L-aspartate alpha-decarboxylase Mutant and Application thereof

Abstract

The disclosure discloses an L-aspartate α-decarboxylase mutant and application thereof, and belongs to the technical field of enzyme engineering. In the disclosure, lysine at position 221 of L-aspartate α-decarboxylase is mutated to arginine, glycine at position 369 is mutated to alanine, and the obtained new mutant enzymes have better temperature tolerance and are beneficial to industrial production. The K221R and G369A recombinant strains are subjected to high-density fermentation, and with sodium L-aspartate as a substrate, a whole cell catalytic reaction is carried out to prepare β-alanine. Compared with a chemical production method, the method has the advantages that the production process is safe and clean, and has no environmental pollution. Compared with a pure enzyme catalysis method, the method has the advantages that the operation is simple and convenient. The yield of the final product β-alanine reaches 91% and 90% respectively, and the concentration reaches 162.15 g/L and 160.42 g/L respectively.

Claims

1. An L-aspartate α-decarboxylase mutant, comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

2. A composition comprising the L-aspartate α-decarboxylase mutant of claim 1 and a protective agent.

3. A cell expressing the L-aspartate α-decarboxylase mutant of claim 1.

4. The cell of claim 3, wherein the cell is an Escherichia coli (E. coli) BL21 cell.

5. The cell of claim 4, wherein pET24a(+) is used as an expression vector.

6. The cell of claim 3, wherein a method for constructing the cell is as follows: a gene encoding the L-aspartate α-decarboxylase mutant is ligated with an expression vector and transformed into Escherichia coli (E. coli).

7. The cell of claim 6, wherein the gene encoding the L-aspartate α-decarboxylase mutant is set forth in SEQ ID NO:4 or SEQ ID NO:5.

8. A method for using the L-aspartate α-decarboxylase mutant of claim 1, comprising using the L-aspartate α-decarboxylase mutant of claim 1 as a catalyst, and using sodium L-aspartate as a substrate to carry out a transformation reaction for producing β-alanine.

9. The method of claim 8, wherein with the sodium L-aspartate as the substrate, Escherichia coli (E. coli) BL21 cell expressing the L-aspartate α-decarboxylase mutant is used for fermentation, and a bacteria solution after the fermentation is used for whole cell transformation to produce β-alanine.

10. The method of claim 9, wherein conditions for the fermentation are as follows: the bacteria solution cultured for 6-8 h is inoculated into a culture medium in a fermenter at an inoculum concentration of 6-8%, and cultured at 35-38° C., when OD.sub.600 reaches 60-70, a temperature is reduced to 28-30° C., Isopropyl β-D-1-Thiogalactopyranoside(IPTG) is added to a final concentration of 0.5-1 mmol/L, and the fermentation is ended after induction culture for 35-40 h, wherein the OD.sub.600 is optical density measured at a wavelength of 600 nm.

11. The method of claim 9, wherein conditions for the whole cell transformation are as follows: in a 10 mL reaction system, recombinant E. coli cells with an OD.sub.600 of 150-250 are added, a final concentration of Pyridoxal 5′-phosphate hydrate(PLP) is 0.5-2 mmol/L, a temperature is adjusted to 35-37° C., and when the substrate reacts completely, a next batch of substrate is added, wherein the OD.sub.600 is optical density measured at a wavelength of 600 nm.

Description

BRIEF DESCRIPTION OF FIGURES

[0025] FIG. 1 is SDS-PAGE electrophoretogram of TcADC wild-type and mutant pure enzymes, in which M is the protein molecular weight standard; 1 is the wild-type purified protein; 2 is the K221R purified protein; and 3 is the G369A purified protein.

[0026] FIG. 2 shows thermal stability curves of enzymes after incubation at different temperatures.

[0027] FIG. 3 shows the catalysis effect of a wild-type strain with high concentration of substrate added once.

[0028] FIG. 4A shows the wild-type strain whole cell catalysis effect after substrates added 3 times.

[0029] FIG. 4B shows the K221R strain whole cell catalysis effect after substrates added 3 times.

[0030] FIG. 4C shows the G369A strain whole cell catalysis effect after substrates added 3 times.

[0031] FIG. 5A shows the K221R strain whole cell catalysis effect after substrates added 4 times.

[0032] FIG. 5B shows the K221R strain whole cell catalysis effect after substrates added 5 times.

[0033] FIG. 5C shows the G369A strain whole cell catalysis effect after substrates added 4 times.

[0034] FIG. 5D shows the G369A strain whole cell catalysis effect after substrates added 4 times.

[0035] FIG. 6A shows the effect of high-density fermentation strain K221R for whole-cell catalytic production of β-alanine.

[0036] FIG. 6B shows the effect of high-density fermentation strain G368A for whole-cell catalytic production of β-alanine.

DETAILED DESCRIPTION

[0037] (I) Determination Method of Enzyme Activity of L-Aspartate α-Decarboxylase

[0038] Determination method of enzyme activity of fermentation broth: 100 μL of recombinant E. coli cells after fermentation are taken, 100 μL of 1 mol/L sodium L-aspartate solution is added, and 800 μL of phosphate buffer with a pH of 6.5 is added. After reacting at 37° C. for 30 min, reaction solution is inactivated at 100° C. for 10 min. The reaction solution is centrifuged at 12000 rpm for 2 min, the supernatant is taken for derivatization and the yield of β-alanine is detected. The reaction solution is filtered through a 0.22 μm microporous filter membrane and loaded onto a C18 chromatographic column for HPLC analysis.

[0039] Determination method of enzyme activity of L-aspartate α-decarboxylase: an appropriate amount of enzyme solution is added to a 1.5 mL centrifuge tube. Sodium L-aspartate at a final concentration of 100 mmol/L is added. The final concentration of PLP is 1 mmol/L, and the enzyme activity is detected after reacting at 37° C. and pH 6.5 for 30 min.

[0040] (II) Culture Medium

[0041] LB culture medium (g/L): peptone 10.0, yeast powder 5.0, NaCl 10.0.

[0042] 2YT culture medium (g/L): peptone 16.0, yeast powder 10.0, NaCl 5.0.

[0043] Fermenter culture medium (g/L): glycerol 8.0, potassium dihydrogen phosphate 13.5, diammonium hydrogen phosphate 4.0, citric acid 1.7, magnesium sulfate heptahydrate 1.7, trace elements 10 mL.

[0044] Feed-batch culture medium (g/L): glycerol 500.0, magnesium sulfate heptahydrate 7.4, yeast powder 4.0, tryptone 4.0.

[0045] Trace elements (g/L): ferrous sulfate heptahydrate 10.0, zinc sulfate heptahydrate 2.3, copper sulfate pentahydrate 10.0, manganese sulfate tetrahydrate 0.5, borax 0.2, calcium chloride 2.0, ammonium molybdate 0.1.

[0046] (III) Method for Detecting the Content of Sodium L-Aspartate and β-Alanine by HPLC

[0047] The reaction solution is derivatized with phenyl isothiocyanate (PITC). The specific steps are: 500 μL of the reaction solution is put into a 2.0 mL centrifuge tube. 250 μL of 0.1 mol/L PITC-acetonitrile solution and 250 μL of 1 mol/L triethylamine-acetonitrile solution are added and mixed thoroughly. The reaction solution is stored in the dark at room temperature for 1.0 h. 750 μL of n-hexane solution is added to terminate the derivatization. The reaction solution is shaken with a vortex shaker for 1 min, and allowed to stand for 30-60 min. The lower layer solution is pipetted and filtered through a 0.22 μm organic filter membrane, and then the sample is injected at a sample volume of 10 μL.

[0048] The derivatized product is determined by HPLC: the chromatographic column is La Chrom C18 (5 μm, 4.6×250 mm). A mobile phase A solution is 80% (V/V) acetonitrile aqueous solution, and a B solution is 97:3 (V/V, pH 6.5) 0.1 mol/L sodium acetate-acetonitrile solution. Gradient elution is adopted: during 0-20 min, the B solution is dropped from 95% to 65%; during 20-30 min, the B solution is raised from 65% to 95%; and during 30-35 min, the B solution gradient is not changed. The detection wavelength is 254 nm, and the column temperature is 40° C.

[0049] (IV) Determination of Temperature Stability

[0050] The wild-type enzyme is used as a control. The wild enzyme and the mutant enzyme are placed in phosphate buffers with a pH of 6.5, and incubated at 0° C., 20° C., 30° C., 40° C., 50° C., and 60° C. respectively for 30 min, and then the residual enzyme activity is determined to obtain the temperature stability results.

EXAMPLE 1

Construction of Recombinant E. coil

[0051] (1) Construction of mutants BL21/pET28a-K221R and BL21/pET28a-G369A: PCR was performed under the conditions shown in Table 1 with pET28a-TcADC plasmids as templates. The sequence information of the forward and reverse primers used to construct the mutant K221R is as set forth in SEQ ID NO:5 and SEQ ID NO:6 respectively. The sequence information of the forward and reverse primers used to construct the mutant G369A is as set forth in SEQ ID NO:7 and SEQ ID NO:8 respectively.

TABLE-US-00001 TABLE 1 Whole plasmid PCR amplification reaction system Reagent Amount (μL) pET-28a-TcADC plasmids 0.5 Forward primer 1.0 Reverse primer 1.0 ddH.sub.2O 22.5 Total 25

[0052] PCR Amplification Reaction Conditions:

TABLE-US-00002 95° C. Initial denaturation  5 min 98° C. Denaturation 10 s 56° C. Annealing 30 s {close oversize brace} 24 cycles 72° C. Extension 75 s 72° C. Extension  5 min

[0053] The PCR products were identified by an agarose gel electrophoresis method. The PCR amplified products were transformed into E. coliJM109 competent cells to obtain recombinant plasmids pET28a-K221R and pET28a-G369A carrying the genes encoding the mutants. The recombinant plasmids pET28a-K221R and pET28a-G369A were respectively transformed into E. coli BL21 cells to obtain recombinant strains BL21/pET28a-K221R and BL21/pET28a-G369A.

[0054] (2) The recombinant E. coli BL21/pET28a-K221R and BL21/pET28a-G369A were respectively inoculated into 5 mL of LB culture medium containing kanamycin at a concentration of 50 μg/mL, and cultured overnight at 37° C. and 200 rpm with shaking.

[0055] (3) Expression and purification of L-aspartate α-decarboxylase

[0056] The recombinant E. coli BL21/pET28a-K221R and BL21/pET28a-G369A were respectively inoculated into 5 mL of LB culture medium containing kanamycin at a concentration of 50 μg/mL, and cultured overnight at 37° C. and 200 rpm with shaking. The overnight culture was inoculated into the 2YT culture medium containing kanamycin at a concentration of 50 μg/mL at an inoculum concentration of 1% (v/v), and cultured with shaking at 37° C. and 200 rpm until the OD.sub.600 of the bacteria solution was 0.6-0.8. IPTG with a final concentration of 0.2 mmol/L was added and induction culture was carried out at 20° C. for about 20 h. Bacterial cells were collected by centrifugation at 6000 rpm, and ultrasonically broken. Protein purification was carried out using a His Trap HP affinity column. The target protein was detected by SDS-PAGE. The results are shown in FIG. 1.

[0057] (4) Determination of thermal stability

[0058] The purified enzyme was diluted to the same concentration, treated at 0° C., 20° C., 30° C., 40° C., 50° C., and 60° C. for half an hour respectively, then reacted at 37° C. for half an hour, and then placed at 100° C. for ten minutes to terminate the reaction. The results are shown in FIG. 2.

EXAMPLE 2

Catalysis Process of Wild-Type Strain with High-Concentration Solid Substrate Added Once

[0059] The cells of the wild-type strain were cultured according to the method of step (3) of Example 1, and the cells induced by IPTG were collected by centrifugation for whole-cell catalysis reaction. In a 10 mL reaction system (including bacterial cells, a phosphate buffer with pH of 6.5, the substrate, and PLP), wherein the OD.sub.600 of the cells was 200, the pH was 6.5, the PLP concentration was 1 mmol/L, and the reaction temperature was 35-37° C., the solid substrate sodium L-aspartate was added once to a final concentration of 1 mol/L. Samples were taken at regular intervals to detect the yield of β-alanine. The results are shown in FIG. 3. The concentration of β-alanine converted after 10 h of reaction was 836 mmol/L, and the molar conversion rate was 83.6%.

EXAMPLE 3

Whole-Cell Catalysis Process of Batch Feeding of Substrate

[0060] The wild-type strain and the cells expressing the recombinant E. coli BL21/pET28a-K221R and BL21/pET28a-G369A were used as cell catalysts, 0.4 mol/L solid substrate was added to the reaction system with a volume of 10 mL and an OD.sub.600 of 200 every 4 h. The solid substrate was added three times and the yield of products was detected. The concentration of β-alanine transformed by the wild-type strain was 1079.98 mmol/L, about 96.22 g/L, and the molar conversion rate was 89.90% (FIG. 4A), with a small amount of substrate remained. After the substrate was added three times, the β-alanine transformed by the K221R mutant strain was 1135.4 mmol/L, about 101.15 g/L, and the molar conversion rate was 94.62% (FIG. 4B), with no substrate remained. After the substrate was added three times, the β-alanine transformed by the G369A mutant strain was 1127.43 mmol/L, about 100.44 g/L, and the molar conversion rate was 93.95% (FIG. 4C), with no substrate remained.

EXAMPLE 4

Optimization of Number of Times of Batch Feeding Addition of Substrate

[0061] The substrate was added to the whole cell catalytic system for different number of times, and the effects of batch feeding of different number of times on the catalytic effect were compared. When the substrate was added four times during the catalytic reaction, the mutant strain K221R could produce 1512.24 mmol/L β-alanine, about 134.72 g/L, and the molar conversion rate was 94.52% (FIG. 5A), with no substrate remained. When the substrate was added five times during the catalytic reaction, the mutant strain K221R could produce 1729.71 mmol/L β-alanine by transformation, about 154.05 g/L, and the molar conversion rate was 86.49% (FIG. 5B), with a small amount of substrate remained. When the substrate was added four times during the catalytic reaction, the mutant strain G369A could produce 1530.27 mmol/L β-alanine by catalysis, about 136.33 g/L, and the molar conversion rate was 95.64% (FIG. 5C), with no substrate remained. When the substrate was added five times during the catalytic reaction, the mutant strain G369A could produce 1698.42 mmol/L β-alanine by transformation, about 151.31 g/L, and the molar conversion rate was 84.92% (FIG. 5D), with a small amount of substrate remained.

Example 5

High-Density Fermentation of Recombinant E. Coil and Whole Cell Catalytic Production of β-Alanine

[0062] Recombinant E. coli BL21/pET28a-K221R and BL21/pET28a-G369A were respectively inoculated into 5 mL of LB culture medium containing kanamycin at a concentration of 50 μg/mL, and cultured overnight at 37° C. and 200 rpm with shaking. The overnight culture was inoculated into an 2YT culture medium containing kanamycin at a concentration of 50 μg/mL at an inoculum concentration of 1%, and cultured with shaking at 37° C. and 200 rpm for 6-8 h. The culture was respectively inoculated into a 2 L fermenter fermentation culture medium containing kanamycin at a concentration of 50 μg/mL at an inoculum concentration of 6-8%, and cultured at 37° C. When the OD.sub.600 reaches 60-70, the temperature was reduced to 28-30° C., IPTG was added to a final concentration of 0.8 mmol/L, and the fermentation was ended after induction culture for 36-40 h.

[0063] The bacteria solution after high-density fermentation was centrifuged, and cells were collected, washed with water, and centrifuged and collected again. In a 50 mL reaction system, wherein the OD.sub.600 of the cells was 200, the pH was 6.5, the PLP concentration was 1 mmol/L, and the reaction temperature was 35-37° C., 0.4 mol/L solid substrate was added in batches and continuously stirred. When the substrate reacted completely, a next batch of substrate was added, and the content of each component in the reaction solution was detected by HPLC. The results are shown in FIG. 6A and FIG. 6B. The strain expressing K221R produced 1820.04 mmol/L β-alanine by transformation, about 162.15 g/L, and the molar conversion rate was 91.00% (FIG. 6A), with no remaining substrate. The strain expressing G369A produced 1800.70 mmol/L β-alanine by transformation, about 160.42 g/L, and the molar conversion rate was 90.04% (FIG. 6B), with no substrate remained.

[0064] Although the disclosure has been disclosed as above in preferred examples, it is not intended to limit the disclosure. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be defined by the claims.