CN106591251A - Oxidase and application thereof - 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)
- C12Y101/03015—(S)-2-Hydroxy-acid oxidase (1.1.3.15)
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Abstract
The invention relates to acquiring and cloning expression of an L-a-hydroxyacid oxidase gene coming from Providencia rettgeri and belongs to the biological engineering field. The substrate specificity of the L-a-hydroxyacid oxidase gene is disclosed, and meanwhile, L-a-hydroxyacid oxidase can oxidize (S)-a-carboxylic acid ester and can be applied to preparation of optics pure (R)-a-carboxylic acid ester.
Description
Technical field
A kind of L- alpha-hydroxy acids oxidase of clonal expression of the present invention, and disclose its nucleotide sequence and aminoacid sequence and
Zymologic property and application, belong to industrial microorganism field.
Background technology
L- alpha-hydroxy acid oxidase (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 oxidase (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 biosensor
Amount, or oxidation Pfansteihl production acetone 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.), Pediococcuses (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 oxidase in antibacterials such as (Acetobacter peroxidans).Pfansteihl oxidase is also one
Detect in a little funguses, 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 present invention goes out a kind of new L- α-hydroxyl from clonal expression in Kerstersia gyiorum DSM 16618 first
Acid oxidase, the enzyme not only can aoxidize (S)-alpha-hydroxy acid, and can aoxidize (S)-alpha-hydroxy acid ester, can be applicable to optical voidness
(R) preparation of-alpha-hydroxy acid ester and (R)-alpha-hydroxy acid.
The content of the invention
Present invention clone from providencia rettgeri (Providencia rettgeri) has obtained a kind of L- α-hydroxyl
The gene of acid oxidase, using colibacillus engineering heterogenous expression, discloses its related enzymatic property, and is applied
Research.
Technical scheme is as follows：
1st, bacterial strain
The source bacterial strain of L- alpha-hydroxy acids lysyloxidase gene of the present invention is：Providencia rettgeri ATCC29944,
Purchased from U.S.'s ATCC strain libraries.
2nd, the clone of L- alpha-hydroxy acids lysyloxidase gene
Extract the phage gene group STb genes of Providencia rettgeri ATCC 29944.Design specific primer, should
With PCR method, L- alpha-hydroxy acid lysyloxidase gene total length encoder block sequences are amplified.And construction recombination plasmid.
3rd, L- alpha-hydroxy acids Oxidase Expression and purification
Recombiant plasmid is imported in E.coli BL21 (DE3), abduction delivering.Crude enzyme liquid is obtained after bacterial cell disruption, after purification
Lyophilization is standby.
4th, the oxidasic characterization analysis of L- alpha-hydroxy acids
Impacts of the pH to L- alpha-hydroxy acids oxidase enzyme activity of the present invention is studied by substrate of Pfansteihl.
Impact of the temperature to L- alpha-hydroxy acids oxidase enzyme activity of the present invention is studied by substrate of Pfansteihl.
The oxidasic substrate specificity analysis of L- alpha-hydroxy acids：Substrate used has Pfansteihl, glycolic, L- phenyllactic acids, L-
Tartaric acid, L MALIC ACID, L- para hydroxybenzene lactic acid, L- danshensus, L- mandelic acid.
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 oxidase splits the alpha-hydroxy acid ester of DL
The method of resolution of alpha-carboxylic esters (alpha-hydroxy esters) is：0.1 gram good of enzyme of purification is taken in 50mL tri-
In the bottle of angle, in adding the phosphate buffer dissolved with the pH 7 of alpha-hydroxy acid ester 5mM, in 30 DEG C, convert 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 formulaRFor the peak area of (R)-enantiomer after reaction, SSFor the liquid chromatograph peak area of (S)-enantiomer after reaction,
S0For (R) before reaction-and the liquid chromatograph peak area sum of (S)-enantiomer.
Product determines liquid phase chromatogram condition：Chiralcel OD-H chiral columns (4.6 × 250mm), mobile phase volume ratio
For normal hexane：Isopropanol：Trifluoroacetic acid=80:20:0.1, flow velocity is 0.5mL/min, 25 DEG C of column temperature, and Detection wavelength 210nm is entered
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, Radix Salviae Miltiorrhizae
Plain Herba Asari alcohol ester, lactic acid norbornene ester, phenyllactic acid Herba Asari alcohol ester, para hydroxybenzene lactic acid Herba Asari alcohol ester.
Described alpha-hydroxy acid ester, according to Chinese patent 200610042787.3,201410180490.8,
201410175950.8 the method synthesis announced with 20140699506.6.
Originally bright usefulness is delivered：Clone has obtained one kind from Providencia rettgeri ATCC 29944
L- alpha-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)-alpha-hydroxy acid ester, with important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of L- alpha-hydroxy acids lysyloxidase gene of the present invention and colibacillus engineering build.
1st, the extraction of Providencia rettgeri ATCC 29944DNA
The bacterial strains of Providencia rettgeri ATCC 29944 are cultivated into 12h in LB culture medium, 12,000rmp/
Min centrifugation 10min obtain thalline, and bacterium is extracted according to its operation using bacterial genomes DNA extraction agent box (TaKaRa companies)
Body genome DNA, puts refrigerator standby.
2nd, prepared by E. coli competent
(1) be inoculated with E.coli DH5 α and BL21 (DE3) in the 250mL shaking flasks containing 20mL LB culture medium, 37
DEG C, 200rpm/min overnight incubations.
(2) it is inoculated in 50mL LB culture medium by 1% inoculum concentration, 37 DEG C are cultivated to OD600About 0.6 (about 2～3h).
(3) bacterium solution is transferred in the centrifuge tube of 50mL pre-coolings, 30min, 8000rpm/min, 4 DEG C of centrifugations is placed on ice
5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl of 5mL pre-coolings 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 pre-coolings are added2Solution (contains 15% glycerol), gently suspension thalline,
Then 100 μ L bacterium solution subpackages are added by each centrifuge tube (1.5mL), -70 DEG C of Storage in refrigerator are standby.
3rd, the clone of L- alpha-hydroxy acids lysyloxidase gene
(1) design of primers
Designing primer sequence is：
Primer 1：5'GCCGGGATCCATGAAAAGCAAAATATTAAAAAC 3'
Primer 2：5'GCCGTCTAGATTATTGATTAAAACTAGCATCAGTA 3'
(2) PCR amplifications
Two primers of synthesis with more than, the genomic DNA with Providencia rettgeri ATCC 2994 is as mould
Plate enters 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 aminoacid 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, enzyme action system is：10×cut buffer 3μl,DNA 4μ
The 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 in E.coli DH5 α competent cells.Linked system：10×DNA ligase buffer
2.5 μ l, the μ l of DNA fragmentation 8, the μ l of carrier DNA 2, the μ l of T4DNA ligase 1, 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 antibacterials in linked system, light to mix, ice bath 30min.
2 are put in 42 DEG C of water-baths of preheating, and placing 90s carries out heat shock process.
3 ice bath 2min immediately.
4 add LB culture fluid of the 1ml without antibiotic, 37 DEG C of culture 1h to make thalline recover.
5 are uniformly coated on thalline on the LB flat boards containing antibiotic.
6 culture 24h grow fine.Choosing single bacterium colony carries out bacterium colony PCR, and recombiant 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 oxidasic abduction delivering of L- alpha-hydroxy acids of the present invention and isolates and purifies.
1st, plus 500 μ l recombinate bacterium solution in 50ml LB culture fluid.37 DEG C of culture 2.5h, at 15 DEG C 0.5h is stood.Plus 20 again
The IPTG of μ l 0.5M, cold-induced culture 24h at 15 DEG C.Fermentation liquid is centrifuged into (8000rmp/min, 10min) and is obtained bacterium
Body, with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (20mmol/L, pH 7.0) thalline is redissolved, 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
Aerofluxuss.Then system flow 1ml/min, flow path (column position 3), delta pressure are set
0.3rd, pre-pressure 0.5, Gradient 0, inset A1, fill pillar after water droplet uniformly flows out, balance ten minutes it
A1 is put into reference in liquid afterwards, B1 is put in eluent, then is exhausted once, balanced 20 minutes, then loading crude enzyme liquid,
With high concentration imidazole buffer (solution residing for B1) the gradient elution destination protein of 500mM, by albumen of the absorption on ion column
Elute the enzyme for obtaining purification.Enzyme after purification is freeze-dried standby.
Embodiment 3
The present embodiment is the oxidasic optimum temperature of L- alpha-hydroxy acids of the present invention.With Pfansteihl as substrate, by substrate with
PH be 4.0 phosphate buffer under 30-60 DEG C of different temperature conditionss water-bath 15min, determine the oxidasic enzyme activity of alpha-hydroxy acid,
The optimal reactive temperature for determining enzyme is 40 DEG C.
Embodiment 4
The present embodiment is the oxidasic optimum pH of L- alpha-hydroxy acids of the present invention.With Pfansteihl as substrate, substrate is existed
PH 3-9,40 DEG C of water-bath 15min determine the enzyme activity of enzyme, as a result find the alpha-hydroxy acid oxidase enzyme activity highest under the conditions of pH 4.0.
Embodiment 5
The present embodiment is that L- alpha-hydroxy acids oxidase of the present invention is listed in table 2 from the response characteristic of different substrates.
Activity of the table 2L- alpha-hydroxy acids oxidase 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 response time, optically pure (the R)-alpha-hydroxy acid ester of all kinds of height can be obtained,
The optics specificity of the enzyme is very good.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of oxidase and its application
<130> No
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1209
<212> DNA
<213> Providencia rettgeri ATCC 29944
<400> 1
atgttgagtt ttacacgaaa aaatacgttt gtgacatcga cattatttgc tgttgcactg 60
actcttggta ccgcacaggc ggcagaatat aaagctagcg ataaagaagg cccaattaaa 120
attgtgaact tggatgaatt agaaagccaa gttgccaaaa gtatggataa aggtgctttt 180
ggttatattc gtggtggtgc tgaagatgaa ttgaatctga agaaaaatac gcaacatttt 240
gataataaat atattatgcc aagagtgatg cagggaattg aaatttcaga tatcgacctt 300
tcaactgatt ttttaggaat taaactaaaa acccctatta ttcaagcgcc gatggcggcc 360
caaggattag cacataaaga cggggaaatt gcgacagcaa aaggtatggc taaagcaggt 420
tctattttct cgttaagtac ctacggtaat aaaacaattg aagaagtggc tgaagtttca 480
ggggaaaacc cattcttttt ccagctttat atgagtaaaa ataatgcatt taacgaattc 540
acgttgaaga gagccaaagc aagtggtgct aaagcgatta ttttaaccgt tgactctcca 600
gttggtgggt atcgtgaaga tgatattcgt aataacttcc aattcccact aggctttgcc 660
aatttagagc tatttgctaa gcaaaatagt gatggttcaa aaacaggtaa aggtgcaggg 720
atcagtgaaa tttatgcgca agcaaaacaa gcctttacac cagcagatat tcaatacgtg 780
aaaaagttat cgggattgcc agtaatagtt aaaggtattc aatcgcccga agatgctgat 840
gttgtgatca aagcaggtgc tgatgcgatt tgggtctcta atcacggtgg tcgccagtta 900
gacagtggtc ccgcgtcttt tgatgtattg ccgtcaattg ctaaggtcgt taataaacgt 960
gttccaattg tatttgacag tggtgtgcgt cgcggttcac acgtatttaa agccttagca 1020
agtggggcag atgttgtggc tgtaggccgt cctattttat atggcttaaa tttaggtggt 1080
gctgaaggcg taaactcggt gatccagcaa ctgaataaag aactttctat caatatgatg 1140
ctaggcgggg ccaaaaatat tgaaggagtt aaagcgacga agttgtatac cgacaaagat 1200
tttcaataa 1209
<210> 2
<211> 402
<212> PRT
<213> Providencia rettgeri ATCC 29944
<400> 2
Met Leu Ser Phe Thr Arg Lys Asn Thr Phe Val Thr Ser Thr Leu Phe
1 5 10 15
Ala Val Ala Leu Thr Leu Gly Thr Ala Gln Ala Ala Glu Tyr Lys Ala
20 25 30
Ser Asp Lys Glu Gly Pro Ile Lys Ile Val Asn Leu Asp Glu Leu Glu
35 40 45
Ser Gln Val Ala Lys Ser Met Asp Lys Gly Ala Phe Gly Tyr Ile Arg
50 55 60
Gly Gly Ala Glu Asp Glu Leu Asn Leu Lys Lys Asn Thr Gln His Phe
65 70 75 80
Asp Asn Lys Tyr Ile Met Pro Arg Val Met Gln Gly Ile Glu Ile Ser
85 90 95
Asp Ile Asp Leu Ser Thr Asp Phe Leu Gly Ile Lys Leu Lys Thr Pro
100 105 110
Ile Ile Gln Ala Pro Met Ala Ala Gln Gly Leu Ala His Lys Asp Gly
115 120 125
Glu Ile Ala Thr Ala Lys Gly Met Ala Lys Ala Gly Ser Ile Phe Ser
130 135 140
Leu Ser Thr Tyr Gly Asn Lys Thr Ile Glu Glu Val Ala Glu Val Ser
145 150 155 160
Gly Glu Asn Pro Phe Phe Phe Gln Leu Tyr Met Ser Lys Asn Asn Ala
165 170 175
Phe Asn Glu Phe Thr Leu Lys Arg Ala Lys Ala Ser Gly Ala Lys Ala
180 185 190
Ile Ile Leu Thr Val Asp Ser Pro Val Gly Gly Tyr Arg Glu Asp Asp
195 200 205
Ile Arg Asn Asn Phe Gln Phe Pro Leu Gly Phe Ala Asn Leu Glu Leu
210 215 220
Phe Ala Lys Gln Asn Ser Asp Gly Ser Lys Thr Gly Lys Gly Ala Gly
225 230 235 240
Ile Ser Glu Ile Tyr Ala Gln Ala Lys Gln Ala Phe Thr Pro Ala Asp
245 250 255
Ile Gln Tyr Val Lys Lys Leu Ser Gly Leu Pro Val Ile Val Lys Gly
260 265 270
Ile Gln Ser Pro Glu Asp Ala Asp Val Val Ile Lys Ala Gly Ala Asp
275 280 285
Ala Ile Trp Val Ser Asn His Gly Gly Arg Gln Leu Asp Ser Gly Pro
290 295 300
Ala Ser Phe Asp Val Leu Pro Ser Ile Ala Lys Val Val Asn Lys Arg
305 310 315 320
Val Pro Ile Val Phe Asp Ser Gly Val Arg Arg Gly Ser His Val Phe
325 330 335
Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Val Gly Arg Pro Ile
340 345 350
Leu Tyr Gly Leu Asn Leu Gly Gly Ala Glu Gly Val Asn Ser Val Ile
355 360 365
Gln Gln Leu Asn Lys Glu Leu Ser Ile Asn Met Met Leu Gly Gly Ala
370 375 380
Lys Asn Ile Glu Gly Val Lys Ala Thr Lys Leu Tyr Thr Asp Lys Asp
385 390 395 400
Phe Gln
Claims (5)
1. one kind derives from the L- alpha-hydroxy acid oxidase of providencia rettgeri (Providencia rettgeri), its amino
Acid sequence is SEQ ID NO:Shown in 2.
2. L- alpha-hydroxy acids oxidase according to claim 1, its nucleotides sequence is classified as SEQ ID NO:Shown in 1.
3. L- alpha-hydroxy acids oxidase according to claim 1, its optimal reactive temperature is 40 DEG C, and optimal reaction pH is 4.
4. L- alpha-hydroxy acids oxidase according to claim 1, oxidable Pfansteihl, glycolic, L- phenyllactic acids, L- are to hydroxyl
Phenyllactic acid, L-TARTARIC ACID, L MALIC ACID, L- mandelic acid, danshensu, generate corresponding keto acid.
5. L- alpha-hydroxy acids oxidase 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：Radix Salviae Miltiorrhizae
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 Herba Asari alcohol ester, phenyllactic acid Herba Asari alcohol ester,
Para hydroxybenzene lactic acid Herba Asari alcohol ester.
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|---|---|---|---|---|
| CN1125961A (en) * | 1993-06-25 | 1996-07-03 | 纳幕尔杜邦公司 | Process for the preparation of pyruvic acid |
| CN101928734A (en) * | 2009-11-13 | 2010-12-29 | 山东大学 | Method for preparing alpha-ketobutyric acid |
| CN102660631A (en) * | 2012-04-13 | 2012-09-12 | 浙江工业大学 | Method for screening stereoselective alpha-hydroxy acid dehydrogenase |
| 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 CN201710006586.6A patent/CN106591251B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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