CN101368174B - Levulose valine oxidizing enzyme of high activity - Google Patents
Levulose valine oxidizing enzyme of high activity Download PDFInfo
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- CN101368174B CN101368174B CN2008101663406A CN200810166340A CN101368174B CN 101368174 B CN101368174 B CN 101368174B CN 2008101663406 A CN2008101663406 A CN 2008101663406A CN 200810166340 A CN200810166340 A CN 200810166340A CN 101368174 B CN101368174 B CN 101368174B
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 43
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 43
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 title claims abstract description 31
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 239000004474 valine Substances 0.000 title claims abstract description 28
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 229960002737 fructose Drugs 0.000 title claims description 24
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims description 21
- 230000001590 oxidative effect Effects 0.000 title claims description 21
- 150000001413 amino acids Chemical group 0.000 claims abstract description 11
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- 238000000034 method Methods 0.000 abstract description 12
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- 238000002360 preparation method Methods 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 241000745841 Arthrobacter sp. 2-4-1 Species 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract description 5
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- 230000002829 reductive effect Effects 0.000 abstract description 4
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- 108090000854 Oxidoreductases Proteins 0.000 abstract 7
- 241000588724 Escherichia coli Species 0.000 abstract 2
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 abstract 2
- 235000001258 Cinchona calisaya Nutrition 0.000 abstract 1
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 abstract 1
- 229960000948 quinine Drugs 0.000 abstract 1
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 241000186216 Corynebacterium Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 238000006479 redox reaction Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ZTQGWROHRVYSPW-UHFFFAOYSA-N 3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 ZTQGWROHRVYSPW-UHFFFAOYSA-N 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 101710176276 SSB protein Proteins 0.000 description 1
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
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- 229940049547 paraxin Drugs 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to high-activity fructose valine oxidase and a preparation method thereof, the amino acid sequence of the fructose valine oxidase is the sequence of SEQ ID. No.2, and the nucleotide sequence is shown in SEQ ID. No.1. The preparation steps are: (1) error-prone PCR amplification is carried out with the FVO gene coded sequence of Corynebacterium sp.2-4-1 as the template to establish the mutation bank of the fructose valine oxidase; (2) the mutation bank is transferred into escherichia coli and the clone, reconstruction, conversion and expression are carried out to the gene of the transferred escherichia coli; and (3) the activity of enzyme is determined by utilizing the quinine method and the high-activity fructose valine oxidase is screened out. The activity of the obtained fructose valine oxidase is about 6 times of the activity of the ordinary fructose valine oxidase, the fructose valine oxidase can be used for testing saccharification Hb kit, the used amount of enzyme is reduced, and the reaction time is shortened.
Description
Technical field
The present invention relates to a kind of levulose valine oxidizing enzyme of high activity.
Background technology
Glycolated hemoglobin in the blood (hereinafter to be referred as: saccharification Hb) reflect biological intravital glucose level in one period, be used as the important indicator of diagnosis of diabetes and treatment in recent years.At present, saccharification Hb can adopt methods such as high performance liquid chromatography, microtrabeculae method, immunization, pigment method to measure, but these methods or need instrumentation, cost an arm and a leg, perhaps detection time is long, measuring accuracy is poor, and the popular enzyme process detects recently, utilize redox reaction, do not need special determining instrument, easy to operation, the precision height, time is short, therefore is widely used in biochemical analysis and clinical examination.
Utilize in the mensuration process of saccharification Hb of above-mentioned redox reaction, need the special enzyme of a class---Fructoamino-acid-oxidase, this enzyme can act on saccharification Hb or glycated amino acid, produce hydrogen peroxide, add peroxidase and reductive agent then, make between hydrogen peroxide and the reductive agent redox reaction takes place, can measure amount of hydrogen peroxide by the colour developing of measuring reductive agent, thus saccharification Hb content in the sample as can be known.
Yet, utilize existing Fructoamino-acid-oxidase catalysis to react, the time still dislikes long slightly, and enzyme amount demand is higher.So require development of new high reactivity Fructoamino-acid-oxidase.
Summary of the invention
The objective of the invention is to overcome above-mentioned deficiency, the levulose valine oxidizing enzyme of high activity that provides a kind of activity to be about active 6 times of common levulose valine oxidizing enzyme, it can be used for saccharification Hb test kit and detects used enzyme amount minimizing, reaction times shortening.
The present invention is based on the encoding sequence of levulose valine oxidizing enzyme, by the fallibility round pcr, set up the sudden change library of levulose valine oxidizing enzyme, the library that will suddenly change changes intestinal bacteria over to, carry out the two-wheeled screening, obtained the active novel high-activity levulose valine oxidizing enzyme that is about active 6 times of common levulose valine oxidizing enzyme.This levulose valine oxidizing enzyme of high activity, its aminoacid sequence are the sequence of SEQ ID.No.2.
The polynucleotide of a kind of levulose valine oxidizing enzyme of high activity of encoding of the present invention, its nucleotides sequence is classified sequence shown in the SEQ ID.No.1 as.
Preparation method's step of levulose valine oxidizing enzyme of high activity of the present invention is as follows:
(1) the FVO gene coded sequence with Corynebacterium sp.2-4-1 is a template, carries out the fallibility pcr amplification, sets up the sudden change library of levulose valine oxidizing enzyme;
(2) library that will suddenly change changes intestinal bacteria over to, to its gene clone, recombinant conversion and expression;
(3) utilize the quinone method to measure enzymic activity, filter out levulose valine oxidizing enzyme of high activity.
Along with the continuous expansion of enzyme catalysis range of application and progressively going deep into of research, the investigator finds, enzymatic accuracy and validity usually can not satisfy the requirement of enzymology and industrial applications well, and the poor stability of natural enzyme, active low make catalytic efficiency very low, also lack the catalysis that commercial value is arranged.For this reason, people utilize the orthogenesis technology of enzyme that natural enzyme is transformed, thereby obtain the evolution enzyme with some characteristic of expectation in advance.
Orthogenesis comprises random mutation and screening, wherein the comparatively general technology of random mutation application is fallibility PCR sudden change, promptly by adjusting the ionic concn in the PCR system, various dNTP content, primer and template concentrations are used the Taq enzyme that easily produces sudden change, use the high methods such as bacterial strain of mutation rate, make the PCR product produce many point mutation, thereby change proteic aminoacid sequence with respect to template.By conditional filtering, obtain to have the purpose enzyme of desired characteristic.
Corynebacterium Corynebacterium sp.2-4-1 bacterial strain contains the encoding gene of FV oxydase (FVO).The present invention is based on the gene coded sequence of FVO, the fallibility PCR primer of two FVO genes of design, the upstream primer sequence is: 5 '-TTGTTCGGATCCATGTCCTCCACCGCTAC-3 ', the downstream primer sequence is: 5 '-TTGTTCAAGCTTCTAGGAGAACCGGCCCG-3 '.With the gene coded sequence of FVO as template, carry out the fallibility pcr amplification, after 35 circulations of increasing, the PCR product purification reclaims, be connected with prokaryotic expression carrier pMD-18T Vector, be transformed among the intestinal bacteria XL1-Red, coating LB flat board, collection contains inserts segmental clone, constitutive mutation body storehouse.
Extract the plasmid DNA in the mutant library, enzyme is cut, reclaim the purpose fragment, connect into carrier pGEX-4T-1m, be transformed into the BL-21 bacterial strain, coat and contain IPTG, fructose Xie Ansuan and N-(carboxymethylamino carbonyl)-4, the LB flat board of 4-two (methylamino)-p-diaminodiphenyl [DA-64], because intestinal bacteria self contain peroxidase, thus 37 ℃ cultivate after 12-24 hour visible obvious metachromatism.Fast and the big clone of variable color circle of picking variable color carries out the liquid nutrient medium cultivation.
Being cloned in 25 ℃ of liquid nutrient mediums of going out of preliminary screening cultivated 48 hours, abduction delivering, the cracking bacterium, utilize the quinone method to measure enzymic activity, finally obtain the high reactivity bacterial strain that a strain enzymic activity is about active 6 times of common levulose valine oxidizing enzyme, its entrained FVO sudden change called after FVO-m-21.Find by sequencing analysis, five base point mutation take place in this mutant clon, be respectively 57 C → T, 374 T → C, 534 G → A, 914 C → A and 1056 G → C in the base sequence, having caused two amino acid mutations altogether, is respectively 125 Ile → Thr and 305 Ala → Glu in the aminoacid sequence.FVO-m-21 is cultivated in a large number, and purifies and separates purpose enzyme behind the abduction delivering is used for the proteic detection of saccharification Hb, can obtain better effects.
Levulose valine oxidizing enzyme of the present invention, active high, be about active 6 times of common levulose valine oxidizing enzyme, it can be used for saccharification Hb test kit and detects used enzyme amount minimizing, reaction times shortening, be suitable for the preparation and the application of test kit, can be widely used in clinical detection.And the preparation method adopts existing conventional means, and is easy to implement.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, but be not limited in following embodiment.
Embodiment 1
FVO gene coded sequence with Corynebacterium sp.2-4-1 is a template, carries out the fallibility pcr amplification.
1. primer sequence:
Forward primer: 5 '-TTGTTCGGATCCATGTCCTCCACCGCTAC-3 '
Reverse primer: 5 '-TTGTTCAAGCTTCTAGGAGAACCGGCCCG-3 '
2. fallibility PCR reaction system and reaction conditions:
The PCR reaction system:
Contain in the 100 μ L systems:
10mM?Tris-HCl?pH8.30，50mM?KCl，6.5mM?MgCl
2，
0.15mM?MnCl
2，0.2mM?dGTP/dATP，0.8mM?dTTP/dCTP，
2.2 μ g SSB Protein, 0.5 μ M forwards/reverse primer, 10ng template DNA
2.5 the TaqDNA of unit polysaccharase.
The PCR reaction conditions:
94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; 72 ℃ of 10min; 4 ℃ of forever.
3. fallibility PCR product is got 3 μ L in 1% agarose gel electrophoresis, about visible 1kb the product band is arranged.Fallibility PCR product is reclaimed the test kit purifying with purifying reclaim, be connected, be transformed among the intestinal bacteria XL1-Red, be coated with Amp resistance LB flat board, can obtain the mutant library of the FVO gene of Corynebacterium sp.2-4-1 with pMD-18T Vector.
Embodiment 2:
The primary dcreening operation of mutants which had:
Extract the plasmid DNA in the mutant library, with BamH I and Hind III double digestion, reclaim the purpose fragment about 1kb, carrier pGEX-4T-1m uses BamH I and Hind III double digestion equally, reclaims, the two 4 ℃ of connection is spent the night, be transformed into the BL-21 bacterial strain, coat and contain IPTG, fructose Xie Ansuan and N-(carboxymethylamino carbonyl)-4, the LB flat board of 4-two (methylamino)-p-diaminodiphenyl [DA-64], cultivate after 12-24 hour visible obviously metachromatism for 37 ℃.Fast and the big clone of variable color circle of picking variable color carries out liquid nutrient medium and cultivates, and picking is cloned 48 altogether.
Embodiment 3:
The active quinone method of mutant enzyme detects:
1. after 4 hours, collect bacterium liquid at abduction delivering, measure and write down the OD600 value of cell culture.
2. preparation detects the bacterial lysate that enzyme is lived.At 374 μ L B-PER
TMAgain suspension cell in the bacterioprotein extraction agent (Pierce product78248) adds 50uL proteolytic enzyme and phosphorglase inhibitor and bacterial cell extract (Sigma product P 8465), the paraxin of 1uL34mg/mL (preparing with methyl alcohol) again.The vortex vibration is one minute fast.Place 5min on ice.Centrifugal 1min is placed on ice.
3. measure protein content with the Bradford method.
4. get the above-mentioned bacterial lysate of 50 μ L and be added in the quinone method reaction mixture, 37 ℃ of temperature are bathed 1-3min, measure 555nm place light absorption value.The reaction mixture composition is: 100mM potassium phosphate buffer (pH8.0), 1purpurogallin unit/ml peroxidase, the amino antipyrine of 0.45mM4-, 0.5mM TOOS (N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-monomethylaniline), 5.0mM fructose Xie Ansuan, cumulative volume are 3mL.
Wherein, but an enzyme activity unit be defined as 37 ℃ of per minute catalysis and produce 0.5 μ M quinone dyestuff.Actual enzyme activity is measured by the formed nmol product of the every mg protein of per minute.
Embodiment 4:
The sequencing of high reactivity sudden change FVO:
By enzyme activity determination, the vigor of filtering out is about the height sudden change alive of 6 times of common levulose valine oxidizing enzyme vigor.Extract plasmid DNA, order-checking finds that this mutant nucleotide sequence is shown in SEQ ID.No.1.
The site of wherein undergoing mutation is 57 C → T, 374 T → C, 534 G → A, 914 C → A and 1056 G → C.
Corresponding amino acid sequence is shown in SEQ ID.No.2.
The amino acid sites of wherein undergoing mutation has two amino acid mutations, is respectively 125 Ile → Thr and 305 Ala → Glu in the aminoacid sequence.
Above embodiment is to the explanation of patent and further explains, rather than limitation of the present invention, and any modification of being made in spirit of the present invention and rights protection scope all falls into protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Meikang Biotech Co., Ltd., Ningbo
＜120〉levulose valine oxidizing enzyme of high activity and preparation method thereof
<130>001
<160>2
<170>PatentIn?version3.3
<210>1
<211>1119
<212>DNA
<213>Corynebacterium?sp.
<220>
<221>CDS
<222>(1)..(1119)
<400>1
<210>2
<211>372
<212>PRT
<213>Corynebacterium?sp.
<400>2
Claims (2)
1. a levulose valine oxidizing enzyme of high activity is characterized in that its aminoacid sequence is the sequence of SEQ ID.No.2.
2. polynucleotide of the described levulose valine oxidizing enzyme of high activity of claim 1 of encoding, its nucleotides sequence is classified sequence shown in the SEQ ID.No.1 as.
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| CN2008101663406A CN101368174B (en) | 2008-09-23 | 2008-09-23 | Levulose valine oxidizing enzyme of high activity |
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Inventor after: Zou Bingde Inventor before: Zou Bingde Inventor before: Jiang Yunfei |
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Address after: 315100 Ningbo science and technology zone, Yinzhou City, Zhejiang, Ma Ma Industrial Park Road, No. 358 Patentee after: NINGBO MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. Address before: 315100 Ningbo science and technology zone, Yinzhou City, Zhejiang, Ma Ma Industrial Park Road, No. 358 Patentee before: Ningbo Medical System Biotechnology Co.,Ltd. |
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Address after: 315104 Zhejiang city of Ningbo province Yinzhou District Qiming Road No. 299 Patentee after: MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. Address before: 315100 Ningbo science and technology zone, Yinzhou City, Zhejiang, Ma Ma Industrial Park Road, No. 358 Patentee before: NINGBO MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. |
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Application publication date: 20090218 Assignee: Ningbo Shengyuan Biotechnology Co., Ltd. Assignor: MEDICAL SYSTEM BIOTECHNOLOGY Co.,Ltd. Contract record no.: X2022330000361 Denomination of invention: Highly active fructose-valine oxidase Granted publication date: 20110427 License type: Common License Record date: 20220809 |
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