CN104830815B - A kind of method that α phenylpyruvic acids are efficiently produced using resting cell - Google Patents

Abstract

The invention discloses a method for efficiently producing α-phenylpyruvate by transforming whole cells, and belongs to the field of fermentation engineering. The invention transforms the L-amino acid deaminase to obtain the L-amino acid deaminase mutant with improved activity. The transformation rate of L-phenylalanine to produce PPA is significantly improved by transforming L-phenylalanine with the whole group of cells of the recombinant bacteria containing the L-amino acid deaminase mutant. The whole-cell transformation system established by the present invention solves the problems of cumbersome steps, low yield, and polluted environment for chemically synthesizing PPA and the low conversion rate of PPA produced by enzymatic transformation, and realizes pollution-free, high-yield, one-step method The production of PPA has laid a certain theoretical foundation for the subsequent industrial production.

Description

A method for efficiently producing α-phenylpyruvate by whole cell transformation

technical field

The invention relates to a method for efficiently producing α-phenylpyruvate by transforming whole cells, belonging to the field of fermentation engineering.

Background technique

α-Phenylpyruvate (PPA) has many applications. It can be used to synthesize heterocyclic compounds of antineoplastic drugs, as an antioxidant and to promote wound healing. It is the raw material for the synthesis of D-phenylalanine, which is a chiral drug intermediate. PPA can also be used to prepare phenyllactic acid, which can be used as an antibacterial substance. Traditional PPA production adopts chemical method. The main disadvantage of chemical method is the lack of selectivity, the use of toxic cyanide and metal copper, low yield, and environmental pollution. Enzymes and whole-cell biocatalysts are increasingly used in industrial production.

L-amino acid deaminase (EC1.4.3.2) catalyzes the oxidative deamination of L-amino acids to generate corresponding α-keto acids and ammonia, mostly flavoproteins with a dimer structure. Proteus mirabilis KCTC2566 has two L-amino acid deaminases, one has broad substrate specificity and can catalyze aliphatic and aromatic L-amino acids, especially for L-phenylalanine with high catalytic activity; the other The species is substrate-limited and only catalytically active towards basic amino acids, especially L-histidine.

Whole-cell transformation has many advantages over isolate enzymes: easy preparation and low cost; more stable and convenient to use; no pollution and less by-products. In previous studies, we have successfully constructed a whole-cell catalyst of E. coli, and transformed E. coli to knock out the three amino acid deaminases that degrade PPA, with a conversion rate of 39%.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a mutant of L-amino acid deaminase, which is obtained by transforming the enzyme encoded by the gene whose nucleotide sequence is shown in SEQ ID NO.1 as a parent, said L-amino acid deaminase - the amino acid deaminase mutant is D165G/S179L/F263V/L336V or D165K/F263M/L336M or D165K or F263M or L336M.

Said mutants can be obtained by error-prone PCR and/or site-directed mutagenesis.

The second technical problem to be solved by the present invention is to provide a whole-cell catalyst containing the L-amino acid deaminase mutant, which is to transform the gene encoding the L-amino acid deaminase mutant into Escherichia coli to obtain a recombinant bacterium, The obtained recombinant bacteria can be used to efficiently transform L-phenylalanine to produce PPA.

In one embodiment of the present invention, the Escherichia coli is E. coli BL21(DE3).

The third technical problem to be solved by the present invention is to provide a method for converting and producing α-phenylpyruvate using the whole-cell catalyst, by mixing L-phenylalanine 10-12g/L and whole-cell catalyst 1.2-1.5g/L , in 20mM Tris-HCl (pH8.0), at 36-37°C, 200-220rpm for 6h.

The whole cell catalyst is wet bacteria.

In one embodiment of the present invention, the whole cell catalyst is obtained by connecting recombinant Escherichia coli containing L-amino acid deaminase mutants into 1.8L fermentation medium according to 1-2% inoculum size, stirring The rotational speed, air flow and temperature were 400rpm, 1.0vvm and 28°C, respectively. When the OD ₆₀₀ reached 0.6-0.8, 0.4mM IPTG was added to induce the expression of L-amino acid deaminase. After induction for 5-7 hours, centrifuge at 8,000 rpm for 10-15 minutes at low temperature, collect the bacteria, and wash the bacteria twice with 20 mM Tris-HCl (pH8.0) buffer solution.

In one embodiment of the present invention, fermentation medium: peptone 12g, yeast extract 24g, glycerin 4mL. The components are dissolved and autoclaved. Cool to 60°C, add 100mL sterilized solution of 17mmol/L KH ₂ PO ₄ and 72mmol/L K ₂ HPO ₄ (dissolve 2.31g of KH ₂ PO ₄ and 12.54g of K ₂ HPO ₄ in water, the final volume is 100 mL, autoclaved).

In one embodiment of the present invention, the whole cell transformation system is: L-phenylalanine 10g/L, whole cell catalyst 1.2g/L, the reaction is carried out in 20mM Tris-HCl (pH8.0), 37 ℃ , 200rpm conversion 6h.

Beneficial effects of the present invention: the present invention successfully realizes the transformation of L-amino acid deaminase, and improves the conversion rate of producing PPA by transforming L-phenylalanine with Escherichia coli recombinant cells. The conversion rate of whole cells of recombinant bacteria containing L-amino acid deaminase mutant D165G/S179L/F263V/L336V to L-phenylalanine to produce PPA is 81%. The conversion rate of whole cells of recombinant bacteria containing L-amino acid deaminase mutant D165K/F263M/L336M to transform L-phenylalanine to produce PPA is 100%. The whole-cell transformation system established by the present invention solves the problems of cumbersome steps, low yield, and polluted environment for chemically synthesizing PPA and the low conversion rate of PPA produced by enzymatic transformation, and realizes pollution-free, high-yield, one-step method The production of PPA has laid a certain theoretical foundation for the subsequent industrial production.

detailed description

Materials and Methods

Seed medium: peptone 1g, yeast powder 0.5g, NaCl 1g, tap water to 100mL.

Fermentation medium: peptone 12g, yeast extract 24g, glycerol 4mL. The components are dissolved and autoclaved. After cooling to 60°C, add 100 mL of a sterilized solution of 17 mmol/L KH ₂ PO ₄ and 72 mmol/L K ₂ HPO ₄ .

Determination of PPA content: Centrifuge the transformation system, take 100 μL of supernatant, add 3 mL of ferric chloride, and measure the absorbance at 640 nm with a spectrophotometer.

Example 1 Error-prone PCR transforms L-amino acid deaminase

Using the genome of P. mirabilisKCTC2566 as a template, the gene pma encoding L-amino acid deaminase was cloned, connected to the vector pET-20b(+), and the obtained recombinant plasmid pET-pma was used as a template, and Mutazyme II DNA polymerase low-fidelity enzyme amplification increase pma gene. After the PCR product was purified and digested, it was ligated with the vector pET-20b(+) to construct a recombinant plasmid, and transformed into Escherichia coli BL21 to construct a mutant library. Pick a single colony grown on the ampicillin-resistant plate and inoculate it into LB, culture it overnight in a 96-well plate with shaking at 37°C, inoculate it into another 96-well plate in TB medium, induce it with IPTG for 5 hours, and measure the PPA production. After the primary screening, the strains with high PPA production were re-screened by shake flask fermentation, and the substrate conversion rate was further tested, (substrate conversion rate=substrate concentration converted into PPA/initial substrate concentration*100%). For the strains with high transformation rate, extract the plasmid and use it as a template for the next round of error-prone PCR or site-directed saturation mutation.

Example 2 Site-directed mutagenesis transforms L-amino acid deaminase

Site-directed saturation mutation was performed on the mutation sites of the mutants with high conversion rate obtained in Example 1 one by one. Using the mutated recombinant plasmid in Example 1 as a template, Prime-STAR HS DNA polymerase high-fidelity enzyme and designed mutation primers were amplified in one step, and the template DNA was digested with DpnI restriction endonuclease, then phosphorylated, and connected into a complete plasmid. Escherichia coli BL21 was transformed.

After two rounds of transformation, among the single mutants obtained, the mutants with higher PPA production were: D165K, F263M, and L336M. These several mutation sites were combined to construct the compound mutant D165K/F263M/L336M. Table 1 shows the conversion rates of whole cells transformed with recombinant bacteria containing wild-type L-amino acid deaminase and various mutants into PPA.

Table 1

Embodiment 3 preparation of whole cell catalyst and whole cell transformation process

The recombinant Escherichia coli BL21 of Example 1 and Example 2 were inoculated with seed medium (chloramphenicol 10 mg/L), and cultivated overnight at 37° C. and 200 rpm. Fermentation was carried out in a 3L NBS fermenter, 1% inoculum was added to 1.8L fermentation medium, the stirring speed, ventilation volume and temperature were 400rpm, 1.0vvm and 28°C, respectively. When the _OD600 reached 0.6, 0.4mM IPTG was added to induce L- Amino acid deaminase expression. After 5 hours of induction, centrifuge at 8,000 rpm for 10 minutes at low temperature, collect the bacterial cells, and wash the bacterial cells twice with 20 mM Tris-HCl (pH 8.0) buffer solution. The whole cell transformation system is: L-phenylalanine 10g/L, whole cell catalyst 1.2g/L, the reaction is carried out in 20mM Tris-HCl (pH8.0), 37°C, 200rpm transformation for 6h.

For the recombinant bacteria containing the D165G/S179L/F263V/L336V (SEQ ID NO.2) mutant, the transformation rate of the whole cell into L-phenylalanine to produce PPA is 81%. For the recombinant bacteria containing the D165K/F263M/L336M (SEQ ID NO.3) mutant, the transformation rate of whole cells into L-phenylalanine to produce PPA is 100%.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. a kind of l-amino acid deaminase mutant, it is characterised in that be the base with nucleotide sequence as shown in SEQ ID NO.1 Because the enzyme of coding is transformed to obtain as parent, the l-amino acid deaminase mutant is D165K/F263M/L336M or D165K Or F263M or L336M.

2. encode the gene of mutant described in claim 1.

3. application of the l-amino acid deaminase mutant described in claim 1 in α-phenylpyruvic acid is produced.

4. the whole-cell catalyst containing l-amino acid deaminase mutant described in claim 1, it is characterised in that be to encode The genetic transformation Escherichia coli of the l-amino acid deaminase mutant obtains recombinant bacterium, using recombinant bacterium as whole-cell catalytic Agent.

5. whole-cell catalyst according to claim 4, it is characterised in that the Escherichia coli are E.coli BL21 (DE3)。

6. the method for the application claim 4 or 5 whole-cell catalyst conversion L-phenylalanine production α-phenylpyruvic acid, it is special Sign is, by L-phenylalanine 10-12g/L, whole-cell catalyst 1.2-1.5g/L, in pH8.0 20mM Tris-HCl, In 36-37 DEG C, 200-220rpm conversions 6h.

7. according to the method for claim 6, it is characterised in that the whole-cell catalyst is wet thallus.

8. according to the method for claim 6, it is characterised in that the acquisition of the whole-cell catalyst, be by containing L- ammonia The recombination bacillus coli of base acid deaminase mutant is connected in 1.8L fermentation mediums by 1-2% inoculum concentrations, speed of agitator, ventilation Amount and temperature are respectively 400rpm, 1.0vvm and 28 DEG C, work as OD₆₀₀Reach 0.6-0.8, add 0.4mM IPTG induction L- amino Sour deamination expression of enzymes；After inducing 5-7h, 8,000rpm low-temperature centrifugation 10-15min, thalline is collected, with 20mM pH8.0 Tris- HCl buffer solutions are washed twice of thalline and produced.

9. according to the method for claim 8, it is characterised in that fermentation medium：Peptone 12g, yeast extract 24g, Glycerine 4mL, autoclaving after each component dissolving, 60 DEG C are cooled to, then add the 17mmol/L KH of 100mL sterilizings₂PO₄With 72mmol/L K₂HPO₄Solution.

10. according to the method for claim 6, it is characterised in that resting cell system is：L-phenylalanine 10g/L, entirely Cell catalyst 1.2g/L, react and carried out in pH 8.0 20mM Tris-HCl, 37 DEG C, 200rpm conversions 6h.