CN107164341A - A kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application - Google Patents

A kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application Download PDF

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CN107164341A
CN107164341A CN201710474277.1A CN201710474277A CN107164341A CN 107164341 A CN107164341 A CN 107164341A CN 201710474277 A CN201710474277 A CN 201710474277A CN 107164341 A CN107164341 A CN 107164341A
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phenylpyruvic
phenylpyruvic acid
acid
genetic engineering
reductase
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李剑芳
袁风娇
刘艳
李雪晴
邬敏辰
李闯
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Jiangnan University
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
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Abstract

The invention discloses a kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application, belong to technical field of bioengineering.Present invention screening first obtains a kind of phenylpyruvic acid reductase that there is catalysis phenylpyruvic acid to produce phenyllactic acid activity, belongs to genetic engineering and field of protein expression.The present invention by the enzyme, express by the successful clone in e. coli bl21, and produces phenyllactic acid applied to whole-cell catalytic phenylpyruvic acid.Make catalytic reaction after 5h, the conversion ratio of substrate reaches that 98.38%, ee values are 99.9%.

Description

A kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application
Technical field
The present invention relates to a kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application, belong to biotechnology neck Domain.
Background technology
Phenyllactic acid (Phenyllactic acid, PLA), also known as the phenylpropionic acid of 2- hydroxyls -3, are class very importantization Compound, has application widely in terms of medicine and biological preservative.Phenyllactic acid have with its derivative danshensu (β- 3,4 one DLA sodium) identical pharmacological function, the level and antiplatelet that can adjust steroids in human body be poly- The activity of collection.Phenyllactic acid is also the important intermediate for synthesizing many medicines, such as：Hypoglycemic agent Englitazone (Englitazone), non-protein amino acid Shi Deding (Statine), anti-AIDS toxin preparation, new antihelmintic PFl022A Deng.In addition, discovered in recent years phenyllactic acid can be as new biological preservative, and there is antimicrobial spectrum widely, it is blue to leather Family name's positive bacteria (such as Hypertrophic Listeria of staphylococcus aureus, monokaryon), Gram-negative bacteria (such as Escherichia coli, Salmonella Bacterium) and eukaryotic microorganisms (such as aspergillus mould) have inhibitory action.And the biological preparation method of phenyllactic acid has high efficiency, height because of it Enantioselectivity, high regioselectivity, reaction condition be gentle, applied widely and environment-friendly etc., and numerous advantages enjoys research Person pays close attention to.Therefore, phenyllactic acid and its enzyme catalyzed and synthesized turn into the focus of Recent study.
, L.plantarum 21B isolated from sour flour dough Lavermicocca etc., the D- benzene breast of generation in 1988 Acid amount is 56mg/L, and this is the report for producing phenyllactic acid on lactic acid bacteria first.Subsequent researcher has found different genera successively Lactic acid bacteria can produce phenyllactic acid, such as Li Xingfeng filters out 112 plants of bacterium from pickles, wherein there is 10 plants of bacterium to produce D- benzene breast The amount of acid is more than 66.4mg/L.But it is due to the limitation of original strain, its amount for producing phenyllactic acid is not general high and different There is larger difference between bacterial strain, it is difficult to reach industrial industrialization requirement.
In the prior art, researcher finds phenylpyruvic acid reductase after catalysis phenylpyruvic acid reaction 12h, phenylpyruvic acid Conversion ratio be 91.3%.Also researcher produces phenyllactic acid using Lactobacillusplantarum CECT-221, excellent Under condition of culture after change, 2L fermentation cylinder for fermentation culture 158h, phenyllactic acid yield is only 0.7g/L.It can thus be seen that The yield for preparing phenyllactic acid using fermentation method is relatively low, and product extracts difficult, and the substrate that biotransformation method prepares phenyllactic acid is uploaded Amount and yield increase, but the reaction time is long, can not still reach the requirement of industrialized production, it is necessary to further exploitation Efficiently reduce the enzyme of ketone acid.
The content of the invention
First purpose of the present invention is to provide a kind of phenylpyruvic acid reductase,Amino acid sequence is such as shown in (a) or (b)：
(a) amino acid sequence is as shown in SEQ ID NO.2；
(b) amino acid sequence in (a) passes through substitution, lacks or adds one or several amino acid and with propiophenone Sour reducing activity as protein derived from (a).
Second object of the present invention is to provide the gene for encoding the phenylpyruvic acid reductase, and its nucleotide sequence is such as Shown in SEQ ID NO.1.
Third object of the present invention is to provide a kind of genetic engineering bacterium, is, using Escherichia coli as host, to express SEQ ID Phenylpyruvic acid reductase gene shown in NO.1.
In one embodiment of the invention, the genetic engineering bacterium with pET-28a (+) be phenylpropyl alcohol described in vector expression Ketone acid reductase gene.
In one embodiment of the present invention, described host is E. coli BL21 (DE3).
In one embodiment of the invention, the genetic engineering bacterium is built as follows：(1) by SEQ ID Genetic fragment shown in NO.1 obtains recombinant plasmid and is named as pUCm- with converting host E.coli JM109 after being connected with pUCm-T T-lpppr；(2) pUCm-T-lpppr and pET-28a (+) plasmid are subjected to double digestion with Nde I and Not I, connected in T4DNA Connect and be attached in the presence of enzyme, obtain recombinant plasmid pET-28a (+)-lpppr；(3) pET-28a (+)-lpppr is transformed into E.coli BL21 (DE3) host.
Fourth object of the present invention is to provide the heterogenetic expression method of the gene, and methods described is by the gene work Journey bacterium is seeded in LB culture mediums, is cultivated in 37 DEG C, rotating speed 220rpm to OD600For 0.6~0.8, plus IPTG is to final concentration of 0.4-0.6mM is induced, and 18~22 DEG C of induced expressions 7~9h。
In one embodiment of the invention, methods described is comprised the following steps that：To contain phenylpyruvic acid reductase base E. coli/pET-28a-Lpppr of cause is production bacterial strain, and fermentation condition is：Picking E.coli/lpppr single bacterium colonies It is inoculated in LB culture mediums of the 2mL containing 100 μ g/mL kanamycins, in 37 DEG C, 220r/min overnight incubations, by 2% inoculum concentration It is inoculated into 100mL LB culture mediums, cultivation temperature is 37 DEG C, rotating speed 220rpm is cultivated to OD600For 0.6-0.8, plus IPTG is extremely Final concentration of 0.4-0.6mM is induced, 18-22 DEG C of induced expression 7-9h.Thalline is collected, 8000rpm centrifugations 5min collects bacterium Body, with 100mM, pH7.0 phosphate buffer carries out washing 2-3 times, is prepared into 100mg/mL bacteria suspension.
The present invention also provides a kind of method for applying the phenylpyruvic acid reductase to prepare phenyllactic acid, be by phenylpyruvic acid also Protoenzyme is added into the reaction system containing glucose and phenylpyruvic acid with the addition of 16~25U/mmol phenylpyruvic acids.
In one embodiment of the invention, the mass ratio of the phenylpyruvic acid and glucose is 1：0.8~1.
In one embodiment of the invention, the phenylpyruvic acid is in the form of enzyme liquid, enzyme powder or genetic engineering bacterium Participate in reaction.
In one embodiment of the invention, the enzyme activity of the genetic engineering bacterium is 2~5U/mg thalline.
In one embodiment of the invention, methods described is using the genetic engineering bacterium as catalyst, with 25mM benzene Pyruvic acid is substrate, and adds 20mM glucose and 50mg wet thallus, is reacted at a temperature of 37 DEG C.
In one embodiment of the invention, the reaction is carried out in pH 7.0~7.5 phosphate buffer.
In one embodiment of the invention, the phosphate buffer is the Na that concentration is 0.1mM2HPO4-NaH2PO4, Buffer solution.
The present invention also provides the phenylpyruvic acid reductase and the genetic engineering bacterium of the expression phenylpyruvic acid reductase exists Food, biology, the application of field of medicaments.
Beneficial effect：The invention provides a kind of phenylpyruvic acid reductase, the conversion effect of phenylpyruvic acid can be significantly improved Rate, makes it in the reaction system of glucose and phenylpyruvic acid substrate, and after 5h reacts, the conversion ratio of substrate reaches 98.38%, e.e. value are 99.9%.
Brief description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of reaction substrate and product；
Fig. 2 is recombinant plasmid pET-28a (+)-Lpppr structure schematic diagram；
Fig. 3 is that conversion reaction is schemed；Wherein, PPA is phenylpyruvic acid；PLA is phenyllactic acid.
Embodiment
Phenylpyruvic acid and phenyllactic acid mark product are purchased from Shanghai sigma companies；Mobile phase is purchased from U.S. TEDIA world reagent Company, performance liquid chromatographic column C-18 (4.6 × 250mm, 5 μm) is purchased from Bischoff Co., Ltds of Germany.Using efficient liquid phase Chromatograph Waters e2695 (Waters, US) are analyzed sample.Mobile phase is containing 0.05% trifluoro second The methanol (B) of acid and the mixed liquor of water (A), gradient elution program is：0~15min by 20%B linear changes to 65%B, 15~ 16min is by 65%B linear changes to 100% and keeps 3min, and 19~20min is by 100%B linear changes to 20%, flow velocity 1mL/min, Detection wavelength 210nm, 30 DEG C of column temperature.Detector is UV-detector, and chromatographic column is C18 posts.Phenylpyruvic acid and benzene The appearance time of lactic acid is as schemed1Shown, the appearance time of phenylpyruvic acid is 11.790min, and the appearance time of phenyllactic acid is 12.831min。
Enzyme activity determination method：Reaction is under the conditions of 30 DEG C, changes of the METHOD FOR CONTINUOUS DETERMINATION 3min in 340nm absorption values.Enzyme activity is determined Justice is：Under conditions of 30 DEG C, p H7.0, the enzyme amount needed for 1 μm of ol NADH of consumption per minute is enzyme-activity unit (1 μ Mol/min=1U)
The acquisition of the Lactobacillus plantarum phenylpyruvic acid reductase gene of embodiment 1 and the structure of expression plasmid
Design specific primer：
LpPPR-F：5’-CATATGATGAAAATTTTAATGTAT-3 ', I containing Nde restriction enzyme site.
LpPPR-R：5’-GCGGCCGCTTAAAAGGCGTGGGCCGT-3 ', I containing Not restriction enzyme site.
Extract the genomic DNA of Lactobacillus plantarum, using Lactobacillus plantarum genomic DNA as template, LpPPR-F and LpPPR- R is primer, enters performing PCR：94 DEG C of denaturation 5min, 30 circulations (94 DEG C of 30s, 50 DEG C of 30s and 72 DEG C of 60s), 72 DEG C of insulations 10min.PCR primer is analyzed through agarose gel electrophoresis, recovery target gene of tapping rubber, and is connected with pUCm-T, Transformed E .coli JM109, through the screening of blue hickie, bacterium solution PCR identifications and DNA sequencing.Correct recombinant plasmid will be sequenced and is named as pUCm-T- lpppr.Correct pUCm-T-lpppr will be sequenced and carries out double digestion with Nde I and Not I with pET-28a (+) plasmid, will tap rubber The digestion products of recovery are attached in the presence of T4DNA ligases, obtain recombinant plasmid pET-28a (+)-lpppr, and right Recombinant plasmid carries out sequencing；PET-28a (+)-lpppr is transformed into E.coli BL21 (DE3).
The induced expression and condition optimizing of the recombinant bacterium of embodiment 2
E.coli BL21-lpppr single bacterium colonies are inoculated in LB culture mediums of the 2mL containing 100 μ g/mL kanamycins, in 37 DEG C, 220r/min overnight incubations；Transferred by 2% inoculum concentration in 100mL LB culture mediums, 37 DEG C of cultures to OD600For 0.6~ When 0.8, the induced expression carried out to destination protein.Inductive condition is：IPTG is added to final concentration 0.4-0.6mmol/L, 18-22 DEG C induction 7-9h.Thalline is collected, with sodium phosphate buffer (Na2HPO4-NaH2PO4, 100mmol/L, pH 7.0) wash 2-3 times, Add same buffer solution suspension and obtain the dense bacteria suspension for 100mg/mL of bacterium.Reacted stupid lactic acid concn is measured, tied Fruit shows that D- phenyllactic acids concentration is up to 7.05~7.79mM.
The preparation of embodiment 3D- phenyllactic acids
400 μ L phenylpyruvic acids (final concentration 25mM) are added in 1.5mL EP pipes, 100 μ L glucose (final concentrations are added For 20mM), add 500 μ L bacteria suspensions (enzyme activity is 19.7U/mg wet thallus), 40 DEG C of reaction 5h.Timing sampling is carried out into methanol Dilution, crosses 0.22 μm of organic filter membrane, enters HPLC and detected after centrifugation.Reaction process curve is as shown in Fig. 2 in reaction 210min Afterwards, phenylpyruvic acid content is less than 5mM, and the conversion ratio of phenylpyruvic acid is 98.38%, e.e. after reaction carries out 5h>99.9%, D- phenyllactic acid yield reaches 4.15g/L.
Reference examples 1
Recombinant bacterium is induced in the way of embodiment 2, difference is that it is 16 DEG C to adjust inducing temperature, in remaining condition Under the same conditions, D- phenyllactic acids concentration only 6.55~6.62mM.
Reference examples 2
Recombinant bacterium is induced in the way of embodiment 2, difference is that it is 25 DEG C to adjust inducing temperature, in remaining condition Under the same conditions, D- phenyllactic acids concentration only 4.35~5.01mM.
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, can do various changes and modification, therefore the protection model of the present invention Enclose what be defined with claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 992
<212> DNA
<213>Artificial sequence
<400> 1
atgaaaattt taatgtatgg tgttcgcgat gatgaacagg atgcgattca agcgtgggct 60
gcggctcatc aaattcaagt agatacgaat gatctggaat tccatccaga aactgccgac 120
ctggtcaagg gttatgacgg cttggtcatt cagcaacgca gtccaattgg tggtgatgct 180
agcttatacc aatcattagc tgcggccggg ttaaaacaac taacaagtcg tacggctggt 240
gtggatacaa tcgatattcc ggcagccaag gcggctggtc ttgtggtcac gaacgtccca 300
gcctattcgc ctaattcagt tgctgagatg tcggtggcac aaaccatgcg cttgattcgc 360
aacttggaaa tgtttgatca acgaatcagc caacagaatt ttcagtgggc gggactgcaa 420
gcacgagaaa ttcgatcatt aaccgttggt attattggtg cgggtcgtat tggtggcacg 480
gccgctcgac ttttccacgg gttaggtgcc aaggtgattg cctatgacgt ggtacgccat 540
cctgaactcg aagacgtttt aacctacgtt gacactaagg aagacctatt gcggcaggcg 600
gatgtcgttg atttacacgt ggatttgaat gagacctctg cggggttgat cgatgcggca 660
gccttgaagt taatgaagac ggatgcctac cttatcaacg cctcacgcgg accagtgatt 720
ctcaccgatg atcttgtggc agcgctaaag gccggtgaaa tcgccggttg tgcactcgat 780
acggttgagg gtgaaaatgc gttatttaac caaaatcatc agggtgaggt gcttcaagat 840
accaacgtgg cacaattaat gcagatgccc aacgtcatta tcacgccaca cgtgggtttc 900
tatacgaatt tagcgatcaa aaacatggtc gatattagcc tggatgacgt cttagaattc 960
taaatggtga aacgacggcc cacgcctttt aa 992
<210> 2
<211> 330
<212> PRT
<213>Artificial sequence
<400> 2
Met Lys Ile Leu Met Tyr Gly Val Arg Asp Asp Glu Gln Asp Ala Ile
1 5 10 15
Gln Ala Trp Ala Ala Ala His Gln Ile Gln Val Asp Thr Asn Asp Leu
20 25 30
Glu Phe His Pro Glu Thr Ala Asp Leu Val Lys Gly Tyr Asp Gly Leu
35 40 45
Val Ile Gln Gln Arg Ser Pro Ile Gly Gly Asp Ala Ser Leu Tyr Gln
50 55 60
Ser Leu Ala Ala Ala Gly Leu Lys Gln Leu Thr Ser Arg Thr Ala Gly
65 70 75 80
Val Asp Thr Ile Asp Ile Pro Ala Ala Lys Ala Ala Gly Leu Val Val
85 90 95
Thr Asn Val Pro Ala Tyr Ser Pro Asn Ser Val Ala Glu Met Ser Val
100 105 110
Ala Gln Thr Met Arg Leu Ile Arg Asn Leu Glu Met Phe Asp Gln Arg
115 120 125
Ile Ser Gln Gln Asn Phe Gln Trp Ala Gly Leu Gln Ala Arg Glu Ile
130 135 140
Arg Ser Leu Thr Val Gly Ile Ile Gly Ala Gly Arg Ile Gly Gly Thr
145 150 155 160
Ala Ala Arg Leu Phe His Gly Leu Gly Ala Lys Val Ile Ala Tyr Asp
165 170 175
Val Val Arg His Pro Glu Leu Glu Asp Val Leu Thr Tyr Val Asp Thr
180 185 190
Lys Glu Asp Leu Leu Arg Gln Ala Asp Val Val Asp Leu His Val Asp
195 200 205
Leu Asn Glu Thr Ser Ala Gly Leu Ile Asp Ala Ala Ala Leu Lys Leu
210 215 220
Met Lys Thr Asp Ala Tyr Leu Ile Asn Ala Ser Arg Gly Pro Val Ile
225 230 235 240
Val Thr Asp Asp Leu Val Ala Ala Leu Lys Ala Gly Glu Ile Ala Gly
245 250 255
Cys Ala Leu Asp Thr Val Glu Gly Glu Asn Ala Leu Phe Asn Gln Asn
260 265 270
His Gln Gly Glu Val Leu Gln Asp Thr Asn Val Ala Gln Leu Met Gln
275 280 285
Met Pro Asn Val Ile Ile Thr Pro His Val Gly Phe Tyr Thr Asn Leu
290 295 300
Ala Ile Lys Asn Met Val Asp Ile Ser Leu Asp Asp Val Leu Ala Ile
305 310 315 320
Leu Asn Gly Glu Thr Thr Ala His Ala Phe
325 330
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
catatgatga aaattttaat gtat 24
<210> 4
<211> 26
<212> DNA
<213>Artificial sequence
<400> 4
gcggccgctt aaaaggcgtg ggccgt 26

Claims (10)

1. a kind of phenylpyruvic acid reductase, it is characterised in that amino acid sequence is such as shown in (a) or (b)：
(a) amino acid sequence is as shown in SEQ ID NO.2；
(b) amino acid sequence in (a) passes through substitution, lacks or adds one or several amino acid and has phenylpyruvic acid also Former activity as protein derived from (a).
2. encode the gene of the phenylpyruvic acid reductase described in claim 1.
3. a kind of genetic engineering bacterium, it is characterised in that using Escherichia coli as host, the propiophenone shown in expression SEQ ID NO.2 Sour reductase.
4. genetic engineering bacterium according to claim 3, it is characterised in that with pET-28a (+) for carrier.
5. the genetic engineering bacterium according to claim 3 or 4, it is characterised in that host is E. coli BL21 (DE3)。
6. build the method for genetic engineering bacterium described in claim 5, it is characterised in that comprise the following steps：(1) by SEQ ID Genetic fragment shown in NO.1 obtains recombinant plasmid and is named as pUCm-T- with being converted after being connected with pUCm-T to E.coli JM109 lpppr；(2) pUCm-T-lpppr and pET-28a (+) plasmid are subjected to double digestion with Nde I and Not I, in T4DNA connections It is attached in the presence of enzyme, obtains recombinant plasmid pET-28a (+)-lpppr；(3) pET-28a (+)-lpppr is transformed into E.coli BL21(DE3)。
7. a kind of method for producing phenylpyruvic acid reductase, it is characterised in that by phenylpyruvic acid reductase described in expression claim 1 Genetic engineering bacterium be seeded in culture medium, culture is to OD600For 0.6~0.8, final concentration of 0.4-0.6mM IPTG is added, In 18~22 DEG C of 7~9h of induced expression.
8. the method that phenylpyruvic acid reductase described in a kind of application claim 1 prepares D- phenyllactic acids, it is characterised in that be by benzene Pyruvic acid reductase is added to the reaction system containing glucose and phenylpyruvic acid with the addition of 16~25U/mmol phenylpyruvic acids In, the mass ratio of the phenylpyruvic acid and glucose is 1：0.8~1.
9. method according to claim 8, it is characterised in that the phenylpyruvic acid is with enzyme liquid, enzyme powder or genetic engineering The form of bacterium participates in reaction.
10. genetic engineering bacterium described in claim 3 food, biology, field of medicaments application.
CN201710474277.1A 2017-06-21 2017-06-21 A kind of phenylpyruvic acid reductase in Lactobacillus plantarum source and application Pending CN107164341A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
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CN113308447A (en) * 2021-05-31 2021-08-27 西南大学 Application of arabidopsis UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactyl glucose
CN113308447B (en) * 2021-05-31 2022-09-30 西南大学 Application of arabidopsis UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactyl glucose
CN114369626A (en) * 2022-01-20 2022-04-19 浙江工业大学 Method for synthesizing phenyllactic acid by catalytic conversion of biocatalyst

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