CN106754790A - A kind of oxidizing ferment and its application - Google Patents
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- CN106754790A CN106754790A CN201710006589.XA CN201710006589A CN106754790A CN 106754790 A CN106754790 A CN 106754790A CN 201710006589 A CN201710006589 A CN 201710006589A CN 106754790 A CN106754790 A CN 106754790A
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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 Cedecea neteri (Cedecea neteri) 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 Cedecea neteri (Cedecea neteri) 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 Cedecea neteri (Cedecea neteri) has obtained a kind of L- alpha-hydroxy acids oxidizing ferment
Gene, using colibacillus engineering heterogenous expression, discloses 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：Cedecea neteri ATCC 33855, purchased from U.S.
State's ATCC strain libraries.
2nd, the clone of L- alpha-hydroxy acids oxidase gene
Extract the phage gene group STb genes of Cedecea neteri ATCC 33855.Design specific primer, using PCR side
Method, amplifies L- alpha-hydroxy acid oxidase gene total length encoder block sequences.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, L- mandelic acids.
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：Clone has obtained a kind of L- α-hydroxyl from Cedecea neteri ATCC 33855
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-hydroxyl
Acid 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 the DNA of Cedecea neteri ATCC 33855
The bacterial strains of Cedecea neteri ATCC 33855 are cultivated into 12h, 12,000rmp/min centrifugations in LB culture mediums
10min obtains thalline, and phage gene group is extracted according to its operation using bacterial genomes DNA extraction agents box (TaKaRa companies)
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'GCCGGGATCCATGATTATTTCCGCAGCAACCGAC 3'
Primer 2：5'GCCGTCTAGATATAAACCCTGGCGAACCAGCAAG 3'
(2) PCR amplifications
Two primers of synthesis, are carried out by template of the genomic DNA of Cedecea neteri ATCC 33855 more than
PCR is expanded.
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 esterase is obtained after Hua Da gene sequencing, such as SEQ ID NO:Shown in 1.According to this
The amino acid sequence that gene order is obtained such as SEQ ID NO: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 8.0 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 8.0
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 below from the response characteristic of different substrates.
Activity of the table 2L- alpha-hydroxy acids oxidizing ferment to different substrates
Embodiment 7
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> 1143
<212> DNA
<213> Cedecea neteri ATCC 33855
<400> 1
atgattattt ccgcagcaac cgactaccgt gccgccgccg agcgcattct gccgcccttt 60
ttattccact acattgacgg tggggcctat gccgagcata cgctgaagcg caatgtggaa 120
gacctggcac aggtggcgct caagcagcgc gtactgaaaa acatgtcggc gctgagtctg 180
gaaaccaggc tgtttaacga aacgttgtct atgccggtgg ccctcggccc cgtggggctt 240
tgtggcatgt atgcgcgccg gggagaagtt caggccgcaa aagccgccgc ggcgaaaggc 300
attcccttta ctctgtcgac ggtgtccgtt tgtccgattg aagaagtcgc ccccgccatc 360
agccgcccga tgtggttcca gctttatgtc ctgaaagatc gcggttttat gcgcaatgcg 420
ctggagcggg cgaaagccgc cggatgctct acgctggtgt ttaccgtcga tatgccaacg 480
ccgggcgctc gctatcgtga tgcacactca ggcatgagcg gccctaacgc agcgatgcgc 540
cgttatcttc aggccgtaac gcacccgctg tgggcatggg atgtcggctt aaacggccgc 600
cctcatgatt tgggcaatat ctccgcctat ctcggcaaac cgaccgggct ggaagattat 660
atcggctggc tggcgaacaa ctttgacccg tccatttcct ggaaagatct ggagtggatc 720
cgcgagttct gggacggccc gatggtgatt aaagggatcc tcgacccgga agacgcccgc 780
gatgcggtgc gttttggcgc cgacgggatc gtggtgtcta accacggcgg ccgccagctg 840
gacggcgtgc tctcttccgc tcgcgccctg cccgctattg ccgatgcggt gaaaggcgat 900
attacgattc tggctgacag cggcatccgc aacggcctgg acgtggtgcg tatgattgcg 960
ctgggtgccg acacggtgct gctgggacgc gctttcctgt atgcgctggc gacccacggc 1020
gaagccggcg tcagcaatct gctgaactta atggagaaag agatgcgcgt ggcgatgacg 1080
ctgaccggcg cgaaatctat tgccgaaatt acgcctgact tgctggttcg ccagggttta 1140
taa 1143
<210> 2
<211> 380
<212> PRT
<213> Cedecea neteri ATCC 33855
<400> 2
Met Ile Ile Ser Ala Ala Thr Asp Tyr Arg Ala Ala Ala Glu Arg Ile
1 5 10 15
Leu Pro Pro Phe Leu Phe His Tyr Ile Asp Gly Gly Ala Tyr Ala Glu
20 25 30
His Thr Leu Lys Arg Asn Val Glu Asp Leu Ala Gln Val Ala Leu Lys
35 40 45
Gln Arg Val Leu Lys Asn Met Ser Ala Leu Ser Leu Glu Thr Arg Leu
50 55 60
Phe Asn Glu Thr Leu Ser Met Pro Val Ala Leu Gly Pro Val Gly Leu
65 70 75 80
Cys Gly Met Tyr Ala Arg Arg Gly Glu Val Gln Ala Ala Lys Ala Ala
85 90 95
Ala Ala Lys Gly Ile Pro Phe Thr Leu Ser Thr Val Ser Val Cys Pro
100 105 110
Ile Glu Glu Val Ala Pro Ala Ile Ser Arg Pro Met Trp Phe Gln Leu
115 120 125
Tyr Val Leu Lys Asp Arg Gly Phe Met Arg Asn Ala Leu Glu Arg Ala
130 135 140
Lys Ala Ala Gly Cys Ser Thr Leu Val Phe Thr Val Asp Met Pro Thr
145 150 155 160
Pro Gly Ala Arg Tyr Arg Asp Ala His Ser Gly Met Ser Gly Pro Asn
165 170 175
Ala Ala Met Arg Arg Tyr Leu Gln Ala Val Thr His Pro Leu Trp Ala
180 185 190
Trp Asp Val Gly Leu Asn Gly Arg Pro His Asp Leu Gly Asn Ile Ser
195 200 205
Ala Tyr Leu Gly Lys Pro Thr Gly Leu Glu Asp Tyr Ile Gly Trp Leu
210 215 220
Ala Asn Asn Phe Asp Pro Ser Ile Ser Trp Lys Asp Leu Glu Trp Ile
225 230 235 240
Arg Glu Phe Trp Asp Gly Pro Met Val Ile Lys Gly Ile Leu Asp Pro
245 250 255
Glu Asp Ala Arg Asp Ala Val Arg Phe Gly Ala Asp Gly Ile Val Val
260 265 270
Ser Asn His Gly Gly Arg Gln Leu Asp Gly Val Leu Ser Ser Ala Arg
275 280 285
Ala Leu Pro Ala Ile Ala Asp Ala Val Lys Gly Asp Ile Thr Ile Leu
290 295 300
Ala Asp Ser Gly Ile Arg Asn Gly Leu Asp Val Val Arg Met Ile Ala
305 310 315 320
Leu Gly Ala Asp Thr Val Leu Leu Gly Arg Ala Phe Leu Tyr Ala Leu
325 330 335
Ala Thr His Gly Glu Ala Gly Val Ser Asn Leu Leu Asn Leu Met Glu
340 345 350
Lys Glu Met Arg Val Ala Met Thr Leu Thr Gly Ala Lys Ser Ile Ala
355 360 365
Glu Ile Thr Pro Asp Leu Leu Val Arg Gln Gly Leu
370 375 380
Claims (5)
1. one kind derives from the L- alpha-hydroxy acid oxidizing ferment of Cedecea neteri (Cedecea neteri), and its amino acid sequence 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 8.
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, L- danshensus, 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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|---|---|---|---|---|
| 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 |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| Title |
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| 无: "WP_061275456.1", 《NCBI》 * |
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