D-carbamoylase mutants with improved thermal stability and their application in the synthesis of D-amino acids

Abstract

The present invention discloses a D-carbamoyl hydrolase mutant with improved thermal stability and its application in the synthesis of D-amino acids. Based on D-carbamoyl hydrolase, mutants with different degrees of improved thermal stability are obtained by site-directed mutagenesis. The best mutant S202P / E208D / R277L obtained by combinatorial mutagenesis has a t 1 / 2 value at 40 °C that is 28.5 times that of M4, approximately 36.5 h. It has a catalytic efficiency comparable to that of M4. When M4 catalyzes the reaction of 100 mM N-carbamoyl-D-tryptophan at 40 °C for 24 h, the conversion rate is only 60.8%, while S202P / E208D / R277L can complete 99.4% conversion in 24 h. This indicates that the mutant S202P / E208D / R277L can carry out reactions at a higher temperature and has good industrial application prospects.

Description

Technical Field

[0001] The present invention relates to a D-carbamoylase mutant with improved thermal stability and its application in the synthesis of D-amino acids, belonging to the technical field of enzyme engineering. Background Art

[0002] D-amino acids are important unnatural amino acids, widely distributed in microorganisms, plants and animals. D-amino acids can be used to synthesize various antibiotic drugs, and some of these drugs such as amoxicillin, ampicillin, penicillin, etc. are defined as essential drugs by the World Health Organization. Moreover, D-amino acids have extensive applications in the food, agriculture and chemical industries.

[0003] There are mainly three types of biocatalysts that can be used to synthesize optically pure D-amino acids, namely hydrolases, oxidoreductases and D-amino acid transaminases. Among them, hydrolases are a relatively wide range of enzymes for the preparation of D-amino acids. They usually use racemic compounds as substrates and can theoretically achieve a yield of 50% through resolution. Combining with racemase can achieve a theoretical yield of 100%. They have the advantages of a wide substrate range, high product yield and few by-products. Currently reported hydrolases for the synthesis of D-amino acids mainly include the coupled hydantoinase process of D-hydantoinase and D-carbamoylase, N-acyl-D-amino acid amide hydrolase, D-amino acid amide hydrolase, D-aminopeptidase and D-peptidase.

[0004] D-carbamoylase (HyuC) belongs to the 6th class (EC3.5.1.77) of the nitrilase superfamily. It can hydrolyze N-carbamoyl-D-amino acids to obtain D-amino acids and is often used to form a cascade reaction with hydantoin racemase and hydantoinase to prepare optically pure D-amino acids. However, the thermal stability of the identified wild-type enzymes and their mutants is generally low. To meet the requirements of industrial applications, extensive research has been carried out on the directional mining and thermal stability analysis of D-carbamoylases with different properties, as well as the molecular modification of the thermal stability of D-carbamoylases based on directed evolution and rational design. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a D-carbamoylase mutant with improved thermal stability. Based on the D-carbamoylase with the amino acid sequence shown in SEQ ID NO.1 in the previous research of the inventors, appropriate mutations are made to obtain a mutant with improved thermal stability, enabling it to have extensive applications in the food and pharmaceutical fields.

[0006] The first object of the present invention is to provide a D-carbamoyl hydrolase mutant with improved thermal stability, wherein the D-carbamoyl hydrolase mutant is based on the D-carbamoyl hydrolase with the amino acid sequence shown in SEQ ID NO.1 as the parent, and the amino acids at positions 138, 202, 204, 208, 277 and 284 are mutated respectively.

[0007] Further, the D-carbamoyl hydrolase mutant is obtained by mutating the glutamic acid (Glu) at the 138th position of the parent into tryptophan (Trp) or serine (Ser); or,

[0008] mutating the serine (Ser) at the 202nd position of the parent into proline (Pro); or,

[0009] mutating the serine at the 204th position of the parent into aspartic acid (Asp) or asparagine (Asn); or,

[0010] mutating the glutamic acid (Glu) at the 208th position of the parent into aspartic acid (Asp); or,

[0011] mutating the arginine (Arg) at the 277th position of the parent into leucine (Leu), glutamine (Gln) or glutamic acid (Glu); or,

[0012] mutating the histidine (His) at the 284th position of the parent into threonine (Thr) or asparagine (Asn).

[0013] Further, the D-carbamoyl hydrolase mutant is obtained by mutating the serine (Ser) at the 202nd position of the parent into proline (Pro), mutating the glutamic acid (Glu) at the 208th position into aspartic acid (Asp), and mutating the arginine (Arg) at the 277th position into leucine (Leu).

[0014] The second object of the present invention is to provide a coding gene for the D-carbamoyl hydrolase mutant.

[0015] The third object of the present invention is to provide an expression vector carrying the coding gene.

[0016] The fourth object of the present invention is to provide a genetically engineered bacterium expressing the D-carbamoyl hydrolase mutant.

[0017] Further, the genetically engineered bacterium uses Escherichia coli as the host.

[0018] Further, the Escherichia coli is E. coli BL21(DE3).

[0019] Further, the genetically engineered bacterium uses pET-28a(+) as the expression vector.

[0020] The fifth object of the present invention is to provide the use of the D-carbamoyl hydrolase mutant or the genetically engineered bacterium in the preparation of D-amino acids.

[0021] Furthermore, in the above application, N-carbamoyl-D-amino acid is used as a substrate, and the D-carbamoyl hydrolase mutant or the genetically engineered bacterium is used as a catalyst to catalytically generate D-amino acids.

[0022] Furthermore, the conditions for the catalysis are 38-42 °C and 150-250 rpm.

[0023] The beneficial effects of the present invention are as follows:

[0024] Based on the D-carbamoyl hydrolase (NiHyuC-M4) derived from Nitratireductor indicus, mutants with different degrees of improved thermal stability were obtained by site-directed mutagenesis in the present invention. Among them, the t 1 / 2 values of the single mutants E138W, E138S, S202P, S204D, S204N, E208D, R277L, R277Q, R277E, H284T, H284N at 40 °C increased from 1.3 h of M4 to 2.2 h, 1.7 h, 2.3 h, 1.9 h, 1.7 h, 3.3 h, 8.0 h, 2.3 h, 1.6 h, 2.2 h, 1.8 h respectively. The best mutant S202P / E208D / R277L obtained by combinatorial mutagenesis had a t 1 / 2 value 28.5 times that of M4 at 40 °C, about 36.5 h. It had a catalytic efficiency equivalent to that of M4 (288 min -1 ·mM -1 ), which was 302 min -1 ·mM -1 . When M4 catalyzed 100 mM N-carbamoyl-D-tryptophan at 40 °C for 24 h, the conversion rate was only 60.8%, while S202P / E208D / R277L could complete 99.4% conversion in 24 h. This indicates that the mutant S202P / E208D / R277L can carry out reactions at a higher temperature and has good industrial application prospects. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments do not limit the present invention.

[0026] The culture media involved in the following embodiments are as follows:

[0027] LB liquid medium: peptone 10 g·L -1 , yeast extract 5 g·L -1, 10 g·L of NaCl -1 .

[0028] LB solid medium: 10 g·L of peptone -1 , 5 g·L of yeast extract -1 , 10 g·L of NaCl -1 , 15 g·L of agar powder -1 .

[0029] The protein purification buffer involved in the following examples is as follows:

[0030] Binding solution A: 20 mM imidazole, 500 mM sodium chloride, 20 mM tris(hydroxymethyl)aminomethane (Tris), 5% glycerol, adjusted to pH 7.4 with HCl;

[0031] Elution solution B: 500 mM imidazole, 500 mM sodium chloride, 20 mM Tris, 5% glycerol, adjusted to pH 7.4 with HCl.

[0032] The detection methods involved in the following examples are as follows:

[0033] Detection of D-carbamoylhydrolase activity: The reaction is carried out in a 1.5 mL EP tube. Take 10 μL of pure enzyme solution with a protein concentration of 1 mg·mL -1 and add it to a mixture containing 200 μL of 10 mM substrate N-carbamoyl-D-tryptophan in Tris-HCl (pH 8.0, 100 mM) buffer. The total reaction system is 1 mL. Place the reaction system at 30 °C for 10 min. After the reaction, add methanol in a ratio of 1:3 to terminate the reaction. The reaction product is centrifuged and filtered through a membrane (0.22 μm) and then analyzed by HPLC to determine the enzyme activity. Each group of data is repeated three times.

[0034] HPLC analysis conditions: The mobile phase is KH2PO4 (20 mM, pH 2.5): acetonitrile = 75:25, and the flow rate is 1 mL·min -1 , and the chromatographic column is ZORBAX SB-C 18 column, the column temperature is 30 °C, and the detection wavelength is 210 nm.

[0035] Definition of enzyme activity unit: At 30 °C, the amount of enzyme required to catalyze the conversion of N-carbamoyl-D-amino acid to 1 μmol of amino acid per minute is defined as one enzyme activity unit (U).

[0036] Example 1: Primary screening of thermostable D-carbamoylhydrolase mutant enzyme

[0037] The specific steps are as follows:

[0038] 1. (1) Construction of mutants:

[0039] The mutant primers are shown below, with the mutation sites underlined:

[0040] Table 1 Mutant Primers

[0041]

[0042]

[0043] Taking the plasmid construction of mutating the 202nd position of D-carbamoyl hydrolase with the amino acid sequence shown in SEQ ID NO.1 to proline (S202P) as an example, using the pET-28a(+) vector ligated with the nucleotide sequence shown in SEQ ID NO.2 as a template, and using S202P-F and S202P-R as primers, PCR amplification was carried out to obtain the nucleotide sequence encoding the mutant (S202P) with the 202nd serine in the amino acid sequence mutated to proline;

[0044] The PCR product containing the recombinant gene obtained in the previous step was digested with DpnI to remove the template, and the digested product was transformed into competent cells of Escherichia coli E.coli BL21(DE3) to obtain a transformation solution; The transformation solution was spread on LB solid medium containing 50 ng·mL -1 kanamycin, and cultured inverted at 37°C for 12 h. Single colonies were picked into LB liquid medium containing 50 ng·mL -1 kanamycin, cultured at 37°C for 8 - 10 h. After plasmid extraction and DNA sequencing, the correct mutant plasmid S202P was obtained.

[0045] Using the primers in Table 1 and the same method as in step (1), recombinant plasmids of mutants E138W / S, S202P, S204D / N, E208D, R277L / Q / E, and H284T / N were constructed.

[0046] PCR amplification: The reaction system was referred to Table 2, with a total volume of 20 μL:

[0047] Table 2 PCR Reaction System

[0048]

[0049] Table 3 PCR Amplification Program

[0050]

[0051] (2) Primary Screening of Thermostable D-Carbamoyl Hydrolase

[0052] Taking D-carbamoyl hydrolase S202P as an example, the steps for primary screening of D-carbamoyl hydrolase mutants with improved thermostability are as follows:

[0053] Transfer the recombinant Escherichia coli containing the D-carbamoyl hydrolase mutant S202P constructed in step (1) to 40 mL of LB liquid medium containing kanamycin at a final concentration of 50 ng·mL -1 and culture it at 37 °C and 120 rpm until the OD 600 reaches 0.6 - 0.8. Then add IPTG with a final concentration of 0.2 mM and induce culture at 16 °C for 20 h. Centrifuge the above-mentioned bacterial cells at 8000 rpm for 5 min to collect them. After adding an appropriate amount of Tris-HCl (100 mM, pH 8.0) to each 50 mL centrifuge tube for collecting the bacterial cells, perform ultrasonic disruption. Take 1 mL of the disrupted solution, centrifuge it at 12000 rpm for 3 min at 4 °C, and transfer 200 μL of the supernatant to 3 clean 1.5 mL centrifuge tubes and place them on ice for standby.

[0054] Incubate the crude enzyme solution of mutant S202P at 40 °C and 30 °C for 15 min respectively, then ice-bath for 2 min, and then carry out the enzyme reaction. The ratio of the specific activity measured after incubating the crude enzyme solution at 40 °C for 15 min to the specific activity measured after incubating at 30 °C for 15 min is recorded as the residual activity, and this is used to preliminarily screen for mutants with improved thermal stability (the residual activity of M4 is about 82%).

[0055] Initial screening reaction system for D-carbamoyl hydrolase: Using N-carbamoyl-D-tryptophan at a final concentration of 2 mM as the substrate, add 20 μL of the crude enzyme solution (M4 or mutant enzyme), and make up to 1 mL with 100 mM Tris-HCl buffer (pH 8.0). React at 30 °C for 10 min, and add methanol in a ratio of 1:3 to terminate the reaction.

[0056] Example 2: Rescreening of the thermostable D-carbamoyl hydrolase mutant enzyme

[0057] (1) Purification of the D-carbamoyl hydrolase mutant enzyme

[0058] Transform the plasmid of the mutant with improved thermal stability screened in Example 1, culture it upside down at 37 °C overnight, pick a single colony into 40 mL of LB liquid medium containing kanamycin at a final concentration of 50 ng·mL -1 and culture it at 37 °C and 120 rpm for 8 - 10 h. Take 1 mL of the above bacterial solution into 100 mL of LB liquid medium containing kanamycin at a final concentration of 50 ng·mL -1 and culture it at 37 °C and 120 rpm until the OD 600 reaches 0.6 - 0.8. Then add IPTG with a final concentration of 0.2 mM and induce culture at 16 °C for 16 - 20 h. Centrifuge the above-mentioned bacterial cells at 4 °C and 8000 rpm for 5 min to collect them.

[0059] Add an appropriate amount of binding solution A to each 50 mL centrifuge tube for collecting bacterial cells. After ultrasonic disruption, centrifuge at 8000 rpm for 30 min at 4°C, filter through a membrane, and purify through a nickel affinity chromatography column. After the sample loading is completed, use imidazole elution solutions with different concentration gradients prepared from binding solution A and elution solution B for protein elution and collection. The elution volume for each concentration gradient is approximately 5 - 10 column volumes. After the elution is completed, perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on the collected solution for verification. According to the results of SDS-PAGE, collect proteins with higher purity and perform ultrafiltration and concentration to remove imidazole. Quick-freeze in liquid nitrogen and store at -80°C.

[0060] (2) Rescreening of thermostable D-carbamoyl hydrolase

[0061] Detection method for thermostability: Dilute the preserved mutant pure enzyme to 1 mg·mL -1 , take an appropriate amount of enzyme solution and incubate it in a 40°C water bath. Use the incubation at 40°C for 5 min as the starting point 0 min, and take samples of the enzyme solution at different incubation times to measure the enzyme activity. Take the enzyme activity at 0 min as 100%, and measure the half-life (t 1 / 2 ). The half-life (t 1 / 2 ) refers to the time required to lose half of the enzyme activity at a given temperature. Repeat each set of data three times.

[0062] Rescreening reaction system for D-carbamoyl hydrolase: Use N-carbamoyl-D-tryptophan with a final concentration of 2 mM as the substrate, add 10 μL of pure enzyme (M4 or mutant enzyme) at 1 mg·mL -1 , and make up to 1 mL with 100 mM Tris-HCl buffer (pH 8.0). React at 30°C for 10 min, and add methanol in a 1:3 ratio to terminate the reaction.

[0063] The thermostability of the mutants from high to low is R277L, E208D, S202P, R277Q, E138W, H284T, S204D, H284N, S204N, E138S, R277E.

[0064] After that, use the same method as in Example 1 and Example 2 to finally obtain the combined mutant enzymes S202P / E208D, S202P / R277L, E208D / R277L, S202P / E208D / R277L with significantly improved thermostability (Table 4). Among them, S202P / E208D / R277L has the best thermostability, and t 1 / 2 (40°C) is 36.5 h, which is 28.5 times that of M4 (1.3 h).

[0065] Table 4 Specific activity and half-life of thermostable mutants

[0066]

[0067] Example 3: Study on the Properties of Thermostable D-Carbamoyl Hydrolase Mutant Enzymes

[0068] 1. Optimal Temperature

[0069] The enzyme reactions of M4 and mutant enzymes (S202P, E208D, R277L, S202P / E208D, S202P / R277L, E208D / R277L, S202P / E208D / R277L) were carried out at different temperatures (25 - 55 °C) respectively. The temperature at which each enzyme showed the highest activity was defined as the optimal temperature.

[0070] As shown in Table 5, the optimal temperatures of mutant enzymes S202P, E208D, and S202P / E208D were 50 °C, which was 5 °C higher than that of M4; however, the optimal temperatures of mutant enzymes R277L, S202P / R277L, E208D / R277L, and S202P / E208D / R277L decreased to 40 °C, which was 5 °C lower than that of M4.

[0071] 2. Thermodynamic Stability Experiment

[0072] The thermodynamic stabilities of M4 and mutant enzymes (S202P, E208D, R277L, S202P / E208D, S202P / R277L, E208D / R277L, S202P / E208D / R277L) were characterized by measuring the unfolding temperature (T m ) through Nano-DSC. The specific operation steps are as follows:

[0073] (1) Baseline scanning: After degassing the buffer Tris-HCl (100 mM, pH 8.0) for 10 min, add it to the sample cell and reference cell. After removing the bubbles, perform baseline scanning. Set the running program as: temperature scanning range 25 - 85 °C, heating rate 1 °C·min -1 , and the number of repetitions is 5 times.

[0074] (2) Sample scanning: Dilute M4 or mutant enzyme to 1 mg·mL -1 and then perform degassing treatment for 10 min. Completely suck out the buffer in the sample cell, add the degassed M4 or mutant enzyme, and perform sample scanning. Set the running program as: temperature scanning range 25 - 85 °C, heating rate 1 °C·min -1 .

[0075] (3) Result processing: Use NanoAnalyze TMThe software processes the operation results, uses the TwoStateScaled model for data fitting, and obtains the T of M4 and mutant enzymes m value (Table 5).

[0076] As shown in Table 5, although the thermal stability of M4Th3 at 40 °C is significantly higher than that of M4, its T m value is only 3.6 °C higher. The ΔT m values of S202P, E208D, and R277L are 1.1 °C, 1.3 °C, and 1.3 °C respectively, and the sum of the three is approximately equal to the corresponding value of M4Th3, indicating that there may be an additive or synergistic effect among these three single mutations.

[0077] Table 5 Characterization of the properties of thermostable mutants

[0078]

[0079] 3. Kinetic stability experiment

[0080] It is measured to characterize the kinetic stability of M4 and mutant enzymes (S202P, E208D, R277L, S202P / E208D, S202P / R277L, E208D / R277L, S202P / E208D / R277L) obtained in Example 2. The pure enzyme is diluted to 1 mg·mL -1 , and 20 μL is aliquoted into PCR tubes, and the enzyme reaction is carried out by incubating at different temperature gradients in a PCR for 15 min. The temperature at which the relative enzyme activity loses half is

[0081] As shown in Table 5, for S202P and E208D, the increment is only 1 - 2 °C; the of R277L is 2.2 °C. However, it is noted that the values of S202P, E208D, and R277L accumulated together are almost equal to the of M4Th3. This may further indicate an additive or synergistic effect among these three single mutations.

[0082] 4. Determination of kinetic parameters

[0083] Under the standard enzyme activity determination conditions, the initial reaction rate of a certain concentration of D-carbamoyl hydrolase on different substrate concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 5.0 mM) is measured, and the Michaelis equation curve of the relationship between the initial reaction rate and the substrate concentration is fitted with Origin 2019 software to obtain the K M and V max values.

[0084] Table 6 Kinetic parameters

[0085]

[0086] The results are shown in Table 6. Although the V max value (2.97 U·mg –1 ) of S202P / E208D / R277L is lower than that of M4 (3.40 U·mg –1 ), the K M value of S202P / E208D / R277L is also lower. Therefore, finally, S202P / E208D / R277L obtains a catalytic efficiency comparable to that of M4 (288 min –1 ·mM –1 ), which is 302 min –1 ·mM –1 .

[0087] Example 4: Evaluation of the application of thermostable D-carbamoyl hydrolase mutants in D-tryptophan synthesis

[0088] To further explore the application of the thermostable D-carbamoyl hydrolase mutant S202P / E208D / R277L in D-tryptophan synthesis, the reaction of M4 and S202P / E208D / R277L with 100 mM N-carbamoyl-D-tryptophan at 40 °C was measured.

[0089] D-carbamoyl hydrolase (5 kU·L –1 ) was added to Tris-HCl buffer (pH 8.0, 100 mM) containing 100 mM N-carbamoyl-D-tryptophan, and the reaction was carried out at 40 °C and 200 rpm for 24 h with a constant pH of 8.0. The total volume was 500 mL.

[0090] The results are shown in Table 7. Although the conversion rate of S202P / E208D / R277L was lower than that of M4 within the first 2 h due to its slightly lower specific activity, it was noted that the conversion rate of S202P / E208D / R277L was significantly higher than that of M4 after that. The conversion rate of M4 remained basically unchanged after 12 h of reaction, and the conversion rate at 24 h was 60.8%; while the conversion rate of S202P / E208D / R277L reached 94.5% at 12 h and completed 99.4% of the conversion at 24 h. This shows that S202P / E208D / R277L has significantly better thermostability than M4, which is beneficial to its industrial application.

[0091] Table 7 Reaction process of D-carbamoyl hydrolase catalyzing 100 mM N-carbamoyl-D-tryptophan

[0092]

[0093] The above-described embodiments are merely preferred embodiments given to fully illustrate 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 shall be subject to the claims.

Claims

1. A D-carbamoylase mutant with improved thermal stability, characterized in that, The D-carbamoylase mutant uses the D-carbamoylase with the amino acid sequence shown in SEQ ID NO.1 as the parent, and mutates the amino acids at positions 202, 208, and 277 respectively; The D-carbamoylase mutant is obtained by mutating the serine (Ser) at position 202 of the parent into proline (Pro), mutating the glutamate (Glu) at position 208 into aspartic acid (Asp), and mutating the arginine (Arg) at position 277 into leucine (Leu).

2. A coding gene for the D-carbamoylase mutant according to claim 1.

3. An expression vector carrying the coding gene according to claim 2.

4. A genetically engineered bacterium expressing the D-carbamoylase mutant according to claim 1.

5. The genetically engineered bacterium according to claim 4, characterized in that, The genetically engineered bacterium uses Escherichia coli as the host.

6. Use of the D-carbamoylase mutant according to claim 1 or the genetically engineered bacterium according to claim 4 in the preparation of D-amino acids.

7. The use according to claim 6, characterized in that, The application uses N-carbamoyl-D-amino acid as the substrate, and uses the D-carbamoylase mutant or the genetically engineered bacterium as the catalyst to catalytically produce D-amino acid.

8. The use according to claim 7, characterized in that, The conditions for the catalysis are 38 - 42 °C and 150 - 250 rpm.

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