CN106754793A - A kind of oxidizing ferment and its application - Google Patents
A kind of oxidizing ferment and its application Download PDFInfo
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Abstract
Acquisition and its clonal expression the present invention relates to a kind of D Lactate oxidase genes from Carnimonas nigrificans, belong to bioengineering field.Its substrate specificity is disclosed, while the D LOs can aoxidize (R) alpha hydroxy acid ester, the preparation of optical voidness (S) alpha hydroxy acid ester is can be applied to.
Description
Technical field
A kind of D-ALPHA-Hydroxypropionic acid oxidizing ferment of clonal expression of the present invention, and disclose its nucleotide sequence and amino acid sequence and enzyme
Property and application are learned, belongs to industrial microorganism field.
Background technology
D-ALPHA-Hydroxypropionic acid oxidizing ferment (D-lactate oxidase) is a kind of alpha-hydroxy acid oxidizing ferment with FAD (FMN) as coenzyme
(being traditionally referred to as D-ALPHA-Hydroxypropionic acid oxidizing ferment).D-ALPHA-Hydroxypropionic acid oxidizing ferment can be used in biology sensor determine the content of lactic acid, or
Oxidation D-ALPHA-Hydroxypropionic acid production pyruvic acid.Also have and be used for the preparation (Chinese patent 201210109290.4) of optical voidness alpha-hydroxy acid
So far, in edwardsiella tarda (Edwardsiella tarda) and zymomonas mobilis
D-ALPHA-Hydroxypropionic acid oxidizing ferment is found that in (Zymomonas mobilis) etc..(Kalnenieks U,Galinina N,Bringer-
Meyer S,et al.Membrane D-lactate oxidase in Zymomonas mobilis:evidence for a
branched respiratory chain[J].FEMS microbiology letters,1998,168(1):91-97)
Clonal expression goes out a kind of new D-ALPHA-Hydroxypropionic acid oxidizing ferment to the present invention from Carnimonas nigrificans first,
The enzyme can not only aoxidize (R)-alpha-hydroxy acid, and can aoxidize (R)-alpha-hydroxy acid ester, and the reaction is coenzyme with NAD (NADP)
The reaction that lactic dehydrogenase is participated in is atomic weak compared to back reaction, can be applied to optical voidness (S)-alpha-hydroxy acid ester with (S)-alpha-hydroxy acid
Prepare.
The content of the invention
Present invention clone from Carnimonas nigrificans has obtained a kind of D-ALPHA-Hydroxypropionic acid oxidation with FAD as coenzyme
The gene of enzyme, 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 D-ALPHA-Hydroxypropionic acid oxidase gene of the present invention is:Carnimonas nigrificans ATCC BAA-
78, purchased from U.S.'s ATCC strain libraries.
2nd, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract Carnimonas nigrificans ATCC BAA-78 phage gene group STb genes.Design specific primer,
Using PCR method, D-ALPHA-Hydroxypropionic acid oxidase gene total length encoder block sequence is amplified.And construction recombination plasmid.
3rd, D-ALPHA-Hydroxypropionic acid 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment
Influence with D-ALPHA-Hydroxypropionic acid as substrate research pH to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention.
Influence with D-ALPHA-Hydroxypropionic acid as substrate research temperature to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention.
The substrate specificity analysis of D-ALPHA-Hydroxypropionic acid oxidizing ferment:Substrate used have D-ALPHA-Hydroxypropionic acid, glycolic, D- phenyllactic acids, D- pairs
Hydroxyphenyl lactic acid, D- tartaric acid, D-malic acid, D- mandelic acids, D- 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, D-ALPHA-Hydroxypropionic acid 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.(R) Alpha-hydroxy in-alpha-hydroxy acid ester is dehydrogenated and is oxidized to corresponding 2-ketoacid
Ester, (S)-alpha-hydroxy acid ester is not oxidized.
The optical purity of product (S)-alpha-hydroxy acid ester is evaluated by enantiomeric excess value (%e.e):
Enantiomeric excess value %e.e=[(SS-SR)/(SS+SR)] × 100%
(S)-alpha-hydroxy acid ester yield (%)=(SS/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:Clonal expression goes out one from Carnimonas nigrificans ATCC BAA-78
New D-ALPHA-Hydroxypropionic acid oxidizing ferment is planted, the enzyme can aoxidize (R)-alpha-hydroxy acid and (R)-alpha-hydroxy acid ester, can be used for prepare with scale hand
Pure (the S)-alpha-hydroxy acid ester of property, with important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of D-ALPHA-Hydroxypropionic acid oxidase gene of the present invention and colibacillus engineering build.
1st, the extraction of Carnimonas nigrificans ATCC BAA-78DNA
Carnimonas nigrificans ATCC BAA-78 bacterial strains are cultivated into 12h in LB culture mediums, 12,
000rmp/min centrifugations 10min obtains thalline, is operated according to it using bacterial genomes DNA extraction agents box (TaKaRa companies)
Phage gene group STb gene is extracted, refrigerator is put 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 D-ALPHA-Hydroxypropionic acid oxidase gene
(1) design of primers
Designing primer sequence is:
Primer 1:5'GCCGGGATCCCTGTCAACCGAGGCTCGTCAGCAAT 3'
Primer 2:5'GCCGTCTAGACAGCTCTGCTGGTGATTGGCCTGC 3'
(2) PCR amplifications
Two primers of synthesis more than, with the genomic DNA of Carnimonas nigrificans ATCC BAA-78
For template 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 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 D-ALPHA-Hydroxypropionic acid 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.With D-ALPHA-Hydroxypropionic acid as substrate, by substrate and pH
For 9.0 phosphate buffer under 30-60 DEG C of different temperature conditionss water-bath 15min, determine D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity, really
The optimal reactive temperature for determining enzyme is 35 DEG C.
Embodiment 4
The present embodiment is the optimum pH of D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.With D-ALPHA-Hydroxypropionic acid as substrate, by substrate in pH
3-9,35 DEG C of water-bath 15min determine the enzyme activity of enzyme, as a result find the D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity highest under the conditions of pH 9.0.
Embodiment 5
The present embodiment is D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention and the response characteristic of different substrates, is listed in Table 2 below.
Activity of the D-ALPHA-Hydroxypropionic acid 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 S)-alpha-hydroxy acid ester of all kinds of height can be obtained,
The optics selectivity of the enzyme is very good.
SEQUENCE LISTING
<110>J Southern Yangtze University
<120>A kind of oxidizing ferment and its application
<130> No
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1725
<212> DNA
<213> Carnimonas nigrificans ATCC BAA-78
<400> 1
ctgtcaaccg aggctcgtca gcaattaatc aaccgtcttg gcagcatcgt cggtaacaag 60
cacacgctga gcgatgagcg cagcaccagt cgtttccgca aaggatttcg tttcggtgat 120
ggccccgcgc tggccgttgt gcagcctgcc tcgctggtag agcagtggcg ggtgctgaaa 180
gcgtgtgaag aagccgatgt gatcgtgatc atgcaggctg ccaataccgg cctcactggc 240
ggctcgaccc cggatggcga tgattatgac cgcgacatcg tgattatcag caccctgcgc 300
atggatcgca ttcatctgat tcatgacgga aaacaggtgg tctgtctgcc tggcgcaacg 360
ctttataacc ttgaaaaagc tctcacgccg ctgggacgcg agccgcactc cgtgatcggc 420
tcctcctgta tcggggcgtc ggtatttggc ggtgtctcca ataattccgg cggtgcattg 480
gtaagacgcg gccccgccta taccgagttc gcgatttacg cccagcgcga tagcgaaggc 540
gagctgcgtc tggtcaatca tctgggcatt gcgctgggtg acgatcccga agagattctc 600
actcggctgg aaaacggcga tttcagcgaa agtgatatct ccgacaacgg cgcagcttcc 660
gatcaccact atcagaacca cgttcgccac gtcgatgagg acacccccgc gcgcttcaat 720
gccgacccta cgcgtctgca cgaagcgtcg ggcaacgccg gcaagctggc gctctttgcg 780
gtgcgcctgg ataccttcga agcgatatca tccaaggtgt tttatatcgg caccaactct 840
atcgacaagc tcacaaagct gcggcgcgag ctgttggctg aaggtcgtga actgccgatc 900
gccggcgaat acatgcaccg cgagtgtttc gatatcgccg acgtatacgg caaggacacc 960
ttcgtgatga tcgagaaatt cggcaccgac aagatgccgc agatttttgc gatcaaaggc 1020
ggggttgatt catttttatc gcgtttcaat attcaaggct tcacggatcg cgccagccag 1080
tggctatcac ggttgtttcc cgatcatctg ccaaagcgca tgcgcgaata ccgcgacaag 1140
tatgagcact acctgatgct gcgcgtagcg accgatcagc ttgaagagac ccaggggcta 1200
ctgaaggagc tgtttgccga cggtgatggc gactacttcg aatgcaccga ggaggaggga 1260
aacaaagcct tcctgcaccg ctttgctgct gcgggcgcgg ctattcgcta ccgcaccgtg 1320
cacaagcaaa cctccgaagg catggtggcc ctcgacgtgg cactgcggcg taacgatacc 1380
gactggttcg agcaccttcc caaggagatc gacgacaaat tagagaaaaa gctctattac 1440
ggccacttcc tgtgccatgt gatgcaccag gattatgtcg tcaagaaagg caacgactgc 1500
atggacgttg aacatgagat gtggaaggag ctggacgccc gtggcgccga ataccccgcc 1560
gagcacaatg ttgggcatct gtatcacgcc aaacccgccc tcgccgagca ctatcagcag 1620
cttgacccct gcaactgctt caaccccggc atcggccaga cctcgaagct gaagtattgg 1680
ggcaaacagt cgcagcaaac gcaggccaat caccagcaga gctga 1725
<210> 2
<211> 574
<212> PRT
<213> Carnimonas nigrificans ATCC BAA-78
<400> 2
Met Ser Thr Glu Ala Arg Gln Gln Leu Ile Asn Arg Leu Gly Ser Ile
1 5 10 15
Val Gly Asn Lys His Thr Leu Ser Asp Glu Arg Ser Thr Ser Arg Phe
20 25 30
Arg Lys Gly Phe Arg Phe Gly Asp Gly Pro Ala Leu Ala Val Val Gln
35 40 45
Pro Ala Ser Leu Val Glu Gln Trp Arg Val Leu Lys Ala Cys Glu Glu
50 55 60
Ala Asp Val Ile Val Ile Met Gln Ala Ala Asn Thr Gly Leu Thr Gly
65 70 75 80
Gly Ser Thr Pro Asp Gly Asp Asp Tyr Asp Arg Asp Ile Val Ile Ile
85 90 95
Ser Thr Leu Arg Met Asp Arg Ile His Leu Ile His Asp Gly Lys Gln
100 105 110
Val Val Cys Leu Pro Gly Ala Thr Leu Tyr Asn Leu Glu Lys Ala Leu
115 120 125
Thr Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser Ser Cys Ile
130 135 140
Gly Ala Ser Val Phe Gly Gly Val Ser Asn Asn Ser Gly Gly Ala Leu
145 150 155 160
Val Arg Arg Gly Pro Ala Tyr Thr Glu Phe Ala Ile Tyr Ala Gln Arg
165 170 175
Asp Ser Glu Gly Glu Leu Arg Leu Val Asn His Leu Gly Ile Ala Leu
180 185 190
Gly Asp Asp Pro Glu Glu Ile Leu Thr Arg Leu Glu Asn Gly Asp Phe
195 200 205
Ser Glu Ser Asp Ile Ser Asp Asn Gly Ala Ala Ser Asp His His Tyr
210 215 220
Gln Asn His Val Arg His Val Asp Glu Asp Thr Pro Ala Arg Phe Asn
225 230 235 240
Ala Asp Pro Thr Arg Leu His Glu Ala Ser Gly Asn Ala Gly Lys Leu
245 250 255
Ala Leu Phe Ala Val Arg Leu Asp Thr Phe Glu Ala Ile Ser Ser Lys
260 265 270
Val Phe Tyr Ile Gly Thr Asn Ser Ile Asp Lys Leu Thr Lys Leu Arg
275 280 285
Arg Glu Leu Leu Ala Glu Gly Arg Glu Leu Pro Ile Ala Gly Glu Tyr
290 295 300
Met His Arg Glu Cys Phe Asp Ile Ala Asp Val Tyr Gly Lys Asp Thr
305 310 315 320
Phe Val Met Ile Glu Lys Phe Gly Thr Asp Lys Met Pro Gln Ile Phe
325 330 335
Ala Ile Lys Gly Gly Val Asp Ser Phe Leu Ser Arg Phe Asn Ile Gln
340 345 350
Gly Phe Thr Asp Arg Ala Ser Gln Trp Leu Ser Arg Leu Phe Pro Asp
355 360 365
His Leu Pro Lys Arg Met Arg Glu Tyr Arg Asp Lys Tyr Glu His Tyr
370 375 380
Leu Met Leu Arg Val Ala Thr Asp Gln Leu Glu Glu Thr Gln Gly Leu
385 390 395 400
Leu Lys Glu Leu Phe Ala Asp Gly Asp Gly Asp Tyr Phe Glu Cys Thr
405 410 415
Glu Glu Glu Gly Asn Lys Ala Phe Leu His Arg Phe Ala Ala Ala Gly
420 425 430
Ala Ala Ile Arg Tyr Arg Thr Val His Lys Gln Thr Ser Glu Gly Met
435 440 445
Val Ala Leu Asp Val Ala Leu Arg Arg Asn Asp Thr Asp Trp Phe Glu
450 455 460
His Leu Pro Lys Glu Ile Asp Asp Lys Leu Glu Lys Lys Leu Tyr Tyr
465 470 475 480
Gly His Phe Leu Cys His Val Met His Gln Asp Tyr Val Val Lys Lys
485 490 495
Gly Asn Asp Cys Met Asp Val Glu His Glu Met Trp Lys Glu Leu Asp
500 505 510
Ala Arg Gly Ala Glu Tyr Pro Ala Glu His Asn Val Gly His Leu Tyr
515 520 525
His Ala Lys Pro Ala Leu Ala Glu His Tyr Gln Gln Leu Asp Pro Cys
530 535 540
Asn Cys Phe Asn Pro Gly Ile Gly Gln Thr Ser Lys Leu Lys Tyr Trp
545 550 555 560
Gly Lys Gln Ser Gln Gln Thr Gln Ala Asn His Gln Gln Ser
565 570
Claims (5)
1. a kind of D-ALPHA-Hydroxypropionic acid oxidizing ferment from Carnimonas nigrificans, its amino acid sequence is SEQ ID NO:
Shown in 2.
2. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, its nucleotides sequence is classified as SEQ ID NO:Shown in 1.
3. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, its optimal reactive temperature is 35 DEG C, and optimal reaction pH is 9.
4. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, oxidable D-ALPHA-Hydroxypropionic acid, glycolic, D- phenyllactic acids, D- para hydroxybenzenes
Lactic acid, D- tartaric acid, D-malic acid, D- mandelic acids, D- danshensus, generate corresponding ketone acid.
5. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, (the R)-alpha-hydroxy acid ester in oxidable racemic ' alpha '-carboxylic esters, tear open
Divide and prepare corresponding optical voidness (S)-alpha-hydroxy acid ester and alpha-keto ester, described alpha-hydroxy acid ester is one of following:Danshensu
Norbornene ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene lactic acid
It is isopropyl ester, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu asarum alcohol ester, phenyllactic acid asarum alcohol ester, right
Hydroxyphenyl lactic acid asarum alcohol ester.
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CN201710006599.3A CN106754793B (en) | 2017-01-05 | 2017-01-05 | A kind of oxidizing ferment and its application |
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