CN104830815B

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

Info

Publication number: CN104830815B
Application number: CN201510295252.6A
Authority: CN
Inventors: 陈坚; 刘龙; 李江华; 堵国成; 侯颖; 顾汉章; 徐堃
Original assignee: JIANGSU HAN KUANG BIOLOGICAL ENGINEERING Co Ltd; Jiangnan University
Current assignee: JIANGSU HAN KUANG BIOLOGICAL ENGINEERING Co Ltd; Jiangnan University
Priority date: 2015-06-02
Filing date: 2015-06-02
Publication date: 2018-02-23
Anticipated expiration: 2035-06-02
Also published as: CN104830815A

Abstract

The invention discloses a kind of method that α phenylpyruvic acids are efficiently produced using resting cell, belong to field of fermentation engineering.The present invention is transformed L amino acid deaminases, obtains the L amino acid deaminase mutants that activity improves.Cell transformation L phenylalanines production PPA is organized with the recombinant bacterium containing L amino acid deaminase mutants entirely, L phenylalanines production PPA conversion ratio significantly improves.The resting cell system that the present invention establishes, solve the problems, such as that the problems such as the step of chemical method synthesizes PPA is cumbersome, yield is low, pollution environment and enzymatic conversion method production PPA conversion ratio are low, pollution-free, high yield, One-step production PPA are realized, certain theoretical foundation has been established for follow-up industrialized production.

Description

A kind of method that α-phenylpyruvic acid is efficiently produced using resting cell

Technical field

The present invention relates to a kind of method that α-phenylpyruvic acid is efficiently produced using resting cell, belongs to Fermentation Engineering neck Domain.

Background technology

α-phenylpyruvic acid (PPA) has many applications, can be used to the heterocyclic compound of synthesizing antineoplastic medicament, can be used as antioxygen Agent and promotion wound healing.It is the raw material for synthesizing D-phenylalanine, D-phenylalanine is chirality pharmaceutical intermediate compound.PPA is also Available for phenyllactic acid is prepared, phenyllactic acid can be used as antibacterial material.Traditional PPA productions use chemical method, the major defect of chemical method Selectivity is a lack of, using poisonous cyanide and metallic copper, low yield, and pollutes environment.Enzyme and whole-cell biocatalyst More and more it is used for industrialized production.

L-amino acid deaminase (EC1.4.3.2) is catalyzed l-amino acid oxidative deamination, generates corresponding 2-ketoacid and ammonia, more For flavoprotein, dimeric structure.Proteus mirabilis KCTC2566 have two kinds of l-amino acid deaminases, and one kind has Extensive substrate specificity, aliphatic and aromatic l-amino acid can be catalyzed, especially there is greater catalytic to L-phenylalanine Activity；Another kind have it is substrate limiting, only there is catalytic activity, especially L-Histidine to basic amino acid.

Resting cell has many advantages compared to separation enzyme：Easily prepare, cost is low；It is more stable, it is easy to use；Without dirt Dye, accessory substance are few.We have successfully constructed E. coli whole cell catalyst in research before, and Escherichia coli are entered Row transformation, has knocked out degraded PPA three kinds of amino acid deaminases, conversion ratio 39%.

The content of the invention

The invention solves first technical problem be to provide a kind of l-amino acid deaminase mutant, be with nucleotides The enzyme of gene code of the sequence as shown in SEQ ID NO.1 is transformed to obtain as parent, the l-amino acid deaminase mutant It is D165G/S179L/F263V/L336V or D165K/F263M/L336M or D165K or F263M or L336M.

The mutant can be obtained by fallibility PCR and/or rite-directed mutagenesis.

The invention solves second technical problem be to provide it is complete thin containing the l-amino acid deaminase mutant Born of the same parents' catalyst, it is that the genetic transformation Escherichia coli for encoding the l-amino acid deaminase mutant is obtained into recombinant bacterium, gained weight Group bacterium can be used for Efficient Conversion L-phenylalanine production PPA.

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

The invention solves the 3rd technical problem be to provide using the whole-cell catalyst conversion production α-phenylpropyl alcohol The method of ketone acid, by L-phenylalanine 10-12g/L, whole-cell catalyst 1.2-1.5g/L, at 20mM Tris-HCl (pH8.0) In, in 36-37 DEG C, 200-220rpm conversions 6h.

The whole-cell catalyst is wet thallus.

In one embodiment of the invention, the acquisition of the whole-cell catalyst, it is by containing l-amino acid deamination The recombination bacillus coli of enzyme mutant is connected in 1.8L fermentation mediums by 1-2% inoculum concentrations, speed of agitator, throughput and temperature Respectively 400rpm, 1.0vvm and 28 DEG C, work as OD₆₀₀Reach 0.6-0.8, add 0.4mM IPTG induction l-amino acid deaminases Expression.After inducing 5-7h, 8,000rpm low-temperature centrifugation 10-15min, thalline is collected, buffered with 20mM Tris-HCl (pH8.0) Liquid is washed twice of thalline and produced.

In one embodiment of the invention, fermentation medium：Peptone 12g, yeast extract 24g, glycerine 4mL. Autoclaving after each component dissolving.60 DEG C are cooled to, then adds the 17mmol/L KH of 100mL sterilizings₂PO₄And 72mmol/L K₂HPO₄Solution (2.31g KH₂PO₄With 12.54g K₂HPO₄It is soluble in water, final volume 100mL, autoclaving).

In one embodiment of the invention, resting cell system is：L-phenylalanine 10g/L, whole-cell catalytic Agent 1.2g/L, react and carried out in 20mM Tris-HCl (pH8.0), 37 DEG C, 200rpm conversions 6h.

Beneficial effects of the present invention：The present invention is successfully realized the transformation of l-amino acid deaminase, improves and utilizes large intestine Bacillus recombinant cell conversion L-phenylalanine production PPA conversion ratio.Contain l-amino acid deaminase mutant D165G/ S179L/F263V/L336V recombinant bacterium resting cell L-phenylalanine production PPA conversion ratio is 81%.Contain L- amino Sour deaminase mutant D165K/F263M/L336M recombinant bacterium resting cell L-phenylalanine production PPA conversion ratio be 100%.The resting cell system that the present invention establishes, solve chemical method synthesis PPA the step of it is cumbersome, yield is low, pollution ring The problem of the problems such as border and enzymatic conversion method production PPA conversion ratio are low, realizes pollution-free, high yield, One-step production PPA, Certain theoretical foundation has been established for follow-up industrialized production.

Embodiment

Materials and methods

Seed culture medium：Peptone 1g, dusty yeast 0.5g, NaCl1g, running water are settled to 100mL.

Fermentation medium：Peptone 12g, yeast extract 24g, glycerine 4mL.Autoclaving after each component dissolving.Cooling To 60 DEG C, then add the 17mmol/L KH of 100mL sterilizings₂PO₄With 72mmol/L K₂HPO₄Solution.

PPA assays：Transformation system is centrifuged, takes the μ L of supernatant 100, adds 3mL ferric trichlorides, spectrophotometer Determine 640nm absorbance.

The fallibility PCR of embodiment 1 is transformed l-amino acid deaminase

Using P.mirabilisKCTC2566 genome as template, clone obtains the gene of encoded L-amino acids deaminase Pma, connection carrier pET-20b (+), using gained recombinant plasmid pET-pma as template, Mutazyme II DNA polymerase Lo-Fi enzymatic amplification pma genes.After PCR primer purifying, digestion, it is connected, construction recombination plasmid, converts with carrier pET-20b (+) E. coli bl21 builds mutant library.The single bacterium colony grown in picking ammonia benzyl resistant panel is inoculated into LB, in 96 orifice plates 37 DEG C of concussions are incubated overnight, and are inoculated into the TB culture mediums of another piece of 96 orifice plates, IPTG induction 5h, determine PPA yield.Primary dcreening operation Afterwards, the high bacterial strain shake flask fermentation secondary screening of PPA yield, further tests substrate conversion efficiency, (substrate conversion efficiency=be converted into PPA's Concentration of substrate/initial substrate concentration * 100%).The bacterial strain of high conversion rate, plasmid is extracted, as next round fallibility PCR or fixed point The template of saturation mutation.

The rite-directed mutagenesis of embodiment 2 is transformed l-amino acid deaminase

The mutational site of the mutant of the high conversion rate obtained to embodiment 1 carries out fixed point saturation mutation one by one.To implement Recombinant plasmid after example 1 is mutated is template, and the mutation of Prime-STAR HS DNA polymerase high-fidelities enzymes and design is drawn Thing one step amplification, DpnI digestion with restriction enzyme template DNAs, then phosphorylation, is connected as full plasmid, converts Escherichia coli BL21。

After two-wheeled transformation, in obtained single mutant, the higher mutant of PPA yield is：D165K, F263M, L336M. These mutational sites are combined, structure complex mutation body D165K/F263M/L336M.L-amino acid containing wild type takes off The recombinant bacterium resting cell PPA of ammonia enzyme and each mutant conversion ratio is as shown in table 1.

Table 1

The preparation of the whole-cell catalyst of embodiment 3 and resting cell process

The recombination bacillus coli BL21 of embodiment 1 and embodiment 2 inoculation seed culture mediums (chloramphenicol 10mg/L), 37 DEG C, 200rpm is incubated overnight.Fermentation carried out in 3LNBS fermentation tanks, 1% inoculum concentration into 1.8L fermentation mediums, speed of agitator, Throughput and temperature are respectively 400rpm, 1.0vvm and 28 DEG C, work as OD₆₀₀Reach 0.6, add 0.4mM IPTG induction L- amino Sour deamination expression of enzymes.After inducing 5h, 8,000rpm low-temperature centrifugation 10min, thalline is collected, it is slow with 20mM Tris-HCl (pH8.0) Fliud flushing washes twice of thalline.Resting cell system is：L-phenylalanine 10g/L, whole-cell catalyst 1.2g/L, reacts in 20mM Carried out in Tris-HCl (pH8.0), 37 DEG C, 200rpm conversions 6h.

Recombinant bacterium containing D165G/S179L/F263V/L336V (SEQIDNO.2) mutant, resting cell L- phenylpropyl alcohol ammonia Acid production PPA conversion ratio is 81%.Recombinant bacterium containing D165K/F263M/L336M (SEQ ID NO.3) mutant, full cell The conversion ratio for converting L-phenylalanine production PPA is 100%.

Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention Enclose being defined of being defined by claims.

Claims

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.