CN113122527A - Aspartase mutant with improved enzyme activity and changed optimal pH - Google Patents
Aspartase mutant with improved enzyme activity and changed optimal pH Download PDFInfo
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- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
Abstract
The invention provides an aspartase mutant with improved enzyme activity and changed optimal pH, belonging to the technical field of genetic engineering; the amino acid sequence of the aspartase mutant is shown in SEQ ID NO. 1. The aspartase mutant is obtained from Bacillus sp.YM55-1 aspartase, wherein leucine at the 12 th site is mutated into aspartic acid, tyrosine at the 166 th site is mutated into glutamic acid, glutamine at the 169 th site is mutated into glutamic acid, and arginine at the 314 th site is mutated into methionine, so that the pH adaptability of the aspartase is improved, the capability of the aspartase in synthesizing beta-aminobutyric acid is enhanced, and the industrial production of the beta-aminobutyric acid is facilitated.
Description
Technical Field
The invention relates to an aspartase mutant with improved enzyme activity and changed optimal pH, belonging to the technical field of genetic engineering.
Background
Beta-amino acids are widely used target molecules in the pharmaceutical industry as building blocks for biologically active compounds or natural products and drugs. Wherein beta-aminobutyric acid is a potent initiator, providing broad spectrum disease protection in at least 40 plant species. In addition, the beta-aminobutyric acid can be used as a precursor of a medical intermediate beta-aminobutanol, and the beta-aminobutyrate is a key intermediate of an antitumor drug 4-methyl cyclophosphamide, a penem antibiotic and an acquired immune deficiency syndrome integrase inhibitory drug durovir (Dolutegravir).
The natural substrate for aspartase is aspartic acid, which is selectively engineered for the preparation of β -amino acids with high substrate specificity and the properties of the secondary carboxylate binding pocket. However, the existing aspartase has strict requirements on the pH value condition of the reaction, depends on the reaction environment of high temperature and strong alkali, and has low enzyme activity under the mild condition, and the defect causes high production cost of the beta-amino acid.
Disclosure of Invention
The invention aims to provide an aspartase mutant, a recombinant expression vector containing the aspartase mutant, a recombinant bacterium and application.
In the invention, 12 th leucine of aspartase is mutated into aspartic acid, 166 th tyrosine is mutated into glutamic acid, 169 th glutamine is mutated into glutamic acid, and 314 th arginine is mutated into methionine. The invention changes the conformation state of the enzyme by modifying the surface charge of the aspartase, is beneficial to the enzyme to adjust the flexible structure and present new pH activity, and enhances the capability of the enzyme to synthesize beta-aminobutyric acid.
The invention provides an aspartase mutant, which comprises an amino acid shown as SEQ ID NO. 1.
The present invention provides a gene encoding the mutant aspartate.
In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
The invention provides a recombinant plasmid carrying the gene.
In one embodiment, the recombinant plasmid uses a pET series vector as a starting vector.
The invention provides microbial cells expressing the mutants, or carrying the recombinant plasmids.
In one embodiment, the microbial cell is an E.coli host cell.
The invention provides a method for producing beta-aminobutyric acid, which is characterized in that the microbial cells are used for transforming crotonic acid to produce beta-aminobutyric acid.
In one embodiment, the microbial cells are added to the reaction system such that the OD of the microbial cells in the reaction system60040-60, and the content of crotonic acid in the reaction system is maintained to be not less than 80 g/L.
In one embodiment, the reaction time is not less than 8 hours at 40-60 ℃.
The invention provides an application of the aspartase mutant, the gene or the recombinant bacterium in preparation of beta-amino acid and derivative products.
The invention has the beneficial effects that:
the invention provides an aspartase mutant, wherein the amino acid sequence of the aspartase mutant is shown in SEQ ID No. 1. The aspartase mutant is obtained by mutating leucine at position 12 into aspartic acid, tyrosine at position 166 into glutamic acid, glutamine at position 169 into glutamic acid and arginine at position 314 into methionine on the basis of aspartase derived from Bacillus sp.YM55-1 (the amino acid sequence is shown as SEQ ID NO. 3). According to the invention, through modifying the original enzyme, the optimum pH of the aspartase mutant is transferred from 9.0 to 8.0, the specific enzyme activity is remarkably improved, the specific enzyme activity can reach 477mU/mg at the pH of 8.0, the capability of the enzyme for synthesizing beta-amino acid is enhanced, and a practical and effective strategy is provided for the industrial production of the enzyme. The mutant is applied to the whole cell transformation production of beta-aminobutyric acid, the transformation is carried out for 12 hours in a 500g/L whole cell transformation system, and the yield can reach 590.8 g/L.
Drawings
FIG. 1 is a diagram showing the synthesis of beta-aminobutyric acid of an original strain and a mutant strain; a: wild type AspB at pH 8.0; b: wild type AspB at pH 9.0; c: mutant strains are under the condition of pH 8.0; d: the mutant strain was at pH 9.0.
Detailed Description
LB culture medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH 7.2.
TY fermentation medium: 0.3g/L ferric ammonium citrate, 0.3g/L anhydrous magnesium sulfate, 2.1g/L citric acid monohydrate, 2.5g/L ammonium sulfate, 4g/L tripotassium phosphate, 8g/L yeast powder, 10g/L glycerol and 12g/L tryptone.
The method for measuring the activity of the aspartase comprises the following steps: the reaction system (200. mu.L) is 300mM ammonia, 100mM Na2HPO4, 300mM crotonic acid, pH is adjusted to 8 by 5M NaOH, a proper amount of enzyme solution is added to start the reaction, the reaction is carried out for 1h at 55 ℃, and the enzyme activity is calculated according to the yield of beta-aminobutyric acid in the reaction.
Definition of enzyme activity: the amount of enzyme required to convert 1. mu. mol crotonic acid per minute into beta-aminobutyric acid is defined as one unit of enzyme activity U. The unit of enzyme activity is U/mL. The specific activity of the enzyme is defined as the unit enzyme activity U/mg of the protein.
TABLE 1 primers required for PCR
Example 1: recombinant expression vector containing gene for coding aspartase mutant and construction of recombinant bacterium
Mutating leucine at position 12 into aspartic acid, mutating tyrosine at position 166 into glutamic acid, mutating glutamine at position 169 into glutamic acid, and mutating arginine at position 314 into methionine, the specific method comprises: the gene shown in SEQ ID No.2 is obtained by constructing mutant plasmids (the reaction system is shown in Table 2 and the reaction conditions are shown in Table 3) by a whole plasmid two-step PCR method by using pET-21a recombinant plasmids (enzyme cutting sites EcoR I and BamH I) containing nucleotide sequences shown in SEQ ID No.4 as a template and sequences shown in Table 1 as primers.
TABLE 2 PCR reaction System
TABLE 3 PCR reaction conditions
And (3) carrying out gel electrophoresis detection on the PCR product, then adding 1 mu L of Dpn I restriction enzyme into 20 mu L of the PCR product to digest the template plasmid, and incubating for 3-4 h at 25 ℃ overnight or 37 ℃. Sucking 5 mul of enzyme digestion products and transforming the enzyme digestion products into escherichia coli BL21(DE3) to obtain corresponding recombinant escherichia coli, coating the recombinant escherichia coli on an LB plate containing ampicillin (100mg/L), culturing overnight at 37 ℃, randomly picking out clones and carrying out colony PCR identification and sequencing verification, wherein the result shows that the recombinant expression vector containing the gene coding the aspartase mutant is successfully transformed into an expression host escherichia coli BL21(DE3) and is named as pET21 a-L12D-Y166E-Q169E-R314M. The strain which is successfully mutated through sequencing verification is pET21a-L12D-Y166E-Q169E-R314M/E.coli BL21, and the bacterial liquid is added with glycerol and preserved in a refrigerator at the temperature of-70 ℃. Sequencing work was done by the Suzhou Kingzhi.
Example 2: expression of recombinant bacterium pET21a-L12D-Y166E-Q169E-R314M/E. coli BL21
Constructed in example 1The recombinant strain pET21a-L12D-Y166E-Q169E-R314M/E.coli BL21 and a control strain pET21a-AspB/E.coli BL21 expressing an unmutated primase BsAspB (primal type, amino acid sequence shown in SEQ ID NO. 3) are respectively inoculated in 10mL LB culture medium containing ampicillin, and shake culture is carried out at 37 ℃ overnight until OD600The strain is transferred to 50mL LB culture medium containing ampicillin according to the inoculum size of 1% on 0.6-0.9 day, cultured for 2-3 h at 37 ℃, and then added with 0.5mM IPTG to induce for 12-16 h at 16 ℃. The cells were collected and disrupted by centrifugation at 8000rpm for 10min at 4 ℃ and the cell disruption supernatant (crude enzyme solution) was collected for subsequent purification.
The purification of aspartase or aspartase mutants was carried out by hot water bath at 60 ℃ for 30min, followed by centrifugation at 12000rmp for 90min to obtain purified enzyme. The resulting purified enzyme was stored at 4 ℃ until use. The purified enzyme solution is analyzed by SDS-PAGE, and the result shows that the electrophoretically pure recombinant aspartase and the electrophoretically pure mutant thereof are obtained.
Example 3: enzyme activity determination of aspartase and HPLC (high Performance liquid chromatography) detection of beta-aminobutyric acid
The original enzyme and the mutant enzyme are tested for enzyme activity under different conditions of 70 ℃ and pH 7.0, 8.0, 9.0 and 10.0, and the content of the product beta-aminobutyric acid is tested by an HPLC method after 1 hour of reaction.
HPLC: mu.L of the reaction mixture was taken and 40. mu.L of 1M NaHCO was added3After mixing, 160. mu.L of 2, 4-dinitrofluorobenzene (20.48mg dissolved in 3mL of acetone) was added, and the mixture was reacted at 60 ℃ in the dark for 1 hour, centrifuged, and filtered through a 0.22. mu.M membrane. Then, sample injection is carried out. A chromatographic column: dimosoil C18(5 μ L, 250mm × 4.6mm), mobile phase: a: 0.1% aqueous formic acid solution, B: 100% acetonitrile, detector: UV Detector, detection wavelength: 360nm, column temperature: 25 ℃, sample introduction: 10 μ L, flow rate: 1.0 mL/min. The process is as follows: 0-22 min, 15% B → 50% B; 22-22.1 min, 50% B → 15% B; 15% B for 22.1-26 min.
The specific enzyme activities of the original enzyme and the mutant enzyme L12D-Y166E-Q169E-R314M are calculated and shown in Table 4. The results showed that the mutant strain had a specific enzyme activity 4.1 times higher than that of the original enzyme at pH 7.0, which was 149.4 mU/mg. The pH of 8.0 becomes the optimum reaction pH of the mutant enzyme, and the multi-site mutation enables the enzyme to have remarkably improved specific enzyme activity under the neutral condition (pH of 8.0), which is about 3.1 times of the original enzyme.
TABLE 4 specific enzyme Activity (mU/mg) of original enzyme and mutant enzyme at different pH
Example 4: kinetic parameters of aspartase original type and mutant thereof
Under standard conditions, enzyme activity is measured by changing the concentration of a substrate in a reaction system, and curve fitting is carried out on enzyme activity data according to a Michaelis-Menten equation in prism software to obtain a corresponding kinetic constant. Kinetic constants in a mixture containing different concentrations of crotonic acid and 2mM MgCl2In 50mM Tris-HCl (pH 8.0) at a substrate concentration of 100mM to 1M. The results show that the Kcat/Km of the original enzyme at pH 9.0 is higher than at low pH, 0.18s-1M-1. The Kcat/Km of the mutant enzyme showed absolute advantage at pH 8.0, 2.6 times the original enzyme pH 9.0 and 5.8 times the original enzyme pH 8.0, which also corresponds to the specific enzyme activity data.
TABLE 5 kinetic parameters of the original and mutant enzymes
Example 5: preparation of beta-aminobutyric acid from aspartase original type and mutant engineering bacteria thereof
Recombinant Escherichia coli was cultured at 100 mg.L-1Streaking ampicillin-resistant LB plate, selecting single colony, inoculating 10mL LB culture medium, culturing at 37 deg.C for 11-12h, inoculating 120mL LB culture medium with 3% (V/V) inoculum size, culturing for 6-8h, inoculating the cultured bacterial liquid as seed into 2L 5L fermentation tank, fermenting at 37 deg.C, pH 7.0, and ventilation 2vvm, gradually increasing rotation speed, and waiting for OD of thallus600Cooling to 25 deg.C at 15-20 deg.C, adding IPTG for induction, culturing for 12-14 hr, terminating fermentation, and centrifuging to collect thallus.
The AspB primitive type and mutant L12D-Y166E-Q169E-R314M engineering bacteria are subjected to pH 8.0 and 9.0 conditionsConversion of crotonic acid substrate. Transformation System 1L initial substrate concentration is 200g/L, the cells obtained are OD in final concentration60050 parts of the mixture was added to the conversion system and reacted at 50 ℃ and 200 rpm. As the reaction proceeds, the substrate crotonic acid gradually decreases, and the product beta-aminobutyric acid is generated. Adding 100g/L of crotonic acid substrate at 5h, 7h and 9h respectively to make the substrate content not less than 100 g/L. The buffer solution is composed of MgCl containing 2mM2The pH of the solution is adjusted to 9.0 by adding 25% ammonia water, and the solution is reacted at 50 ℃ for 12 hours, wherein the yield of beta-aminobutyric acid is shown in figure 1. The results showed that the yield of AspB at pH 8.0 and pH 9.0 for 12h of transformation was 493.3g/L and 458.8g/L, respectively, while the yield of mutant at pH 8.0 and pH 9.0 for 12h of transformation was 590.8g/L and 529.1g/L, 97.5g/L and 70.3g/L higher than the original strain, respectively. This means that the mutant strain achieves lower cost consumption and more efficient and durable transformation, and the aspartase mutant has wide industrial application prospect.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> aspartase mutant with improved enzyme activity and changed optimum pH
<130> BAA210028A
<160> 4
<170> PatentIn version 3.3
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Claims (10)
1. An aspartase mutant, wherein the aspartase mutant comprises an amino acid shown in SEQ ID No. 1.
2. A gene encoding the aspartic acid mutant of claim 1.
3. A recombinant plasmid carrying the gene of claim 2.
4. The recombinant expression vector of claim 3, wherein the recombinant plasmid uses pET series vectors as starting vectors.
5. A microbial cell expressing the mutant of claim 1, or carrying the recombinant plasmid of claim 3.
6. The recombinant bacterium according to claim 5, wherein the microbial cell is Escherichia coli as a host cell.
7. A method for producing beta-aminobutyric acid, wherein the microbial cell according to claim 5 or 6 is used to transform crotonic acid to produce beta-aminobutyric acid.
8. The method according to claim 7, wherein the microbial cells are added to the reaction system such that the OD of the microbial cells in the reaction system60040-60, and the content of crotonic acid in the reaction system is maintained to be not less than 80 g/L.
9. The method of claim 8, wherein the reaction is carried out at 40-60 ℃ for not less than 8 hours.
10. Use of the aspartase mutant according to claim 1, the gene according to claim 2, or the recombinant bacterium according to any one of claims 5 to 7 in the preparation of beta-amino acids and derivatives thereof.
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