CN106754796B - A kind of oxidizing ferment and its application - Google Patents
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
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Abstract
The present invention relates to a kind of acquisition of L- alpha-hydroxy acid oxidase gene from yellowish-brown pseudomonad (Pseudomonas fulva) and its clonal expressions, belong to bioengineering field.Its substrate specificity is disclosed, while the L- alpha-hydroxy acid oxidizing ferment can aoxidize (S)-alpha-hydroxy acid ester, can be applied to the preparation of optical voidness (R)-alpha-hydroxy acid ester.
Description
Technical field
A kind of L- alpha-hydroxy acid oxidizing ferment of clonal expression of the present invention, and disclose its nucleotide sequence and amino acid sequence and
Zymologic property and application belong to industrial microorganism field.
Background technique
L- alpha-hydroxy acid oxidizing ferment (L- α-hydroxyacid oxidase) is a kind of dehydrogenase with FMN (FAD) for coenzyme
(Aliphatic l-α-hydroxyacid oxidase from rat livers purification and
Properties. Biochimica et Biophysica Acta (BBA)-Enzymology 1968,167:9-22), usually
It include glycolate oxidase (glycolate oxidase) (Preparation and some properties of
crystalline glycolic acid oxidase of spinach.J.Biol.Chem.1958,231(1):135–57)、
Pfansteihl oxidizing ferment (L-lactate oxidase) (Conversion of L-lactate oxidase to a long
chain alpha- hydroxyacid oxidase by site-directed mutagenesis of alanine 95to
glycine.J Biol Chem.19968；271(45):28300-28305).It can be used for measuring containing for lactic acid in biosensor
Amount, or oxidation Pfansteihl produce pyruvic acid.Also there is the preparation (Chinese patent for being used for optical voidness alpha-hydroxy acid
201210109290.4)
So far, in pseudomonas putida (Pseudomonas putida), aerococcus viridans
(Aerococcus viridians), streptococcus (Streptococcus sp.), Pediococcus (Pediococcus
Sp.), Lactococcus lactis (Lactococus lactis), edwardsiella tarda (Edwardsiella tarda), shame dirt branch
Bacillus (Mycobacterium smegmatis), zymomonas mobilis (Zymomonas mobilis) and peroxidating acetobacter
Clonal expression has obtained Pfansteihl oxidizing ferment in bacteriums such as (Acetobacter peroxidans).Pfansteihl oxidizing ferment is also one
It is detected in a little fungies, such as geotrichum candidum (Geotrichum candidum) and Yarrowia lipolytica (Yarrowia
lipolytica).(Search for Lactate Oxidase Producer Microorganisms, Applied
Biochemistry and Microbiology,2007,43(2)178–181)
The clonal expression from Kerstersia gyiorum DSM 16618 goes out a kind of novel L- α-hydroxyl to the present invention for the first time
Acid oxidase, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, but also can aoxidize (S)-alpha-hydroxy acid ester, can be applied to optical voidness
(R) preparation of-alpha-hydroxy acid ester and (R)-alpha-hydroxy acid.
Summary of the invention
Present invention clone from yellowish-brown pseudomonad (Pseudomonas fulva) has obtained a kind of L- alpha-hydroxy acid oxidizing ferment
Gene disclose its relevant enzymatic property, and carried out application study using colibacillus engineering heterogenous expression.
Technical scheme is as follows:
1, bacterial strain
The source bacterial strain of L- alpha-hydroxy acid oxidase gene of the present invention are as follows: Pseudomonas fulva ATCC 31418, purchase
From U.S.'s ATCC strain library.
2, the clone of L- alpha-hydroxy acid oxidizing ferment
Extract 31418 phage gene group total DNA of Pseudomonas fulva ATCC.Design specific primer, application
PCR method amplifies L- Α-hydroxy acid oxidase full length gene encoder block sequence.And construction recombination plasmid.
3, L- alpha-hydroxy acid Oxidase Expression and purifying
Recombinant plasmid is imported in E.coli BL21 (DE3), inducing expression.Crude enzyme liquid is obtained after bacterial cell disruption, after purification
It is freeze-dried spare.
4, the characterization analysis of L- alpha-hydroxy acid oxidizing ferment
Influence of the pH to L- alpha-hydroxy acid oxidizing ferment enzyme activity of the present invention is studied by substrate of Pfansteihl.
Influence of the temperature to L- alpha-hydroxy acid oxidizing ferment enzyme activity of the present invention is studied by substrate of Pfansteihl.
The substrate specificity of L- alpha-hydroxy acid oxidizing ferment is analyzed: substrate used has Pfansteihl, glycolic, L- phenyllactic acid, L-
Tartaric acid, L MALIC ACID, L- para hydroxybenzene lactic acid, L- danshensu.
Enzyme activity determination method are as follows: according to Characterization of a Lactate Oxidase from a
Strain of Gram Negative Bacterium from Soil, Applied Biochemistry and
Biotechnology,56, 1996,278-288.The method carries out.
5, L- alpha-hydroxy acid oxidizing ferment splits the alpha-hydroxy acid ester of mixed
The method of resolution of alpha-carboxylic esters (alpha-hydroxy esters) are as follows: take 0.1 gram of purified enzyme in 50 mL tri-
In the bottle of angle, it is added dissolved in the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, is converted in 30 DEG C, 150rpm shaking bath
16h, liquid-phase chromatographic analysis supernatant after conversion.(S) Alpha-hydroxy in-alpha-hydroxy acid ester, which is dehydrogenated, is oxidized to corresponding 2-ketoacid
Ester, (R)-alpha-hydroxy acid ester are not oxidized.
Product (R)-alpha-hydroxy acid ester optical purity is evaluated by enantiomeric excess value (%e.e):
Enantiomeric excess value %e.e=[(SR-SS)/(SR+SS)] × 100%
(R)-alpha-hydroxy acid ester yield (%)=(SR/S0) × 100%
S in formulaRFor the peak area of (R)-enantiomer after reaction, SSFor reaction after (S)-enantiomer liquid chromatogram peak area,
S0For the sum of the liquid chromatogram peak area of (R)-and (S)-enantiomer before reaction.
Product measures liquid phase chromatogram condition are as follows: Chiralcel OD-H chiral column (4.6 × 250mm), mobile phase volume ratio
For n-hexane: isopropanol: trifluoroacetic acid=80:20:0.1, flow velocity 0.5mL/min, 25 DEG C of column temperature, Detection wavelength 210nm,
20 μ L of sample volume.
The alpha-hydroxy acid ester is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, benzene cream
Isopropyl propionate, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene isopropyl lactate, mandelic acid norbornene ester, almond isopropyl propionate, Radix Salviae Miltiorrhizae
Plain asarum alcohol ester, lactic acid norbornene ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
The alpha-hydroxy acid ester, according to Chinese patent 200610042787.3,201410180490.8,
201410175950.8 the method synthesis announced with 20140699506.6.
Originally deliver bright usefulness: clone has obtained a kind of L- α-from Pseudomonas fulva ATCC 31418
Hydroxy acid oxidase, the enzyme can aoxidize (S)-alpha-hydroxy acid and (S)-alpha-hydroxy acid ester, can be used for prepare with scale chiral purity (R)-α-
Carboxylic esters have important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of L- alpha-hydroxy acid oxidase gene of the present invention and colibacillus engineering construct.
1, the extraction of Pseudomonas fulva ATCC 31418DNA
31418 bacterial strain of Pseudomonas fulva ATCC is cultivated into 12h, 12,000 rmp/min in LB culture medium
Centrifugation 10min obtains thallus, operates using bacterial genomes DNA extraction agent box (TaKaRa company) according to it and extracts thallus base
Because of a group total DNA, it is spare to put refrigerator.
2, prepared by E. coli competent
(1) inoculation E.coli DH5 α and BL21 (DE3) is respectively in the 250mL shaking flask containing 20mL LB culture medium, and 37
DEG C, 200rpm/min overnight incubation.
(2) it is inoculated in 50mL LB culture medium by 1% inoculum concentration, 37 DEG C of cultures to OD600About 0.6 (about 2~3h).
(3) bacterium solution is transferred in the centrifuge tube of 50mL pre-cooling, places 30min, 8000 rpm/min, 4 DEG C of centrifugations on ice
5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl of 5mL pre-cooling is added2Solution makes thallus suspend, and places 20min on ice,
8000rpm/min, 4 DEG C of centrifugation 5min.It is repeated 2 times.
(5) supernatant is abandoned, the 0.1mol/L CaCl of 1.5mL pre-cooling is added2Solution (contains 15% glycerol), gently suspension thalline,
Then the packing of 100 μ L bacterium solutions is added by each centrifuge tube (1.5mL), -70 DEG C of Storage in refrigerator are spare.
3, the clone of L- alpha-hydroxy acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCGTGAAAGAATCATCGACTCCCCGGC 3'
Primer 2: 5'GCCGTCTAGATGCGTCGAAGAACGGCTTGTGCA 3'
(2) PCR amplification
With two primers synthesized above, using the genomic DNA of Pseudomonas fulva ATCC 31418 as template
Carry out PCR amplification.
Amplification system in this step are as follows:
Amplification program are as follows:
98 DEG C, 10min
98 DEG C, 10sec；55 DEG C, 15sec；72 DEG C, 2min reacts 30 circulations
72 DEG C, 10min
PCR product obtains the gene order of the enzyme after sending Hua Da gene sequencing, as shown in SEQ ID NO:1.According to the base
The amino acid sequence obtained by sequence is as shown in SEQ ID NO:2.
(3) double digestion and connection
II plasmid of pCold and PCR product are subjected to double digestion, digestion system are as follows: 10 × cut buffer, 3 μ l, DNA 4
Each 0.5 μ l of μ l, enzyme BamHI and XbaI, 2 μ l of sterile water totally 30 μ l.Double digestion 1h under 37 DEG C of water-baths.DNA fragmentation is cloned into
On II carrier of pCold, and it is transformed into E.coli DH5 α competent cell.Linked system: 10 × DNA ligase buffer
2.5 μ l, 8 μ l of DNA fragmentation, 2 μ l, T4DNA ligase of carrier DNA 1 μ l, 11.5 μ l of sterile water totally 25 μ l.Under 16 DEG C of water-baths
Connect 12h-16h.
(4) it converts
Step:
1 is added 100 μ l DH5 α competent bacterias in linked system, light to mix, ice bath 30min.
2 are put into 42 DEG C of water-baths of preheating, place 90s and carry out heat shock processing.
3 ice bath 2min immediately.
4 are added the not antibiotic LB culture solution of 1ml, and 37 DEG C of culture 1h make thallus recover.
5 are uniformly coated on thallus on antibiotic LB plate.
6 cultures are grown fine for 24 hours.It chooses single colonie and carries out bacterium colony PCR, recombinant plasmid is extracted in nucleic acid electrophoresis verifying.It will recombination
Plasmid imports in BL21 E. coli competent, saves backup.
Embodiment 2
The present embodiment is the inducing expression of L- alpha-hydroxy acid oxidizing ferment of the present invention and isolates and purifies.
1, plus 500 μ l recombination bacterium solution is into 50ml LB culture solution.37 DEG C of culture 2.5h stand 0.5h at 15 DEG C.Again plus 20
The IPTG of μ l 0.5M, cold-induction culture is for 24 hours at 15 DEG C.Fermentation liquid is centrifuged (8000rmp/min, 10min) and obtains bacterium
Body redissolves thallus with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (20mmol/L, pH 7.0), and Ultrasonic Cell Disruptor is broken,
Centrifugation (8000rmp/min, 10 min) collects supernatant and obtains crude enzyme liquid.
2, the crude enzyme liquid for obtaining step 1 carries out ni-sepharose purification using the operation of 150 protein purification system of AKTA avant,
Elution process are as follows: all put the tetra- root canal road A1, A2, B1, B2 into water, system flow 20ml/min flow velocity is set, carry out
Exhaust.Then system flow 1ml/min, flow path (column position 3), delta pressure are set
0.3, pre-pressure 0.5, Gradient 0, inset A1, fill pillar after water droplet uniformly flows out, balance ten minutes it
A1 is put into conjunction in liquid afterwards, B1 is put into eluent, then primary, balance 20 minutes is exhausted, then loading crude enzyme liquid,
With high concentration imidazole buffer (solution locating for B1) gradient elution destination protein of 500mM, the albumen that will be adsorbed on ion column
Elute the enzyme purified.Enzyme after purification is freeze-dried spare.
Embodiment 3
The present embodiment is the optimum temperature of L- alpha-hydroxy acid oxidizing ferment of the present invention.Using Pfansteihl as substrate, by substrate with
The phosphate buffer that pH is 7.0 is lauched bath 15min in 30-60 DEG C of different temperature condition, measures the enzyme of L- alpha-hydroxy acid oxidizing ferment
It is living, determine that the optimal reactive temperature of enzyme is 30 DEG C.
Embodiment 4
The present embodiment is the optimum pH of L- alpha-hydroxy acid oxidizing ferment of the present invention.Using Pfansteihl as substrate, substrate is existed
PH 3-9, the enzyme activity of 30 DEG C of water-bath 15min measurement enzymes, as a result, it has been found that L- alpha-hydroxy acid oxidizing ferment enzyme activity is most under the conditions of 7.0 pH
It is high.
Embodiment 5
The present embodiment is listed in table 1 from the response characteristic of different substrates for L- alpha-hydroxy acid oxidizing ferment of the present invention.
Activity of the 1 L- alpha-hydroxy acid oxidizing ferment of table to different substrates
Embodiment 6
Various racemic ' alpha '-carboxylic esters are split according to the method in summary of the invention, as a result as shown in the table:
Table 2 splits the effect of various racemic ' alpha '-carboxylic esters
As can be seen from the above table, when the reaction time is abundant, available all kinds of optically pure (R)-α-hydroxy acids of height
The optics specificity of ester, the enzyme is very good.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of oxidizing ferment and its application
<130> No
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1083
<212> DNA
<213> Pseudomonas fulva ATCC 31418
<400> 1
gtgaaagaat catcgactcc ccggcaagcg ccgaaaacct acaacctttt ggagctggaa 60
gcgctcgcat cagaggtcat tcctcgcccg gccttcgatt acatcgcacg tggcgccggt 120
gacgagcaga ccttgcggga gaaccgtacg gcattttccc gagccttcct cgaccagcgc 180
atcctgaccg gcaagacggt caagagcctg gagacgaaaa tcctcggcag cccactacgt 240
gcacctgtcg tagtgtccgc gatggcagcg cacggtctgg cgcacccatc ggcagaatcc 300
ggcacggcaa aaggcgccgc tgcctacgga acactgctgg gtgtcagcac cgtctcgacc 360
cagaacctcg agcaagttgc ttcggccagc aacggtgaca agtggttcca gtgctacctg 420
acgcgcgaca gcggcttcaa ccgcgaactg ctgcagcgcg cgcacgcggc gggttacaaa 480
gccatcgtcc tgactgcaga cgtgacggtc ggcggtaacc gcgagcagga ccggcgcaat 540
aacctgcgca tgccaacccc cggcaacttt ctagatacca gtaatcgacc tcgtaagatc 600
gagttcgcgt tcgacagcag tattggcctg gacagcctgg atttcgtgcg cgagcacagt 660
ggtggtctgc cgttgatcgt caagggcgtg accactgcgc tggatgcgcg agcgctgctg 720
caacacggtg tcgatgccat ccaggtttcc aaccacggcg gccgccaact ggacggctct 780
ccggcagcat tcgactcatt gcaacgagtg gccgccgagg taaagggtcg cgtcccgctc 840
atcttcgaca gcggcatccg gcgcggcctc gatgtgttca aggccatcgc cgccggcgct 900
gatctggtcg cagtcggtcg tcccgtgctc tacggcctgg ccctcaatgg ctcgcaaggg 960
gtgcagtggg tgctcgaaca gctcgagcag gaactgcgca tcgtcatgca actttccggc 1020
gctgccacgg ttgccgacat ccgcaccacg cctttgctgc acaagccgtt cttcgacgca 1080
tga 1083
<210> 2
<211> 360
<212> PRT
<213> Pseudomonas fulva ATCC 31418
<400> 2
Met Lys Glu Ser Ser Thr Pro Arg Gln Ala Pro Lys Thr Tyr Asn Leu
1 5 10 15
Leu Glu Leu Glu Ala Leu Ala Ser Glu Val Ile Pro Arg Pro Ala Phe
20 25 30
Asp Tyr Ile Ala Arg Gly Ala Gly Asp Glu Gln Thr Leu Arg Glu Asn
35 40 45
Arg Thr Ala Phe Ser Arg Ala Phe Leu Asp Gln Arg Ile Leu Thr Gly
50 55 60
Lys Thr Val Lys Ser Leu Glu Thr Lys Ile Leu Gly Ser Pro Leu Arg
65 70 75 80
Ala Pro Val Val Val Ser Ala Met Ala Ala His Gly Leu Ala His Pro
85 90 95
Ser Ala Glu Ser Gly Thr Ala Lys Gly Ala Ala Ala Tyr Gly Thr Leu
100 105 110
Leu Gly Val Ser Thr Val Ser Thr Gln Asn Leu Glu Gln Val Ala Ser
115 120 125
Ala Ser Asn Gly Asp Lys Trp Phe Gln Cys Tyr Leu Thr Arg Asp Ser
130 135 140
Gly Phe Asn Arg Glu Leu Leu Gln Arg Ala His Ala Ala Gly Tyr Lys
145 150 155 160
Ala Ile Val Leu Thr Ala Asp Val Thr Val Gly Gly Asn Arg Glu Gln
165 170 175
Asp Arg Arg Asn Asn Leu Arg Met Pro Thr Pro Gly Asn Phe Leu Asp
180 185 190
Thr Ser Asn Arg Pro Arg Lys Ile Glu Phe Ala Phe Asp Ser Ser Ile
195 200 205
Gly Leu Asp Ser Leu Asp Phe Val Arg Glu His Ser Gly Gly Leu Pro
210 215 220
Leu Ile Val Lys Gly Val Thr Thr Ala Leu Asp Ala Arg Ala Leu Leu
225 230 235 240
Gln His Gly Val Asp Ala Ile Gln Val Ser Asn His Gly Gly Arg Gln
245 250 255
Leu Asp Gly Ser Pro Ala Ala Phe Asp Ser Leu Gln Arg Val Ala Ala
260 265 270
Glu Val Lys Gly Arg Val Pro Leu Ile Phe Asp Ser Gly Ile Arg Arg
275 280 285
Gly Leu Asp Val Phe Lys Ala Ile Ala Ala Gly Ala Asp Leu Val Ala
290 295 300
Val Gly Arg Pro Val Leu Tyr Gly Leu Ala Leu Asn Gly Ser Gln Gly
305 310 315 320
Val Gln Trp Val Leu Glu Gln Leu Glu Gln Glu Leu Arg Ile Val Met
325 330 335
Gln Leu Ser Gly Ala Ala Thr Val Ala Asp Ile Arg Thr Thr Pro Leu
340 345 350
Leu His Lys Pro Phe Phe Asp Ala
355 360
Claims (2)
1. a kind of method of resolution of alpha-carboxylic esters (alpha-hydroxy esters), which is characterized in that the method are as follows: take pure
0.1 gram of the enzyme changed is added in 50mL triangular flask dissolved in the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, in 30 DEG C,
16h is converted in 150rpm shaking bath, liquid-phase chromatographic analysis supernatant after conversion；The enzyme is from yellowish-brown pseudomonad
The L- alpha-hydroxy acid oxidizing ferment of (Pseudomonas fulva), amino acid sequence are shown in SEQ ID NO:2；α-the hydroxyl
Acid esters is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene cream
Sour norbornene ester, para hydroxybenzene isopropyl lactate, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu kakuol
Ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
2. the method according to claim 1, wherein the nucleotides sequence of the L- alpha-hydroxy acid oxidizing ferment is classified as SEQ
Shown in ID NO:1.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1125961A (en) * | 1993-06-25 | 1996-07-03 | 纳幕尔杜邦公司 | Process for the preparation of pyruvic acid |
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| CN102660470B (en) * | 2012-04-13 | 2013-07-31 | 浙江工业大学 | Sinorhizobium fredii and its application in producing chiral alpha-hydroxy acid by biologically splitting alpha-hydroxy acid raceme |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1125961A (en) * | 1993-06-25 | 1996-07-03 | 纳幕尔杜邦公司 | Process for the preparation of pyruvic acid |
Non-Patent Citations (2)
| Title |
|---|
| D-Lactate Dehydrogenase Substrate Specificity and Use as a Catalyst in the Synthesis of Homochiral 2-Hydroxy Acids;ETHAN S.SIMON et al.;《Applied Biochemistry and Biotechnology》;19891231;第22卷;第169-179页 * |
| NCBI：KIP89088.1;NCBI;《NCBI》;20150209;第1-2页 * |
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