity equivalent to the commonly used clinical antidepressant diazepam, L-carnosine (β-alanine-L-histidine) widely used in anti-aging cosmetics, and alistair (isoserine-leucine), a representative molecule for the treatment of adult acute non-lymphocytic leukemia.

[0003] The chemical synthesis reaction of amide bonds requires the use of toxic reagents and expensive coupling reaction catalysts, and in most cases there are multiple active groups. The process of "group protection-coupling-deprotection" makes the synthesis route complicated and the yield is low. Therefore, more efficient, low-cost, environmentally friendly and single-product enzymatic reactions have become a better choice for amide bond synthesis. These enzymes have unique substrate specificity and catalytic properties. Most of the dipeptide synthetases reported so far recognize natural amino acid substrates and have a relatively simple configuration. The continued discovery and characterization of amide synthases in nature will expand the range of substrates used by these enzymes, enrich the dipeptide compound library, and provide effective tools for drug discovery and molecular design, and for the production of industrially and pharmaceutically valuable peptide products.

[0004] Therefore, technicians in this field are committed to developing a dipeptide synthetase with broad substrate and configuration tolerance, and its catalysis is simple and easy to operate, with low raw material cost, high catalytic efficiency and low time cost. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to develop a dipeptide synthetase with broad substrates and configuration tolerance, and its catalysis is simple and easy to operate, with low raw material cost, high catalytic efficiency and low time cost.

[0006] To achieve the above object, the present invention provides a novel dipeptide synthetase, the nucleotide sequence of which is shown in SEQ ID NO: 1; and the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] The present invention also provides a method for recombining a dipeptide synthetase, comprising the following steps:

[0008] Step 1, using Streptomyces albogriseolus 1-36 as a template to perform polymerase chain reaction, and purifying the amplified product to obtain gene alb28;

[0009] Step 2: connect the gene alb28 obtained in step 1 to the NcoI and XhoI restriction sites of the high-efficiency expression vector pET-28a(+), transform it into Escherichia coli DH5α competent cells, and extract the recombinant plasmid;

[0010] Step 3, transforming the recombinant plasmid obtained in step 2 into Escherichia coli to obtain a genetically engineered strain that efficiently expresses dipeptide synthetase;

[0011] Step 4: Induce the expression of the genetically engineered strain obtained in step 3, and obtain the dipeptide synthetase through purification.

[0012] Furthermore, step 1 uses primer 1 and primer 2, the base sequence of primer 1 is shown as SEQ ID NO.3, and the base sequence of primer 2 is shown as SEQ ID NO.4.

[0013] Furthermore, step 2 also includes PCR verification of the recombinant plasmid, using primer 3 and primer 4 for verification, the base sequence of primer 3 is shown as SEQ ID NO.5, and the base sequence of primer 4 is shown as SEQ ID NO.6.

[0014] The invention also provides an application of a dipeptide synthetase in a synthesis reaction of dipeptide compounds.

[0015] Furthermore, the dipeptide synthetase can recognize 19 different configurations of carboxyl donors and 7 amino donors derived from non-natural amino acids and react in pairs.

[0016] Further, 19 different configurations of carboxyl donors derived from non-natural amino acids include L / D-isoserine, β-alanine, L-2,3-diaminopropionic acid, L / D-3-aminoisobutyric acid, L / D-3-aminobutyric acid, 3-amino-4-hydroxybutyric acid, 4-aminobutyric acid, 2-hydroxy-4-aminobutyric acid, D-2,4-diaminobutyric acid, 4-amino-2-methylbutyric acid, L / D-4-amino-3-hydroxybutyric acid, 3,4-diaminobutyric acid, 3-aminovaleric acid, 4-aminovaleric acid and 5-aminovaleric acid.

[0017] Further, the seven amino donors include L-2b, L-7b, L-12b, L-13b, L-14b, L-15b and L-16b; L-2b is shown in structural formula 1, L-7b is shown in structural formula 2, L-12b, L-13b, L-14b, L-15b and L-16b are shown in structural formula 3; when n in structural formula 3 is 2, 3, 4, 5, 6, they are the structural formulas of L-12b, L-13b, L-14b, L-15b, L-16b, respectively.

[0018]

[0019]

[0020] Furthermore, the optimum reaction pH for the synthesis reaction is 9.0, and the optimum reaction temperature is 35°C.

[0021] The present invention also provides an application of a dipeptide synthetase in the synthesis of a compound using a dipeptide compound as a precursor.

[0022] Furthermore, the dipeptide synthetase provides a more economical and practical tool enzyme for the synthesis of dipeptide compounds or the synthesis of compounds using dipeptide compounds as precursors.

[0023] Furthermore, the dipeptide synthetase, protein purification and enzyme catalysis process are simple and easy to operate, with low raw material cost, high catalytic efficiency and low time cost.

[0024] Furthermore, dipeptide synthetase can achieve a higher conversion efficiency under the conditions of pH 9, 35°C, and 5 minutes.

[0025] In preferred embodiment 1 of the present invention, the polymerase chain reaction (PCR) amplification of the alb28 gene and the purification of the amplified product are described in detail;

[0026] In another preferred embodiment 2 of the present invention, the construction process of the expression vector is described in detail;

[0027] In another preferred embodiment 3 of the present invention, the inducible expression process of the recombinant plasmid in Escherichia coli is described in detail;

[0028] In another preferred embodiment 4 of the present invention, the purification process of the recombinant dipeptide synthetase is described in detail;

[0029] In another preferred embodiment 5 of the present invention, the substrate specificity analysis process of the recombinant dipeptide synthetase is described in detail;

[0030] In another preferred embodiment 6 of the present invention, the effect of reaction temperature on the activity of alb28 is described in detail;

[0031] In another preferred embodiment 7 of the present invention, the effect of reaction pH on the activity of alb28 is described in detail.

[0032] The beneficial technical effects of the present invention are as follows:

[0033] The present invention successfully cloned the dipeptide synthetase gene from Streptomyces albogriseolus 1-36 by PCR technology. The full length of the gene (from the start codon to the stop codon) is 1323 bp, the G+C content is 64%, and it encodes 440 amino acids.

[0034] The dipeptide synthetase fragment containing the vector homology arm at the end was amplified by PCR technology, connected to the NcoI and XhoI restriction sites of the efficient expression vector pET-28a(+), transformed into E. coli DH5α competent cells, and the plasmid was extracted. The verified correct plasmid was transformed into the expression host strain BL21(DE3) to obtain a genetically engineered strain that efficiently expressed dipeptide synthetase.

[0035] The dipeptide synthetase produced by the engineering strain induced by IPTG can use L / D-isoserine, β-alanine, L-2,3 diaminopropionic acid, L / D-3-aminoisobutyric acid, L / D-3-aminobutyric acid, 3-amino-4-hydroxybutyric acid, 4-aminobutyric acid, 2-hydroxy-4-aminobutyric acid, D-2,4-diaminobutyric acid, 4-amino-2-methylbutyric acid, L / D-4-amino-3-hydroxybutyric acid, 3,4-diaminobutyric acid, 3-aminopentanoic acid, 4-aminopentanoic acid and 5-aminopentanoic acid as carboxyl substrates and L-2b, L-7b, L-12b, L-13b, L-14b, L-15b and L-16b as amino substrates to prepare a series of dipeptide compounds by two-by-two combination reactions. It will provide a more economical and practical new tool for the synthesis of dipeptide compounds or the synthesis of compounds using dipeptide compounds as precursors.

[0036] The dipeptide synthetase has an optimum reaction pH of 9.0 and an optimum reaction temperature of 35° C., and is suitable for use in industrial production.

[0037] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a map of the pET-28a(+)-alb28 recombinant plasmid of a preferred embodiment 2 of the present invention;

[0039] Figure 2 This is a separation and purification diagram of a recombinant dipeptide synthetase according to a preferred embodiment 4 of the present invention;

[0040] Figure 3 is the structural formula of a compound that is a carboxyl donor that can be accepted by the recombinant dipeptide synthetase of a preferred embodiment 5 of the present invention;

[0041] Figure 4 This is a graph showing the optimum reaction temperature of the recombinant dipeptide synthetase of a preferred embodiment 6 of the present invention;

[0042] Figure 5 This is a graph showing the most suitable reaction pH result of the recombinant dipeptide synthetase of a preferred embodiment 7 of the present invention. DETAILED DESCRIPTION

[0043] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0044] Example 1: Polymerase chain reaction (PCR) amplification of alb28 gene and purification of amplified product

[0045] Streptomyces albogriseolus 1-36, collection location: soil of Zhangjiangkou Mangrove Nature Reserve, Yunxiao, Zhangzhou, Fujian.

[0046] Streptomyces albogriseolus 1-36 was obtained, total DNA of Streptomyces albogriseolus 1-36 was extracted, and PCR was performed using the genomic DNA (SEQ ID NO.1) as a template under the action of primer 1 (base sequence shown in SEQ ID NO.3) and primer 2 (base sequence shown in SEQ ID NO.4).

[0047] The PCR amplification system was as follows: 1 μL of primer 1 (5 nmol), 1 μL of primer 2 (5 nmol), 1 μL of 30 ng / μL Treptomyces albogriseolus 1-36 total DNA genomic DNA, 25 μL prime STARmax, add sterile water to 50 μL, and mix well;

[0048] The parameters of the PCR amplification reaction were: denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 10 s, and after 30 cycles, a final extension at 72°C for 5 min.

[0049] The amplified product was detected by 0.8% agarose gel electrophoresis, and an obvious positive amplification band was found, that is, a DNA fragment with a size close to the theoretical size. The gel block containing the target gene was cut out and placed in an enzyme-free 1.5mL EP tube, and the PCR amplified product was purified according to the instructions of the OMEGA PCR product purification kit.

[0050] Example 2: Construction of expression vector

[0051] The vector pET-28a(+) was digested with NcoI and XhoI at 37℃ for 1h, and the vector fragment was recovered after complete digestion. The digestion system was: 10x Green buffer, 1.5μg alb28 fragment, 1.5μL each of NcoI and XhoI, and made up to 50μL with sterile water, mixed and reacted at 37℃ for 1h;

[0052] 30 ng of alb28 recovery fragment and vector recovery fragment, 10 μL of 2xHieffclone multis Enzyme premix, add sterile water to 20 μL, mix well and place at 50°C for 50 min. The ligation product was transformed into competent E. coli DH5α cells;

[0053] Five transformants were selected and cultured on an LB plate containing 50 mg / L Kan, and plasmids were extracted and identified according to the instructions of the OMEGA plasmid extraction kit. PCR verification was performed using primer 3 (base sequence as shown in SEQ ID NO.5) and primer 4 (base sequence as shown in SEQ ID NO.6). The recombinant plasmid was identified by PCR to obtain a gene fragment of about 1652 bp in size, and the theoretically correct plasmid was sequenced. Software analysis proved that the alb28 gene (SEQ ID NO.1) had been successfully recombined into the plasmid pET-28a(+). The map of the pET-28a(+)-alb28 recombinant plasmid is shown in Figure 1 shown.

[0054] Example 3: Inducible expression of recombinant plasmid in Escherichia coli

[0055] A single DH5α E. coli containing the recombinant plasmid was picked and inoculated into 3 mL LB liquid culture medium (containing 50 μg / L Kan), and cultured in a shaker at 37°C overnight. The plasmid was extracted and transformed into the competent cells of the expression host bacteria BL21 (DE3), and evenly spread on the surface of the LB plate containing Kan (final concentration of 50 μg / L), and cultured inverted at 37°C overnight.

[0056] Positive recombinants were selected and inoculated into 10 mL of LB culture medium containing Kan and Cm and cultured at 37°C overnight. The next day, the inoculation volume was transferred to 1 L of LB culture medium containing Kan, Cm and arabinose at 1% and cultured at 37°C until OD 600 The concentration of IPTG was 0.6-0.8, and a final concentration of 0.2 mM IPTG was added. The culture was continued at 16°C for 16-20 h. The cells were collected by centrifugation and stored at -80°C for later use.

[0057] Example 4. Purification of recombinant dipeptide synthetase

[0058] Each 1L of fermented bacteria was resuspended with 90mL binding buffer (containing 20Mm Tris, 300mM NaCl, 10% glycerol, pH8), and the bacteria were ultrasonically disrupted on ice. The lysed bacteria were centrifuged at 9500rpm, 4℃ for 60min, and the supernatant was passed through a Ni-NTA affinity chromatography column. The column was washed with washing buffer containing 0mM, 20mM, 50mM and 100mM imidazole (containing 20Mm Tris, 300mM NaCl, 10% glycerol, pH8) in sequence, and the target protein was eluted with 250mM and 500mM imidazole elution buffer (containing 20Mm Tris, 300mM NaCl, 10% glycerol, pH8). Each component was identified by 12% SDS-PAGE electrophoresis. The separation and purification map of the recombinant dipeptide synthetase is shown in FIG. Figure 2 As shown, lane 1 is the standard protein molecular weight Marker, and lane 2 is the purified dipeptide synthetase; the band corresponding to 55KDa is the target protein band, the protein is alb28, and the amino acid sequence is shown in SEQ ID NO.2. The target component is dialyzed with a dialysis buffer (containing 10MmTris, 150mMNaCl, pH8) to obtain a purified dipeptide synthetase. It is quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0059] Example 5: Analysis of substrate specificity of recombinant dipeptide synthetase

[0060] Taking L-2b as the amino substrate, different types of carboxyl substrates were selected, namely L / D-isoserine, β-alanine, L-2,3-diaminopropionic acid, L / D-3-aminoisobutyric acid, L / D-3-aminobutyric acid, 3-amino-4-hydroxybutyric acid, 4-aminobutyric acid, 2-hydroxy-4-aminobutyric acid, D-2,4-diaminobutyric acid, 4-amino-2-methylbutyric acid, L / D-4-amino-3-hydroxybutyric acid, 3,4-diaminobutyric acid, 3-aminopentanoic acid, 4-aminopentanoic acid and 5-aminopentanoic acid, to test the specificity of alb28 for carboxyl substrates. Taking D-isoserine as the carboxyl substrate, different types of amino substrates were selected to test the specificity of alb28 for amino substrates. The compound structures of the carboxyl donors that the recombinant dipeptide synthetase can accept are as follows: Figure 3 The amino donor compounds that the recombinant dipeptide synthetase can accept are L-2b, L-7b, L-12b, L-13b, L-14b, L-15b and L-16b.

[0061] The reaction system was 100 μL: 20 mM Tris-HCl ((PH9.0), 5 mM ATP, 24 mM MgCl2, 24 mM carboxyl substrate, 1 mM amino substrate and 20 μM alb28. After reacting at 35°C for 5 min, an equal volume of acetonitrile was added to terminate the reaction. The supernatant was centrifuged and tested by HPLC.

[0062] HPLC detection conditions: XSelect-HSS T3 column (Waters, 150×4.6mm, 5μm), flow rate of 0.8mL / min, detection wavelength of 190-800nm. Mobile phase A is water containing 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid. Elution conditions are: 0-5min 2% mobile phase B; 5.01-30min 2%-80% mobile phase B; 30.01-35min 100% mobile phase B; 35.1-43min 2% mobile phase B.

[0063] Example 6: Effect of reaction temperature on alb28 activity

[0064] The reaction system was 50 μL: 20 mM Tris-HCl ((PH9.0), 3 mM ATP, 5 mM MgCl2, 2 mM D-isoserine, 1 mM L-2b and 10 μM alb28. The reactants were placed at 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C and 55°C for 5 minutes. An equal volume of acetonitrile was added to terminate the reaction, and the supernatant was centrifuged for HPLC detection. HPLC detection conditions: XSelect-HSS T3 column (Waters, 150×4.6 mm, 5 μm), flow rate of 0.8 mL / min, detection wavelength of 190-800 nm. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. Elution conditions were: 0-5 min 2% mobile phase B; 5.01-17 min 2%-40% mobile phase B; 17.1-22min 100% mobile phase B; 22.1-30min 2% mobile phase B. The optimal reaction temperature of recombinant dipeptide synthetase was shown in the figure below, using D-isoserine and L-2b as substrates. Figure 4 As shown, it can be seen that the optimal reaction temperature is 35°C.

[0065] Example 7: Effect of reaction pH on alb28 activity

[0066] According to the enzyme reaction system and detection method described in Example 6, the reaction solution was reacted in pH 6.5, pH 7, pH 7.5, pH 8, pH 8.5, pH 9, pH 9.5, and pH 10 buffers. Taking D-isoserine and L-2b as substrates for the reaction, the most suitable reaction pH results of the recombinant dipeptide synthetase are shown in the figure below: Figure 5 As shown, it can be seen that the optimal reaction pH is 9.

[0067] It can be seen that the dipeptide synthetase derived from Streptomyces albogriseolus 1-36 provided by the present invention can use L / D-isoserine, β-alanine, L-2,3-diaminopropionic acid, L / D-3-aminoisobutyric acid, L / D-3-aminobutyric acid, 3-amino-4-hydroxybutyric acid, 4-aminobutyric acid, 2-hydroxy-4-aminobutyric acid, D-2,4-diaminobutyric acid, 4-amino-2-methylbutyric acid, L / D-4-amino-3-hydroxybutyric acid, 3,4-diaminobutyric acid, 3-aminopentanoic acid, 4-aminopentanoic acid and 5-aminopentanoic acid as carboxyl substrates and L-2b, L-7b, L-12b, L-13b, L-14b, L-15b and L-16b as amino substrates to carry out two-by-two combination reactions to prepare a series of dipeptide compounds, and is an effective tool for synthesizing dipeptide compounds. The dipeptide synthetase has an optimum reaction pH of 9.0 and an optimum reaction temperature of 35° C., and is suitable for use in industrial production.

[0068] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A novel dipeptide synthetase, characterized in that: The nucleotide sequence of the enzyme is shown in SEQ ID NO: 1; the amino acid sequence of the enzyme is shown in SEQ ID NO:

2.

2. A method for recombining a dipeptide synthetase as claimed in claim 1, characterized in that: The method comprises the following steps: Step 1, using Streptomyces albogriseolus 1-36 as a template to perform polymerase chain reaction, and purifying the amplified product to obtain gene alb28; Step 2: connect the gene alb28 obtained in step 1 to the NcoI and XhoI restriction sites of the high-efficiency expression vector pET-28a(+), transform it into Escherichia coli DH5α competent cells, and extract the recombinant plasmid; Step 3, transforming the recombinant plasmid obtained in step 2 into Escherichia coli to obtain a genetically engineered strain that efficiently expresses dipeptide synthetase; Step 4: Induce the expression of the genetically engineered strain obtained in step 3, and obtain the dipeptide synthetase through purification.

3. The recombination method according to claim 2, characterized in that The step 1 uses primer 1 and primer 2, the base sequence of primer 1 is shown as SEQ ID NO.3, and the base sequence of primer 2 is shown as SEQ ID NO.

4.

4. The recombination method according to claim 2, characterized in that The step 2 also includes PCR verification of the recombinant plasmid, and the verification uses primer 3 and primer 4. The base sequence of primer 3 is shown in SEQ ID NO.5, and the base sequence of primer 4 is shown in SEQ ID NO.

6.

5. Use of the dipeptide synthetase as claimed in claim 1 in the synthesis reaction of dipeptide compounds.

6. The use according to claim 5, characterized in that The dipeptide synthetase can recognize 19 carboxyl donors of different configurations and 7 amino donors from non-natural amino acids and react in pairs.

7. The use according to claim 6, characterized in that The 19 carboxyl donors of different configurations from the non-natural amino acid source include L / D-isoserine, β-alanine, L-2,3-diaminopropionic acid, L / D-3-aminoisobutyric acid, L / D-3-aminobutyric acid, 3-amino-4-hydroxybutyric acid, 4-aminobutyric acid, 2-hydroxy-4-aminobutyric acid, D-2,4-diaminobutyric acid, 4-amino-2-methylbutyric acid, L / D-4-amino-3-hydroxybutyric acid, 3,4-diaminobutyric acid, 3-aminovaleric acid, 4-aminovaleric acid and 5-aminovaleric acid.

8. The use according to claim 6, characterized in that The seven amino donors include L-2b, L-7b, L-12b, L-13b, L-14b, L-15b and L-16b; the L-2b is shown in structural formula 1, the L-7b is shown in structural formula 2, and the L-12b, L-13b, L-14b, L-15b and L-16b are shown in structural formula 3; When n in the structural formula 3 is 2, 3, 4, 5, or 6, it is the structural formula of L-12b, L-13b, L-14b, L-15b, or L-16b, respectively.

9. The use according to any one of claims 5 to 8, characterized in that: The optimum reaction pH of the synthesis reaction is 9.0, and the optimum reaction temperature is 35°C.

10. Use of the enzyme as claimed in claim 1 in a synthesis reaction using dipeptide compounds as precursor compounds.

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