A phenylpyruvate reductase mutant with high enzyme activity and its application

Abstract

The present invention discloses a phenylpyruvate reductase mutant with high enzyme activity and its application. Lactobacillus The wild-type phenylpyruvate reductase of sp. CGMCC 9967 is molecularly modified, and the 193rd position of the amino acid sequence shown in SEQ ID NO.1 is mutated from asparagine to aspartic acid, and the 203rd position is mutated from histidine to arginine, so as to obtain a phenylpyruvate reductase mutant with high enzyme activity. The 193rd and 203rd positions are located on the enzyme surface and are related to NADH and substrate binding activity. The present invention proposes for the first time to carry out site-directed mutation of these two positions, and obtains a mutant with significantly improved enzyme activity, which provides more possibilities for the mutation design of phenylpyruvate reductase with improved enzyme activity. The present invention has important practical significance for the industrial catalysis of phenylpyruvate to produce phenyllactic acid and the industrial application of phenylpyruvate reductase.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and relates to a phenylpyruvate reductase mutant, in particular to a phenylpyruvate reductase mutant with high enzyme activity and application thereof in catalytic production of phenyllactic acid. Background Art

[0002] Phenylactic acid (PLA), also known as 2-hydroxy-3-phenylpropionic acid, is a natural small molecule organic acid with a chemical formula of C9H 10 O3 was first found in fermented foods and honey. It is a white crystal at high purity, has good water solubility, is acid-resistant and heat-resistant, and has stable properties. At present, food safety has gradually become a hot topic, and phenyllactic acid has attracted widespread attention as a natural and safe new food preservative. Phenyllactic acid mainly inhibits and kills bacteria by destroying cell walls, cell membranes and DNA. It has a wide antibacterial spectrum and has good inhibitory effects on Gram-positive bacteria (such as Listeria monocytogenes and Staphylococcus aureus), Gram-negative bacteria (such as Escherichia coli and Salmonella) and eukaryotic microorganisms (such as Aspergillus and Penicillium). Phenyllactic acid also has certain pharmacological functions, which can regulate human steroid levels and resist platelet aggregation. In addition, phenyllactic acid is also a good bio-based material. The polyphenyllactic acid polymerized from it has better thermal stability and ultraviolet absorption performance than polylactic acid. In summary, phenyllactic acid has good applications in the fields of medicine, food, and materials, so the synthesis of phenyllactic acid has also become a hot topic in research.

[0003] Phenylpyruvate reductase can catalyze the asymmetric reduction of prochiral phenylpyruvic acid (PPA) to generate phenyllactic acid. Compared with chemical synthesis and biological fermentation, this enzyme-catalyzed method for producing phenyllactic acid has the characteristics of mild conditions, high yield, simple process and environmental friendliness. Ketoacid reductase is widely available, and reductase-producing strains have been screened out in microorganisms such as Escherichia coli, Saccharomyces cerevisiae, and lactic acid bacteria. However, the expression level of most reductases in wild-type strains is low, which cannot meet the needs of industrial production. A small part of them has shown good enzymatic properties after multiple rounds of mutagenesis and screening, but there is little room for improvement due to the blindness and randomness of random mutations. Therefore, the key to achieving industrial-scale catalytic production of phenyllactic acid is to obtain phenylpyruvate reductase with high catalytic activity through genetic engineering technology. Summary of the invention

[0004] The purpose of the present invention is to provide a phenylpyruvate reductase mutant, which has important practical significance for the industrial catalysis of phenylpyruvate to produce phenyllactic acid and the industrial application of phenylpyruvate reductase. At present, there is no research on the 193rd and 203rd sites, which are located on the enzyme surface and are related to NADH and substrate binding activity. The present invention proposes for the first time to carry out site-directed mutation of these two sites and obtain a mutant with significantly improved enzyme activity, which provides more possibilities for the design of mutations to improve the enzyme activity of phenylpyruvate reductase.

[0005] The invention uses a site-directed mutagenesis method to molecularly transform a wild-type phenylpyruvate reductase derived from Lactobacillus sp. CGMCC 9967 to obtain a phenylpyruvate reductase mutant with high enzyme activity.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a phenylpyruvate reductase mutant with high enzymatic activity, wherein the 193rd position of the amino acid sequence shown in SEQ ID NO.1 is mutated from asparagine to aspartic acid, and the 203rd position is mutated from histidine to arginine.

[0008] In a second aspect, the present invention provides a phenylpyruvate reductase gene, which encodes the phenylpyruvate reductase mutant. The gene is obtained by base mutation of the nucleotide sequence shown in SEQ ID NO.2.

[0009] In a third aspect, the present invention provides an expression vector, wherein the expression vector comprises the nucleotide sequence of the second aspect. The expression vector is a pET series expression vector.

[0010] In a fourth aspect, the present invention provides a recombinant engineered bacterium, which is an Escherichia coli comprising the expression vector of the third aspect.

[0011] In a fifth aspect, the present invention provides a method for constructing a recombinant engineered bacterium, comprising the following steps: (1) obtaining a mutant sequence: obtaining a mutant sequence by point mutation using a wild-type sequence as a template; (2) constructing an expression vector: homologously recombining the mutant sequence with an enzyme-cut plasmid to obtain a recombinant expression vector; (3) constructing a recombinant bacterium: transferring the constructed recombinant expression vector into competent Escherichia coli cells, culturing and screening to obtain the recombinant engineered bacterium.

[0012] In a sixth aspect, the present invention provides a catalyst, wherein the catalyst comprises the phenylpyruvate reductase mutant.

[0013] In a seventh aspect, the present invention also provides the use of the mutant or the recombinant engineered bacteria in the fields of pharmaceutical production and food, such as the use in the catalytic synthesis of phenyllactic acid.

[0014] Compared with the prior art, the present invention has the following significant advantages:

[0015] 1. The present invention provides a phenylpyruvate reductase mutant with high enzyme activity.

[0016] 2. The present invention provides a catalyst comprising a phenylpyruvate reductase mutant with high enzyme activity, which has mild catalytic conditions, high enzyme activity efficiency, single product chirality, and has potential for industrial production application.

[0017] 3. The present invention provides a gene encoding the above-mentioned phenylpyruvate reductase mutant, an expression vector comprising the gene encoding the above-mentioned phenylpyruvate reductase mutant, and a recombinant bacterium comprising the expression vector. The present invention provides a method for preparing and using the above-mentioned phenylpyruvate reductase mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The plasmid pET-28a-ppr and plasmid pET-28a-ppr N193D / H203R Agarose gel electrophoresis of PCR.

[0019] Figure 2 BL21(DE3) / pET-28a-ppr N193D / H203R Gel electrophoresis of expressed and purified protein.

[0020] Figure 3 It is the specific enzyme activity of phenylpyruvate reductase and its mutant N193D / H203R.

[0021] Figure 4 It is the optimal pH for phenylpyruvate reductase and its mutant N193D / H203R.

[0022] Figure 5 The optimum temperature of phenylpyruvate reductase and its mutant N193D / H203R

[0023] Figure 6 This is a diagram of the reaction process of phenylpyruvate reductase catalyzing the synthesis of phenyllactic acid.

[0024] Figure 7 This is a diagram of the reaction process of synthesizing phenyllactic acid catalyzed by the phenylpyruvate reductase mutant N193D / H203R. DETAILED DESCRIPTION

[0025] SEQ ID NO.1

[0026] >ALB78224.1 phenylpyruvate reductase[Lactobacillus sp.CGMCC 9967]

[0027] SEQ ID NO.2

[0028] >KP735960.1 Lactobacillus sp. CGMCC 9967 phenylpyruvate reductase(ppr)gene,complete cds

[0029] Example 1

[0030] Construction of strains producing phenylpyruvate reductase and its mutant N193D / H203R

[0031] The wild-type gene of phenylpyruvate reductase was synthesized by a biotechnology company and constructed on pET-28a, named pET-28a-ppr, and the vector was transformed into the E. coli DH5α strain. The recombinant bacteria E. coli DH5α was inoculated into a 5ml LB test tube culture medium and cultured at 37°C and 220rpm for 12 hours. After the culture was completed, the bacteria were collected by centrifugation at 12000rpm for 1min, and the plasmid was extracted with a plasmid mini-extraction kit. The obtained plasmid was transferred into the expression host of E. coli BL21 (DE3) to obtain the wild-type phenylpyruvate reductase-producing strain E. coli BL21 (DE3) / pET-28a-ppr, and a glycerol strain was prepared by adding sterile glycerol with a final concentration of 20% and stored at -80°C.

[0032] Using pET-28a-ppr as a template, site-directed mutations N193D and H203R were introduced by inverse PCR. The PCR products were sent to a biological company for sequencing verification. The sequencing results showed that no random mutations occurred except for the desired mutation sites. Therefore, the mutant plasmid pET-28a-ppr N193D / H203R Build Success

[0033] Upstream mutation primers:

[0034] Downstream mutagenesis primers:

[0035] Note: The underline indicates the homologous part, and the bold indicates the point mutation base site

[0036] The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0037] Table 1 PCR reaction system

[0038]

[0039]

[0040] Table 2 PCR reaction conditions

[0041]

[0042] After the PCR was completed, the amplified product was detected by 1% agarose gel electrophoresis, and the result showed that the amplified product was a single band with a size of about 6289 bp. The amplified product was purified and recovered using a gel recovery kit.

[0043] The purified gene fragment was digested with Dpn I to remove the template and then transferred into E. coli DH5α competent cells, spread on the surface of LB solid medium containing 50ug / ml kanamycin, cultured at 37℃ for 12h, picked a single colony to LB liquid medium for culture, and successfully constructed positive transformants were identified by colony PCR. The plasmid was extracted and the correctness of the mutation site was verified by sequencing. After verification, sterile glycerol with a final concentration of 20% was added to prepare the glycerol strain and stored at -80℃.

[0044] The recombinant expression plasmid pET-28a-ppr N193D / H203R Transform into expression host E.coli BL21(DE3) to construct recombinant mutant expression strain E.coli BL21(DE3) / pET-28a-ppr N193D / H203R .

[0045] Example 2

[0046] Expression and Purification of Phenylpyruvate Reductase and Its Mutant N193D / H203R

[0047] The successfully constructed wild-type and mutant strains were spread onto solid LB plates containing kanamycin at a final concentration of 50ug / ml. Single colonies were picked and inoculated into 5 ml of LB liquid test tube medium containing resistance, and cultured at 37°C, 220rpm, and then transferred to 50 ml of TB medium containing resistance at a 1% (v / v) inoculation volume, and cultured at 37°C, 220rpm, and waited until OD 600 When the cell culture temperature reached 2-4, IPTG was added to a final concentration of 0.6 mM and the culture was induced at 28°C with shaking at 220 rpm for about 18 h.

[0048] 50 ml of bacterial solution was centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the bacterial cells were resuspended in 10 mL of lysis buffer (50 mM Tris, 200 mM NaCl, pH = 7.0), and the cells were ultrasonically disrupted in an ice bath (power 750 W, working time 5 s, interval time 8 s, a total of 99 times), and after the disruption was completed, the supernatant was collected at 6000 rpm for 5 min to obtain the crude enzyme solution. The crude enzyme solution was filtered with a 0.45 μm microporous membrane and set aside.

[0049] Use nickel ion affinity chromatography column for protein purification, rinse the column with deionized water at 4°C (about 10 column volumes), and then balance the column with low salt concentration buffer (500mmol / L NaCl, 50mM Tris, pH=7.0). When the effluent at the lower end of the column is consistent with the pH value of the low salt concentration buffer pumped into the column (about 5 column volumes of buffer), add the reserved membrane-passed crude enzyme solution to the column. First, rinse with a buffer containing a low concentration of imidazole (500mmol / L NaCl, 50mmol / L imidazole, 50mM Tris, pH=7.0) to remove impurities until baseline equilibrium, and then elute the target protein with an eluent containing a high concentration of imidazole (500mmol / L NaCl, 500mmol / L imidazole, 50mM Tris, pH=7.0). Collect the eluent of the absorption peak, which is the pure enzyme solution of the target protein, and perform SDS-PAGE protein gel electrophoresis to verify the band.

[0050] Example 3

[0051] Determination of Enzyme Activity of Phenylpyruvate Reductase and Its Mutant N193D / H203R

[0052] The definition and determination method of enzyme activity refer to "Cloning and Expression of Ketoacid Reductase Gene for Asymmetric Synthesis of Phenyllactic Acid" in the Journal of Food and Biotechnology.

[0053] Definition of enzyme activity (U): The amount of enzyme required to catalyze the oxidation of 1 μmol NADH per unit time (min) under standard reaction conditions.

[0054] Definition of specific enzyme activity (U / mg): The number of units of enzyme activity per unit mass (mg) of enzyme protein under standard reaction conditions.

[0055] The total reaction system was 250 μl, including potassium phosphate buffer (KPB, 100 mmol / L, pH = 7.0), 0.5 mmol / L nicotinamide adenine dinucleotide (NADH), 5 mmol / L PPA, mixed and kept at 30 ° C for 2 min, and 0.5 μmol / L phenylpyruvate reductase and mutant N193D / H203R enzyme solution were added respectively, and the changes in the absorption value of NADH at 340 nm under different pH were detected. The protein quality was detected by Coomassie Brilliant Blue-G250 method.

[0056] Results Figure 3 The enzyme activity of the wild-type phenylpyruvate reductase was 176.3 U / mg, and the enzyme activity of the mutant N193D / H203R was 420.7 U / mg, which was 138.6% higher than that of the wild-type.

[0057] Example 4

[0058] Determination of the Optimal pH of Phenylpyruvate Reductase and Its Mutant N193D / H203R

[0059] The optimal reaction pH is the pH value corresponding to the highest enzyme activity. The enzyme activity at the optimal reaction pH is set as 100% relative enzyme activity, and the relative enzyme activity at other reaction pHs is calculated to compare the effects of different pH on the enzyme activity of phenylpyruvate reductase and mutant N193D / H203R.

[0060] The total reaction system was 250 μl, including potassium phosphate buffer (KPB, 100 mmol / L, pH buffer range 6.0-8.5), 0.5 mmol / L NADH, and 5 mmol / L PPA. After mixing, the mixture was kept at 30°C for 2 min, and 0.5 μmol / L phenylpyruvate reductase and mutant N193D / H203R enzyme solution were added respectively. The changes in the absorbance value of NADH at 340 nm under different pH values ​​were detected.

[0061] Results Figure 4 The optimal pH of the mutant N193D / H203R was consistent with that of the wild type, both of which were 7.0.

[0062] Example 5

[0063] Determination of the Optimal Temperature of Phenylpyruvate Reductase and Its Mutant N193D / H203R

[0064] The optimum reaction temperature is the temperature corresponding to the highest enzyme activity. The enzyme activity at the optimum temperature is set as 100% relative enzyme activity, and the relative enzyme activity at other reaction temperatures is calculated to compare the effects of different temperatures on the enzyme activity of phenylpyruvate reductase and mutant N193D / H203R.

[0065] The total reaction system was 250 μl, including potassium phosphate buffer (KPB, 100 mmol / L, pH = 7.0), 0.5 mmol / L NADH, 5 mmol / L PPA, and was kept warm for 2 min in the temperature range of 18°C-50°C (a temperature point was set every 4°C), and 0.5 μmol / L phenylpyruvate reductase and mutant N193D / H203R enzyme solution were added respectively, and the changes in the absorption value of NADH at 340 nm under different pH were detected. The results are shown in Figure 5 The optimum temperature of mutant N193D / H203R is consistent with that of the wild type, both of which are 30°C, but the relative enzyme activity of mutant N193D / H203R changes less within the gradient temperature range, proving that mutant N193D / H203R has better thermal stability than the wild type.

[0066] Example 6

[0067] Synthesis of phenyllactic acid catalyzed by phenylpyruvate reductase and mutant N193D / H203R in 1L conversion system

[0068] With 80g / L PPA as substrate, 1μmol / L wild-type phenylpyruvate reductase and mutant N193D / H203R pure enzyme solution and 8g / L NADH were added respectively, and the catalytic reaction was carried out in a 1L reactor at 30℃ and pH=7.0 in KPB buffer. Samples were taken every 1h, and the samples were boiled for 10min to inactivate. The supernatant was centrifuged and passed through a membrane for liquid phase detection. The substrate and product were analyzed by reversed-phase HPLC. The detection conditions were: chromatographic column, Agilent Zorbax SB-C18 (150mm×4.6mm, 5μm); mobile phase, 0.5g / L trifluoroacetic acid / water (A) and 0.5g / L trifluoroacetic acid / methanol (B) mixture; column temperature, 30℃; flow rate, 1ml / min. The results of phenylpyruvate reductase and mutant N193D / H203R catalyzing phenyllactic acid synthesis are shown in Figure 6 , Figure 7 After 8 hours of catalytic reaction, the wild-type phenylpyruvate reductase had a residual phenylpyruvate of 43.2 g / L and a phenyllactic acid yield of 32.7 g / L. After 6 hours of catalytic reaction, the phenylpyruvate reductase mutant N193D / H203R completely converted phenylpyruvate and produced 78.6 g / L of phenyllactic acid. With a shorter reaction time, the yield increased by 140.4%.

[0069] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described above are also within the scope of the present application. Therefore, the equalization and modification made without departing from the spirit and scope of the present application should be included in the scope of the present application. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that without certain specific details, the present invention can be implemented equally. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the purport of the present application.

Claims

1. A phenylpyruvate reductase mutant with high enzyme activity, characterized in that: The amino acid sequence shown in SEQ ID NO.1 was mutated from asparagine to aspartic acid at position 193, and from histidine to arginine at position 203.

2. A phenylpyruvate reductase gene, characterized in that: The gene encodes the phenylpyruvate reductase mutant according to claim 1.

3. The gene according to claim 2, characterized in that It is obtained by base substitution of the nucleotide sequence shown in SEQ ID NO.

2.

4. An expression vector, characterized in that: The expression vector comprises the nucleotide sequence of claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a pET series expression vector.

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria is Escherichia coli containing the expression vector according to claim 4.

7. A method for constructing a recombinant engineered bacterium, characterized in that: The steps include: (1) Obtaining mutant sequences: Using the wild-type sequence as a template, point mutations are performed to obtain mutant sequences; (2) constructing an expression vector: homologously recombining the gene described in claim 2 with the enzyme-cut plasmid to obtain a recombinant expression vector; (3) Construction of recombinant bacteria: The constructed recombinant expression vector is transferred into competent Escherichia coli cells, and the recombinant engineered bacteria are obtained by culture and screening.

8. A catalyst, characterized in that It comprises the phenylpyruvate reductase mutant according to claim 1.

9. Use of the phenylpyruvate reductase mutant according to claim 1 in catalyzing the synthesis of phenyllactic acid.

10. The use according to claim 9, characterized in that The optimal reaction conditions included a reaction temperature of 30°C and a pH of 7.0.

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