CN106754796A - A kind of oxidizing ferment and its application - Google Patents
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- CN106754796A CN106754796A CN201710006613.XA CN201710006613A CN106754796A CN 106754796 A CN106754796 A CN 106754796A CN 201710006613 A CN201710006613 A CN 201710006613A CN 106754796 A CN106754796 A CN 106754796A
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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)
- C12Y101/03015—(S)-2-Hydroxy-acid oxidase (1.1.3.15)
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Abstract
The present invention relates to one kind, the acquisition from the L alpha hydroxy acid oxidase genes of yellowish-brown pseudomonad (Pseudomonas fulva) and its clonal expression, belong to bioengineering field.Its substrate specificity is disclosed, while the L alpha hydroxy acids oxidizing ferment can aoxidize (S) alpha hydroxy acid ester, the preparation of optical voidness (R) alpha hydroxy acid ester is can be applied to.
Description
Technical field
A kind of L- alpha-hydroxy acids 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 technology
L- alpha-hydroxy acids oxidizing ferment (L- α-hydroxyacid oxidase) is a kind of dehydrogenase with FMN (FAD) as coenzyme
(Aliphatic l-α-hydroxyacid oxidase from rat livers purification and
properties.Biochimica et Biophysica Acta(BBA)-Enzymology 1968,167:9-22), generally
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).Can be used to determine containing for lactic acid in biology sensor
Amount, or oxidation Pfansteihl production pyruvic acid.Also have and be used for the preparation (Chinese patent of 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), mycobacterium smegmatis
(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
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)
Clonal expression goes out a kind of new L- α-hydroxyl to the present invention from Kerstersia gyiorum DSM 16618 first
Acid oxidase, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, and 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.
The content of the invention
Present invention clone from yellowish-brown pseudomonad (Pseudomonas fulva) has obtained a kind of L- alpha-hydroxy acids oxidizing ferment
Gene, using colibacillus engineering heterogenous expression, disclose its related enzymatic property, and carried out application study.
Technical scheme is as follows：
1st, bacterial strain
The source bacterial strain of L- alpha-hydroxy acids oxidase gene of the present invention is：Pseudomonas fulva ATCC 31418, purchase
From U.S.'s ATCC strain libraries.
2nd, the clone of L- alpha-hydroxy acids oxidizing ferment
Extract the phage gene group STb genes of Pseudomonas fulva ATCC 31418.Design specific primer, application
PCR method, amplifies L- Α-hydroxy acid oxidase full length gene encoder block sequence.And construction recombination plasmid.
3rd, L- alpha-hydroxy acids Oxidase Expression and purifying
Recombinant plasmid is imported in E.coli BL21 (DE3), induced expression.Crude enzyme liquid is obtained after bacterial cell disruption, after purification
Freeze-drying is standby.
4th, the characterization analysis of L- alpha-hydroxy acids oxidizing ferment
Influence with Pfansteihl as substrate research pH to L- alpha-hydroxy acids oxidizing ferment enzyme activity of the present invention.
Influence with Pfansteihl as substrate research temperature to L- alpha-hydroxy acids oxidizing ferment enzyme activity of the present invention.
The substrate specificity analysis of L- alpha-hydroxy acid oxidizing ferment：Substrate used has Pfansteihl, glycolic, L- phenyllactic acids, L-
Tartaric acid, L MALIC ACID, L- para hydroxybenzenes lactic acid, L- danshensus.
Enzyme activity determination method is：According to Characterization of a Lactate Oxidase from a
Strain of Gram Negative Bacterium from Soil, Applied Biochemistry and
Biotechnology,56,1996,278-288.Methods described is carried out.
5th, L- alpha-hydroxy acids oxidizing ferment splits the alpha-hydroxy acid ester of DL
The method of resolution of alpha-carboxylic esters (alpha-hydroxy esters) is：0.1 gram of the enzyme for having purified is taken in 50mL tri-
In the bottle of angle, in adding dissolved with the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, in 30 DEG C, converted in 150rpm shaking baths
16h, liquid-phase chromatographic analysis supernatant after conversion.(S) Alpha-hydroxy in-alpha-hydroxy acid ester is dehydrogenated and is oxidized to corresponding 2-ketoacid
Ester, (R)-alpha-hydroxy acid ester is not oxidized.
The optical purity of product (R)-alpha-hydroxy acid ester 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 formulaRIt is the peak area of (R)-enantiomer after reaction, SSIt is the liquid chromatogram peak area of (S)-enantiomer after reaction,
S0It is the liquid chromatogram peak area sum of (R)-and (S)-enantiomer before reaction.
Product determines liquid phase chromatogram condition：Chiralcel OD-H chiral columns (4.6 × 250mm), mobile phase volume ratio
It is n-hexane：Isopropanol：Trifluoroacetic acid=80:20:0.1, flow velocity is 0.5mL/min, and 25 DEG C of column temperature, Detection wavelength 210nm enters
The μ L of sample amount 20.
Described alpha-hydroxy acid ester is one of following：Tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, benzene breast
Isopropyl propionate, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene isopropyl lactate, mandelic acid norbornene ester, almond isopropyl propionate, the red sage root
Plain asarum alcohol ester, lactic acid norbornene ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
Described alpha-hydroxy acid ester, according to Chinese patent 200610042787.3,201410180490.8,
The 201410175950.8 and 20140699506.6 method synthesis announced.
Originally bright usefulness is delivered：From Pseudomonas fulva ATCC 31418 clone obtained a kind of L- α-
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, with important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of L- alpha-hydroxy acids oxidase gene of the present invention and colibacillus engineering build.
1st, the extraction of Pseudomonas fulva ATCC 31418DNA
The bacterial strains of Pseudomonas fulva ATCC 31418 are cultivated into 12h in LB culture mediums, 12,000rmp/min from
Heart 10min obtains thalline, and phage gene is extracted according to its operation using bacterial genomes DNA extraction agents box (TaKaRa companies)
Group STb gene, puts refrigerator standby.
2nd, prepared by E. coli competent
(1) inoculation E.coli DH5 α and BL21 (DE3) is respectively in the 250mL shaking flasks containing 20mL LB culture mediums, and 37
DEG C, 200rpm/min overnight incubations.
(2) it is inoculated in 50mL LB culture mediums 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 precoolings, 30min, 8000rpm/min, 4 DEG C of centrifugations is placed on ice
5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl of 5mL precoolings are added2Solution, makes thalline suspend, and 20min is placed 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 precoolings are added2Solution (contains 15% glycerine), 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 standby.
3rd, the clone of L- alpha-hydroxy acids oxidase gene
(1) design of primers
Designing primer sequence is：
Primer 1：5'GCCGGGATCCGTGAAAGAATCATCGACTCCCCGGC 3'
Primer 2：5'GCCGTCTAGATGCGTCGAAGAACGGCTTGTGCA 3'
(2) PCR amplifications
Two primers of synthesis more than, the genomic DNA with Pseudomonas fulva ATCC 31418 is as template
Enter performing PCR amplification.
Amplification system is in this step：
Amplification program is：
98 DEG C, 10min
98 DEG C, 10sec；55 DEG C, 15sec；72 DEG C, 2min reacts 30 circulations
72 DEG C, 10min
PCR primer send the gene order that the enzyme is obtained after Hua Da gene sequencing, such as SEQ ID NO:Shown in 1.According to the base
Because of the amino acid sequence such as SEQ ID NO that sequence is obtained:Shown in 2.
(3) double digestion and connection
The plasmids of pCold II and PCR primer are carried out into double digestion, digestion system is：10×cut buffer 3μl,DNA 4μ
Each 0.5 μ l of l, enzyme BamHI and XbaI, the μ l of sterilized water 2 totally 30 μ l.Double digestion 1h under 37 DEG C of water-baths.DNA fragmentation is cloned into
On the carriers of pCold II, and it is transformed into E.coli DH5 α competent cells.Linked system：10×DNA ligase buffer
2.5 μ l, the μ l of DNA fragmentation 8,2 μ l, T4DNA ligase of carrier DNA 1 μ l, the μ l of sterilized water 11.5 totally 25 μ l.Connect under 16 DEG C of water-baths
Meet 12h-16h.
(4) convert
Step：
1 adds 100 μ l DH5 α competence bacteriums in linked system, light to mix, ice bath 30min.
2 are put into 42 DEG C of water-baths of preheating, and placing 90s carries out heat shock treatment.
3 ice bath 2min immediately.
4 add LB nutrient solutions of the 1ml without antibiotic, and cultivating 1h for 37 DEG C makes thalline recover.
5 are uniformly coated on the LB flat boards containing antibiotic thalline.
6 culture 24h grow fine.Choosing single bacterium colony carries out bacterium colony PCR, and recombinant plasmid is extracted in nucleic acid electrophoresis checking.Will restructuring
Plasmid is imported in BL21 E. coli competents, is saved backup.
Embodiment 2
The present embodiment is the induced expression of L- alpha-hydroxy acids oxidizing ferment of the present invention and isolates and purifies.
1st, plus 500 μ l recombinate bacterium solution in 50ml LB nutrient solutions.37 DEG C of culture 2.5h, 0.5h is stood at 15 DEG C.Plus 20 again
The IPTG of μ l 0.5M, cold-induced culture 24h at 15 DEG C.Zymotic fluid is centrifuged (8000rmp/min, 10min) and is obtained bacterium
Body, thalline is redissolved with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (20mmol/L, pH 7.0), and Ultrasonic Cell Disruptor is crushed, from
The heart (8000rmp/min, 10min) collects supernatant and obtains crude enzyme liquid.
2nd, the crude enzyme liquid for obtaining step 1 carries out ni-sepharose purification using the operation of the protein purification systems of AKTA avant 150,
Elution process is：All put tetra- pipelines of A1, A2, B1, B2 into water, system flow 20ml/min flow velocitys are set, carry out
Exhaust.Then system flow 1ml/min, flow path (column position 3), delta pressure are set
0.3rd, pre-pressure 0.5, Gradient 0, inset A1, after filling pillar after water droplet uniformly outflow, balance ten minutes it
A1 is put into reference in liquid afterwards, B1 is put into eluent, then is exhausted once, balance 20 minutes, then loading crude enzyme liquid,
With high concentration imidazole buffer (solution residing for B1) gradient elution destination protein of 500mM, the albumen on ion column will be adsorbed
Elute the enzyme for being purified.Enzyme after purification is freeze-dried standby.
Embodiment 3
The present embodiment is the optimum temperature of L- alpha-hydroxy acids oxidizing ferment of the present invention.With Pfansteihl as substrate, by substrate with
PH be 7.0 phosphate buffer under 30-60 DEG C of different temperature conditionss water-bath 15min, determine L- alpha-hydroxy acid oxidizing ferment enzyme
Living, the optimal reactive temperature for determining enzyme is 30 DEG C.
Embodiment 4
The present embodiment is the optimum pH of L- alpha-hydroxy acids oxidizing ferment of the present invention.With Pfansteihl as substrate, substrate is existed
As a result pH 3-9,30 DEG C of enzyme activity of water-bath 15min measure enzymes find that L- alpha-hydroxy acids oxidizing ferment enzyme activity is most under the conditions of pH 7.0
It is high.
Embodiment 5
The present embodiment is that L- alpha-hydroxy acids oxidizing ferment of the present invention is listed in table 2 from the response characteristic of different substrates.
Activity of the L- alpha-hydroxy acids oxidizing ferment of table 2 to different substrates
Embodiment 6
Method in the content of the invention splits various racemic ' alpha '-carboxylic esters, as a result as shown in the table：
Table 3 splits the effect of various racemic ' alpha '-carboxylic esters
As can be seen from the above table, when abundant in the reaction time, optically pure (the R)-alpha-hydroxy acid ester of all kinds of height can be obtained,
The optics selectivity of 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 (5)
1. one kind derives from the L- alpha-hydroxy acid oxidizing ferment of yellowish-brown pseudomonad (Pseudomonas fulva), its amino acid sequence
It is SEQ ID NO:Shown in 2.
2. L- alpha-hydroxy acids oxidizing ferment according to claim 1, its nucleotides sequence is classified as SEQ ID NO:Shown in 1.
3. L- alpha-hydroxy acids oxidizing ferment according to claim 1, its optimal reactive temperature is 30 DEG C, and optimal reaction pH is 7.
4. L- alpha-hydroxy acids oxidizing ferment according to claim 1, oxidable Pfansteihl, glycolic, L- phenyllactic acids, L- are to hydroxyl
Phenyllactic acid, L-TARTARIC ACID, L MALIC ACID, L- mandelic acids, generate corresponding ketone acid.
5. L- alpha-hydroxy acids oxidizing ferment according to claim 1, (the S)-alpha-hydroxy acid ester in oxidable racemic ' alpha '-carboxylic esters,
Fractionation prepares corresponding optical voidness (R)-alpha-hydroxy acid ester and alpha-keto ester, and described alpha-hydroxy acid ester is one of following：The red sage root
Plain norbornene ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene breast
Isopropyl propionate, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu asarum alcohol ester, phenyllactic acid asarum alcohol ester,
Para hydroxybenzene lactic acid asarum alcohol ester.
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Citations (2)
| 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 |
| CN102660470A (en) * | 2012-04-13 | 2012-09-12 | 浙江工业大学 | Sinorhizobium fredii and its application in producing chiral alpha-hydroxy acid by biologically splitting alpha-hydroxy acid raceme |
-
2017
- 2017-01-05 CN CN201710006613.XA patent/CN106754796B/en active Active
Patent Citations (2)
| 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 |
| CN102660470A (en) * | 2012-04-13 | 2012-09-12 | 浙江工业大学 | Sinorhizobium fredii and its application in producing chiral alpha-hydroxy acid by biologically splitting alpha-hydroxy acid raceme |
Non-Patent Citations (2)
| Title |
|---|
| ETHAN S.SIMON ET AL.: "D-Lactate Dehydrogenase Substrate Specificity and Use as a Catalyst in the Synthesis of Homochiral 2-Hydroxy Acids", 《APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》 * |
| NCBI: "NCBI：KIP89088.1", 《NCBI》 * |
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