CN109706130A - Fructoamino-acid-oxidase mutant, detection kit and detection method - Google Patents
Fructoamino-acid-oxidase mutant, detection kit and detection method Download PDFInfo
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Abstract
This application involves the Fructoamino-acid-oxidase mutant obtained by Fructoamino-acid-oxidase parent mutant.The application further relates to encode the isolated polynucleotides of the Fructoamino-acid-oxidase mutant, the recombinant DNA of polynucleotides comprising the separation, the host cell that is converted with the recombinant DNA.Detection kit and detection method of the application further to the method for expressing the Fructoamino-acid-oxidase mutant and for measuring the glycosylated protein comprising fructosyl lysine.Fructoamino-acid-oxidase mutant of the invention is compared with Fructoamino-acid-oxidase parent, there is significant specificity to the glycosylated protein comprising fructosyl lysine, therefore detection kit and detection method of the invention can carry out specific detection to the glycosylated protein comprising fructosyl lysine.
Description
Technical field
The present invention relates to biochemistry detection technical fields, and in particular to a kind of Fructoamino-acid-oxidase mutant, detection
Kit and detection method, the especially detectable glycosylated protein comprising fructosyl lysine.
Background technique
Diabetes are one of the principal diseases of influence health of people, and are in rejuvenation trend.In the internal of diabetic
In hyperglycemia environment, protein macromolecule is glycosylated with sugar by complicated biochemical reaction generation, forms glycosylation albumen
Matter.Wherein, seralbumin peptide chain the 189th lysine forms macromolecule ketoamine in conjunction with glucose, and there is fructosyl-to rely
Propylhomoserin structure.The half life of albumin, is shorter than hemoglobin, about 17-19 days, can be by measuring serum Effects of advanced glycated bovine serum albumin
Level come the glycemic control situation before reflecting for 2 to 3 weeks.Therefore, Effects of advanced glycated bovine serum albumin be diabetes Blood diagnosis index it
One.
Fructoamino-acid-oxidase (FAOD) is a kind of enzyme that can hydrolyze Fructoamino-acid.At present in prokaryotes and
Fructoamino-acid-oxidase is found in eucaryote such as fungi.Patent literature report in relation to the enzyme is for instance that come from stick
The fructosyl amino of shape Bacillus (Japan Patent 5-33997 and 6-65300) or aspergillus (Japanese Patent Publication 3-155780)
Acid oxidase；Fructosyl amine from candida removes glycosyl enzyme (Japanese Patent Publication 6-46846)；Fruit from Penicillium
Glycosyl amino acid removes glycosyl enzyme (Japanese Patent Publication 4-4874)；From corynebacterium, Fusarium, Acremonium or De Bali
The Ketoamine oxidase (Japanese Patent Publication 5-192193) of family name's saccharomyces.However, above-mentioned enzyme is for Effects of advanced glycated bovine serum albumin (GA)
Specific insufficient, end can provide the Accurate Analysis to Effects of advanced glycated bovine serum albumin.
Summary of the invention
The purpose of the present invention is to provide to fructosyl lysine have high degree of specificity Fructoamino-acid-oxidase,
And detection kit and detection method using the Fructoamino-acid-oxidase.
Therefore, in one aspect, the present invention provides a kind of Fructoamino-acid-oxidase mutant, the Fructoamino-acid
Oxidation enzyme mutant is obtained as the Fructoamino-acid-oxidase parent mutant with amino acid sequence shown in SEQ ID NO:2
It arrives.
Specifically, the Fructoamino-acid-oxidase mutant:
It is amino acid sequence the 41st as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:4
Aspartic acid (D) sport alanine (A) and obtain；
It is amino acid sequence the 51st as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:6
Alanine (A) sport glycine (G) and obtain；
It is amino acid sequence the 62nd as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:8
Arginine (R) sports lysine (K) and obtains；
It is the amino acid sequence the 369th as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:10
The alanine (A) of position sports glycine (G) and obtains.
Above-mentioned Fructoamino-acid-oxidase mutant and the fructosyl with amino acid sequence shown in SEQ ID NO:2
Amino acid oxidase parent compares, and improves to the catalytic activity of fructosyl lysine.
Specifically, above-mentioned Fructoamino-acid-oxidase mutant with have SEQ ID NO:2 shown in amino acid sequence
Fructoamino-acid-oxidase parent compare, at least 63% is improved to the catalytic activity of fructosyl lysine, is more preferably mentioned
Height at least 100% still more preferably improves 150%, still more preferably improves 200% or higher.
In second aspect, the present invention provides a kind of isolated polynucleotides, the polynucleotide encoding present invention of the separation
The Fructoamino-acid-oxidase mutant of one side.
It should be understood that big amino acids can be encoded by more than one codon due to the degeneracy feature of genetic codon,
Therefore the Fructoamino-acid-oxidase mutant of each first aspect present invention can correspond to more than one isolated multicore glycosides
Acid.The polynucleotides of these separation are all covered by the present invention.
Preferably, the polynucleotides of the separation:
With nucleotide sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:4 is encoded；
With nucleotide sequence shown in SEQ ID NO:5, amino acid sequence shown in SEQ ID NO:6 is encoded；
With nucleotide sequence shown in SEQ ID NO:7, amino acid sequence shown in SEQ ID NO:8 is encoded；
With nucleotide sequence shown in SEQ ID NO:9, amino acid sequence shown in SEQ ID NO:10 is encoded.
In the third aspect, the present invention provides a kind of recombinant DNA, which includes carrier and connect this hair with the carrier
The isolated polynucleotides of bright second aspect.It is any commonly employed in connection purpose that carrier used can be molecular biology field
The carrier of DNA molecular.As an example, which is pET22b carrier.
In fourth aspect, the present invention provides a kind of host cell, the recombinant DNA of the host cell third aspect present invention
Conversion.It is any commonly employed in the cell converted with recombinant DNA that host cell used can be molecular biology field, particularly common
Host cell be Escherichia coli (E.coli) cell.
At the 5th aspect, the present invention provides a kind of Fructoamino-acid-oxidase mutant for expressing first aspect present invention
Method, which is characterized in that the expression include make the host cell of fourth aspect present invention in LB liquid medium
After cultivating a period of time at 37 DEG C, IPTG inducing expression is added.It, can be general by molecular biology field after expression of enzymes
Separating and purifying technology is isolated and purified, and pure enzyme preparation is obtained.
At the 6th aspect, the present invention provides a kind of for measuring the detection of the glycosylated protein comprising fructosyl lysine
Kit, the detection kit include the Fructoamino-acid-oxidase mutant of first aspect present invention.The kit may be used also
It is replaced comprising fructosyl lysine FL), N- ethyl-N- (2- hydroxyl -3- sulfopropyl) meta-aminotoluene sodium salt (TOOS) and 4- amino peace
Than woods (4-AAP) reagent.The Fructoamino-acid-oxidase mutant and various reagents of first aspect present invention are divided in reagent
In bottle.
At the 7th aspect, the present invention provides a kind of for measuring the detection of the glycosylated protein comprising fructosyl lysine
Method, the detection method include making sample and first party of the present invention with the glycosylated protein comprising fructosyl lysine
The Fructoamino-acid-oxidase mutant in face contacts, and measures by Fructoamino-acid-oxidase mutant catalysis generation
The amount of hydrogen peroxide.It can make H by the way that TOOS and 4-AAP are added in the reaction system2O2In the presence of peroxidase with
They occur chromogenic reactions, then measure 555nm at light absorption value variation, come measure generation hydrogen peroxide amount, Jin Erji
Calculate the activity of enzyme.The detection method can be used for the nondiagnostic detection of the glycosylated protein comprising fructosyl lysine,
It can be used for diagnostic assays.
Advantageously, can use sixth aspect present invention detection kit, by Fructoamino-acid-oxidase mutant,
TOOS and 4-AAP is configured to reaction reagent mixture in suitable buffer, is then added into the reaction reagent mixture
Sample to be tested with the glycosylated protein comprising fructosyl lysine reacts such as 5 under such as 37 DEG C of reaction temperature
Minute, then measure the variation of light absorption value at 555nm, come measure generation hydrogen peroxide amount, and then calculate the activity of enzyme.
Beneficial effects of the present invention:
Testing through enzyme activity proves, Fructoamino-acid-oxidase mutant of the invention and Fructoamino-acid-oxidase parent
Originally it compares, at least 85% is improved to the catalytic activity of fructosyl lysine, therefore to the glycosylation egg comprising fructosyl lysine
White matter has significant specificity.
The detection kit and detection method for being used to measure the glycosylated protein comprising fructosyl lysine of the invention,
Specific detection can be carried out to the glycosylated protein comprising fructosyl lysine, be expected to use in Biochemical Research, including
The glycosylated protein in blood is measured, can also clinically be used, for detecting the glycosylation of diabetes diagnosis in blood
Protein Index.
Detailed description of the invention
Fig. 1 shows the concentration of the target protein using the measurement of Bradford method, and wherein M indicates molecular weight standard.
Specific embodiment
Below by specific embodiment, invention is further described in detail.
In order to obtain the Fructoamino-acid-oxidase for having high degree of specificity to fructosyl lysine, the present inventor passes through
Numerous studies obtain the Fructoamino-acid oxygen of black spore bacterium of megacanthopore (Periconia macrospinosa) from GeneBank
Change enzyme gene sequence (GeneBank:PVI07354.1), and under the premise of not changing amino acid sequence, optimize its codon,
And His label is added at the end 3' and is named as to obtain the gene order for encoding this Fructoamino-acid-oxidase
PMF(SEQ ID NO:1).The gene of PMF entrusts Jin Weizhi Biotechnology Co., Ltd in Suzhou to synthesize by the present inventor, is connected in
On pET22b carrier, plasmid PMF-pET22b is obtained.
The present inventor carries out point mutation using PMF as Fructoamino-acid-oxidase parental gene sequence, obtains the phase of PMF
It answers the 41st, the 51st, the 62nd or the 369th in amino acid sequence (SEQ ID NO:2) to mutate, obtains four
Fructoamino-acid-oxidase mutant.Point mutation becomes specifically: PMF the 41st aspartic acid (D) sports alanine
(A), the 51st alanine (A) sports glycine (G), the 62nd arginine (R) sports lysine (K) and the 369th
The alanine (A) of position sports glycine (G).
To obtain above-mentioned mutant, the present inventor has four primers in mutational site for the gene order design of PMF
It is right, as shown in table 1 below.
Table 1 is directed to four with the mutational site primer pair of PMF design
The preparation of four Fructoamino-acid-oxidase mutant is illustrated below by way of preparation example, and passes through test example
Illustrate the expression and enzyme activity of these Fructoamino-acid-oxidase mutant, and compares with Fructoamino-acid-oxidase parent
Compared with.In following preparation example and test example, reagent used and instrument are the general reagent and instrument of molecular biology, used
Preparation and test method are the general method of molecular biology, unless otherwise indicated.
41 rite-directed mutagenesis of site of preparation example 1:PMF prepares mutant D41A
It is mutated in order to which the 41st aspartic acid (D) in PMF parent amino acid sequence is sported alanine (A)
Body D41A, using plasmid PMF-pET22b as template, with primer pair PMF-F (SEQ ID NO:11) and 41AR (SEQ ID NO:
14) F-AR segment, is expanded, with primer pair 41AF (SEQ ID NO:13) and PMF-R (SEQ ID NO:12), expands AF-R piece
Section.
The reaction system of PCR amplification are as follows: ddH218 μ L, 2 × Phanta Master Mix of O 25 μ L, 2-41AF and
Each 5 μ L of 1 μ L, 2PAF-pET22b of each 1 μ L or 2-41AR and 2PAF-F of 2PAF-R.PCR response parameter is as follows: 95 DEG C of initial denaturations
5min；95 DEG C of denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min repeat 30 circulations；72 DEG C of extension 5min.Reaction
After, it is separated through 0.8% agarose gel electrophoresis, plastic recovery kit recycling respectively obtains F-AR segment and AF-R piece
Section.
Then full-length gene is expanded.The reaction condition of amplification are as follows: ddH213 μ L, 2 × Phanta Master Mix25 μ of O
Each 1 μ L, F-AR segment of L, PMF-F and PMF-R and each 5 μ L of AF-R segment.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95
DEG C denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeat 30 recycle；72 DEG C of extension 5min.After completion of the reaction,
It is separated through 0.8% agarose gel electrophoresis, plastic recovery kit recycling obtains overall length mutated gene D41A.
Under 37 DEG C of water-baths, D41A segment and carrier pET- are cut with identical restriction enzyme EcoRI and XhoI is bis-
22b, digestion system are as follows: ddH275 each 4 μ L, pET-22b or D41A segments of μ L, XhoI and EcoRI of μ L, 10 × Buffer of O are each
30μL.With 0.8% agarose gel electrophoresis detection digestion it is complete after, with kit recycle endonuclease bamhi.
D41A segment after the recovery is mixed with carrier pET-22b segment, 16 DEG C of companies are placed under the action of T4 ligase
Meet a few hours, enzyme disjunctor system are as follows: 10 × Buffer, 1 μ L, T4 Ligase, 1 μ L, pET-22b carrier segments and D41A segment each 4
μL.Connection product is converted into DH5 α competent cell again, is coated on the LB plate containing Ampr resistance, 37 DEG C were cultivated
Night.Picking monoclonal is cultivated, and is extracted plasmid order-checking and is analysed whether that enzyme is linked to be function.Through sequence verification recombinant plasmid D41A-
PET22b is constructed successfully, and sequence is correct.The nucleotides sequence of gained mutant D41A is classified as SEQ ID NO:3, and amino acid sequence is
SEQ ID NO:4, referring to sequence table.
51 rite-directed mutagenesis of site of preparation example 2:PMF prepares mutant A51G
Mutant is obtained in order to which the 51st alanine (A) in PMF parent amino acid sequence is sported glycine (G)
A51G, using plasmid PMF-pET22b as template, with primer pair PMF-F (SEQ ID NO:11) and 51GR (SEQ ID NO:16),
F-GR segment, primer pair 51GF (SEQ ID NO:15) and PMF-R (SEQ ID NO:12) are expanded, GF-R segment is expanded.
The reaction system of PCR amplification are as follows: ddH218 μ L, 2 × Phanta Master Mix of O 25 μ L, 51GF and PMF-R
Each each 5 μ L of 1 μ L, PMF-pET22b of 1 μ L or 51GR and PMF-F.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95 DEG C of changes
Property 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeat 30 circulation；72 DEG C of extension 5min.After completion of the reaction, it passes through
0.8% agarose gel electrophoresis separation, plastic recovery kit recycling respectively obtain F-GR segment and GF-R segment.
Then full-length gene is expanded.The reaction condition of amplification are as follows: ddH213 μ L, 2 × Phanta Master Mix25 μ of O
Each 2 μ L, F-GR segment of L, PMF-F and PMF-R and each 5 μ L of GF-R segment.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95
DEG C denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeat 30 recycle；72 DEG C of extension 5min.After completion of the reaction,
It is separated through 0.8% agarose gel electrophoresis, plastic recovery kit recycling obtains overall length mutated gene A51G.
Under 37 DEG C of water-baths, A51G segment and carrier pET- are cut with identical restriction enzyme EcoRI and XhoI is bis-
22b, digestion system are as follows: ddH275 each 4 μ L, pET-22b or A51G segments of μ L, XhoI and EcoRI of μ L, 10 × Buffer of O are each
30μL.With 0.8% agarose gel electrophoresis detection digestion it is complete after, with kit recycle endonuclease bamhi.
A51G segment after the recovery is mixed with carrier pET-22b segment, 16 DEG C of companies are placed under the action of T4 ligase
Meet a few hours, enzyme disjunctor system are as follows: 10 × Buffer, 1 μ L, T4 Ligase, 1 μ L, pET-22b carrier segments and A51G segment each 4
μL.Connection product is converted into DH5 α competent cell again, is coated on the LB plate containing Ampr resistance, 37 DEG C were cultivated
Night.Picking monoclonal is cultivated, and is extracted plasmid order-checking and is analysed whether that enzyme is linked to be function.Through sequence verification recombinant plasmid A51G-
PET22b is constructed successfully, and sequence is correct.The nucleotides sequence of gained mutant 2A51G is classified as SEQ ID NO:5, and amino acid sequence is
SEQ ID NO:6, referring to sequence table.
62 rite-directed mutagenesis of site of preparation example 3:PMF prepares mutant R62K
Mutant is obtained in order to which the 62nd arginine (R) in PMF parent amino acid sequence is sported lysine (K)
R62K, using plasmid PMF-pET22b as template, with primer pair PMF-F (SEQ ID NO:11) and 62KR (SEQ ID NO:18),
F-KR segment, primer pair 62KF (SEQ ID NO:17) and PMF-R (SEQ ID NO:12) are expanded, KF-R segment is expanded.
The reaction system of PCR amplification are as follows: ddH218 μ L, 2 × Phanta Master Mix of O 25 μ L, 62KF and PMF-R
Each each 5 μ L of 1 μ L, PMF-pET22b of 1 μ L or 62KR and PMF-F.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95 DEG C of changes
Property 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeat 30 circulation；72 DEG C of extension 5min.After completion of the reaction, it passes through
0.8% agarose gel electrophoresis separation, plastic recovery kit recycling respectively obtain F-KR segment and KF-R segment.
Then full-length gene is expanded.The reaction condition of amplification are as follows: ddH213 μ L, 2 × Phanta Master Mix25 μ of O
Each 1 μ L, F-KR segment of L, PMF-F and PMF-R and each 5 μ L of KF-R segment.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95
DEG C denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeat 30 recycle；72 DEG C of extension 5min.After completion of the reaction,
It is separated through 0.8% agarose gel electrophoresis, plastic recovery kit recycling obtains overall length mutated gene R62K.
Under 37 DEG C of water-baths, R62K segment and carrier pET- are cut with identical restriction enzyme EcoRI and XhoI is bis-
22b, digestion system are as follows: ddH275 each 4 μ L, pET-22b or R62K segments of μ L, XhoI and EcoRI of μ L, 10 × Buffer of O are each
30μL.With 0.8% agarose gel electrophoresis detection digestion it is complete after, with kit recycle endonuclease bamhi.
R62K segment after the recovery is mixed with carrier pET-22b segment, 16 DEG C of companies are placed under the action of T4 ligase
Meet a few hours, enzyme disjunctor system are as follows: 10 × Buffer, 1 μ L, T4 Ligase, 1 μ L, pET-22b carrier segments and R62K segment each 4
μL.Connection product is converted into DH5 α competent cell again, is coated on the LB plate containing Ampr resistance, 37 DEG C were cultivated
Night.Picking monoclonal is cultivated, and is extracted plasmid order-checking and is analysed whether that enzyme is linked to be function.Through sequence verification recombinant plasmid R62K-
PET22b is constructed successfully, and sequence is correct.The nucleotides sequence of gained mutant R62K is classified as SEQ ID NO:7, and amino acid sequence is
SEQ ID NO:8, referring to sequence table.
369 rite-directed mutagenesis of site of preparation example 4:PMF prepares mutant A369G
It is mutated in order to which the 369th alanine (A) in PMF parent amino acid sequence is sported glycine (G)
Body A369G, using plasmid PMF-pET22b as template, with primer pair PMF-F (SEQ ID NO:11) and 369GR (SEQ ID NO:
20) F-GR segment, primer pair 369GF (SEQ ID NO:19) and PMF-R (SEQ ID NO:12), are expanded, GF-R segment is expanded.
The reaction system of PCR amplification are as follows: ddH218 μ L, 2 × Phanta Master Mix of O 25 μ L, 369GF and PMF-R
Each each 5 μ L of 1 μ L, PMF-pET22b of 1 μ L or 369GR and PMF-F.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95℃
It is denaturalized 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeats 30 circulations；72 DEG C of extension 5min.After completion of the reaction, it passes through
0.8% agarose gel electrophoresis separation, plastic recovery kit recycling respectively obtain F-GR segment and GF-R segment.
Then full-length gene is expanded.The reaction condition of amplification are as follows: ddH213 μ L, 2 × Phanta Master Mix25 μ of O
Each 1 μ L, F-GR segment of L, PMF-F and PMF-R and each 5 μ L of GF-R segment.PCR response parameter is as follows: 95 DEG C of initial denaturation 5min；95
DEG C denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min, repeat 30 recycle；72 DEG C of extension 5min.After completion of the reaction,
It is separated through 0.8% agarose gel electrophoresis, plastic recovery kit recycling obtains overall length mutated gene A369G.
Under 37 DEG C of water-baths, A369G segment and carrier pET- are cut with identical restriction enzyme EcoRI and XhoI is bis-
22b, digestion system are as follows: ddH275 each 4 μ L, pET-22b or A369G segments of μ L, XhoI and EcoRI of μ L, 10 × Buffer of O are each
30μL.With 0.8% agarose gel electrophoresis detection digestion it is complete after, with kit recycle endonuclease bamhi.
A369G segment after the recovery is mixed with carrier pET-22b segment, 16 DEG C of companies are placed under the action of T4 ligase
Meet a few hours, enzyme disjunctor system are as follows: 10 × Buffer, 1 μ L, T4 Ligase, 1 μ L, pET-22b carrier segments and A369G segment are each
4μL.Connection product is converted into DH5 α competent cell again, is coated on the LB plate containing Ampr resistance, 37 DEG C of cultures
Overnight.Picking monoclonal is cultivated, and is extracted plasmid order-checking and is analysed whether that enzyme is linked to be function.Through sequence verification recombinant plasmid A369G-
PET22b is constructed successfully, and sequence is correct.The nucleotides sequence of gained A369G mutant is classified as SEQ ID NO:9, and amino acid sequence is
SEQ ID NO:10, referring to sequence table.
Test example: the expression of Fructoamino-acid-oxidase mutant and enzyme activity determination
This test example illustrate preparation example preparation four Fructoamino-acid-oxidase mutant expression and enzyme activity, and with
Fructoamino-acid-oxidase parent is compared.
(1) it converts
By recombinant plasmid D41A-pET22b, R62K-pET22b, A369G-pET22b for being prepared in preparation example 1-4 and
A51G-pET22b is transformed into respectively in competence BL21CodonPlus (DE3)-RIPL coli strain.Concrete operations are as follows:
2 μ L plasmids and 50 μ L competence BL21CodonPlus (DE3)-RIPL coli strains are mixed, place 30min on ice, so
42 DEG C of water-bath thermal shock 90s afterwards are immediately placed in cooled on ice 2-3min after thermal shock, and 1mL LB liquid medium is added (no into pipe
Containing Amp), centrifugal concentrating after mixing is coated on the plate containing Amp resistance, and plate is placed in 37 DEG C of constant incubators
Overnight incubation.
(2) inducing expression
Then, single clone is inoculated in 5mL LB liquid medium (containing Amp), in 37 DEG C, it is small that 200rpm cultivates number
When, as bacterium solution OD600=0.4-0.6, the IPTG overnight induction of final concentration of 0.4mM is added under the conditions of 30 DEG C, 200rpm.
Thalline were collected by centrifugation, and is suspended in 1mL 50mM Tris hydrochloride buffer (pH 7.5).Then ultrasonic treatment cell is used,
12000rpm centrifugation, obtains supernatant and precipitating, after being handled respectively with 4 × SDS-PAGE Buffer, boils sample.It utilizes
Whether the Protein Detection result of SDS-PAGE detection gained sample is consistent with target, analyzes the expression quantity of destination protein.As a result it shows
Show has the expression albumen for detecting about 50KD in supernatant, almost the same with expected size, illustrates four fructosyl ammonia
Base acid oxidase mutant can be in e. coli bl21 CodonPlus as Fructoamino-acid-oxidase parent
(DE3) successful expression in-RIPL.
(3) protein purification
Strain is expressed into aforementioned four recombinant expression strain and parent and carry out protein purification, concrete operations are as follows: take 50 μ L glycerol bacterium
In 5mL LB liquid medium (containing Amp), after 37 DEG C, 200rpm overnight incubation, seed liquor is transferred to the training of 2L LB liquid
It supports in base (containing Amp), 37 DEG C, 200rpm cultivates a few hours, as bacterium solution OD600When=0.4-0.6, at 30 DEG C, under the conditions of 200rpm
The IPTG overnight induction of final concentration of 0.4mM is added.Next day collects thallus, with appropriate distillation aqueous suspension precipitating, is incorporated in 2
In 50mL centrifuge tube, 4 DEG C, 7000rpm is centrifuged 3min, abandons supernatant, thallus weight is weighed, with the 50mM of 4 times of volumes of thallus quality
Tris hydrochloride buffer (pH 7.5) suspends, and ultrasonic disruption 20min lytic cell turns after adjusting pH to 7.5 to get crude enzyme liquid
Enter in centrifuge tube, 17000rpm is centrifuged 20min, collects supernatant, is filtered with 0.45 μm of miillpore filter.It will be arrived on thick enzyme supernatant
In the Ni column balanced, repetition loading is primary, substantially flows to end to sample, successively slow with the 50mM Tris hydrochloric acid of 15 times of column volumes
Fliud flushing (pH 7.5), the 10mM imidazoles of 15 times of column volumes, 15 times of column volumes 50mM imidazoles wash foreign protein, then use 250mM miaow
Azoles affords destination protein, the eluent in collecting pipe is carried out SDS-PAGE electrophoresis, as a result such as Fig. 1.Using Bradford
The concentration of the target protein of method measurement.
(4) enzyme activity determination
The reaction equation of enzyme activity determination is as follows:
In formula: PL is fructosyl lysine, and TOOS is N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3- methylaniline sodium
Salt, 4-AAP are 4-AA.
Simply, the 4 Fructoamino-acid-oxidase mutant and fructosyl amino obtained respectively with above-mentioned expression
Acid oxidase parent is enzyme to be measured, using PL as substrate, generates H through enzymatic to be measured2O2, H2O2It develops the color with TOOS and 4-AAP
Reaction, by the variation of light absorption value at measurement 555nm, to measure the activity of enzyme.
Reaction temperature is 37 DEG C, and total reaction volume is 800 μ L, contains 100mmol/L phosphate (pH in reaction system
8.0), 0.1%4-AAP, 0.02%TOOS, 0.12mg/mL PL, 20U/mL peroxidase；After 37 DEG C of preheating 5min, add
Enter diluted 100 μ L of enzyme solution to be measured (diluting enzyme solution according to the measurement range of spectrophotometer) starting is reacted, and control group is added etc.
Measure ddH2O, quick oscillation mix.With 1cm cuvette colorimetric, the absorbance change at 555nm is read, METHOD FOR CONTINUOUS DETERMINATION 5min takes
2-4min rate is calculated.Enzymatic activity is calculated as follows:
In formula: Δ A/min is absorbance change rate per minute, and D is enzyme solution extension rate, VAlwaysFor reaction system total volume
(mL), VSampleFor sample volume (mL), d is cuvette optical path (cm), and ε is molar extinction coefficient (11.9mM-1·cm-1)。
Measurement result is shown in Table 2.
The enzymatic activity of table 2:PMF parent enzyme and its mutant enzyme to fructosyl lysine
By table as it can be seen that four Fructoamino-acid-oxidase mutant of the invention are relative to its corresponding fructosyl amino
Acid oxidase parent shows to significantly improve the enzymatic activity of fructosyl lysine, therefore to including fructosyl lysine
Glycosylated protein has significant specificity, is expected to be used for carrying out the glycosylated protein comprising fructosyl lysine special
Property detection.
Use above specific example is expounded the present invention, is merely used to help understand the present invention, not to
The limitation present invention.The design of those skilled in the art according to the present invention can also be made and several simply push away
It drills, deform or replaces.These are deduced, deformation or alternative are also fallen into scope of the presently claimed invention.
SEQUENCE LISTING
<110>Shenzhen Shang Tai bioengineering Co., Ltd
<120>Fructoamino-acid-oxidase mutant, detection kit and detection method
<130> 18I27542
<160> 40
<170> PatentIn version 3.3
<210> 1
<211> 1317
<212> DNA
<213>artificial sequence
<400> 1
atgtcttcgt cacgtaccac tacaaaagtc attgttgtcg gtggaggtgg cactataggg 60
tcatctacag cactgcatct tcttcgtgcg ggctacacac cctctaatat aacagttcta 120
gatccgtacc caatcccttc ggcacaatca gcaggaaatg atctcaataa gatcatgggt 180
atccgcgtac aaaacgatgt cgacgtacaa ctcagtttag aggccagaga tatgtggaag 240
aacgatgacg tattcaagca tttcttccac aacactggaa ggcttgattg tgcacattca 300
gaggaagagc ttaagtccct gaaaacggag tatcaaaagc tggttgatgc cggtcttgga 360
gaaactcatg aatggcttga taacgaagat gagattttga aaaaaatgcc attgctagag 420
cgagataaca taaaagggtg gaaagccata ttcagcgcag atggtggctg gttagcagcc 480
gcaaaagcga taaatgtaat cgggattata tcgaaagagc aaggcgttag atttggggtt 540
ggcagcgcag gatcattccg aaagcctcta tttgcaagcg acgacgtgac ttgcgtgggt 600
gttgagacgg tggacggaac gaagtactac ggcgataaag taatcatttc cgcaggcgcg 660
tggactccga cactggtaga cttggaagat cagtgttgtt caaaagcatg ggtttatgct 720
cacattcgtc taacaccaaa cgagattgct gagtatcaga atatgcccgt ggtatacaat 780
ggggatgttg gtttcttttt cgaacccgac gagtatggtg tgatgaaagt ctgcgatgaa 840
tttcccggtt tcacgagatt caaacagcat ctgccctacg gtgctgagac tcctaaacgg 900
gtatcagtgc ctcgctctca cgcgaaacat cccacagata cctaccccga tgcttctgaa 960
aaatcaatac agaatgccat tgacgtttac ctgccacgat tcaagaacaa gaagaagttc 1020
aatcgcacgc tatgctggtg tacagacaca gctgatgcag cgctgttggt ttgcgagcat 1080
ccgaaatgga agaatctcat tatcgcgtca ggtgacagcg gacactcctt caaacttctg 1140
ccgaatattg gaaaacatgt ggtggaactt atggagggtc ggttggctga cgatctaaaa 1200
aatgcatgga agtggagacc tggcggtgat gccctaaagt caacgcggcg aggccacgca 1260
aaagacctgg ctgatatgcc tggctggaac catgacgggg aaaaatcaaa aatgtag 1317
<210> 2
<211> 438
<212> PRT
<213>artificial sequence
<400> 2
Met Ser Ser Ser Arg Thr Thr Thr Lys Val Ile Val Val Gly Gly Gly
1 5 10 15
Gly Thr Ile Gly Ser Ser Thr Ala Leu His Leu Leu Arg Ala Gly Tyr
20 25 30
Thr Pro Ser Asn Ile Thr Val Leu Asp Pro Tyr Pro Ile Pro Ser Ala
35 40 45
Gln Ser Ala Gly Asn Asp Leu Asn Lys Ile Met Gly Ile Arg Val Gln
50 55 60
Asn Asp Val Asp Val Gln Leu Ser Leu Glu Ala Arg Asp Met Trp Lys
65 70 75 80
Asn Asp Asp Val Phe Lys His Phe Phe His Asn Thr Gly Arg Leu Asp
85 90 95
Cys Ala His Ser Glu Glu Glu Leu Lys Ser Leu Lys Thr Glu Tyr Gln
100 105 110
Lys Leu Val Asp Ala Gly Leu Gly Glu Thr His Glu Trp Leu Asp Asn
115 120 125
Glu Asp Glu Ile Leu Lys Lys Met Pro Leu Leu Glu Arg Asp Asn Ile
130 135 140
Lys Gly Trp Lys Ala Ile Phe Ser Ala Asp Gly Gly Trp Leu Ala Ala
145 150 155 160
Ala Lys Ala Ile Asn Val Ile Gly Ile Ile Ser Lys Glu Gln Gly Val
165 170 175
Arg Phe Gly Val Gly Ser Ala Gly Ser Phe Arg Lys Pro Leu Phe Ala
180 185 190
Ser Asp Asp Val Thr Cys Val Gly Val Glu Thr Val Asp Gly Thr Lys
195 200 205
Tyr Tyr Gly Asp Lys Val Ile Ile Ser Ala Gly Ala Trp Thr Pro Thr
210 215 220
Leu Val Asp Leu Glu Asp Gln Cys Cys Ser Lys Ala Trp Val Tyr Ala
225 230 235 240
His Ile Arg Leu Thr Pro Asn Glu Ile Ala Glu Tyr Gln Asn Met Pro
245 250 255
Val Val Tyr Asn Gly Asp Val Gly Phe Phe Phe Glu Pro Asp Glu Tyr
260 265 270
Gly Val Met Lys Val Cys Asp Glu Phe Pro Gly Phe Thr Arg Phe Lys
275 280 285
Gln His Leu Pro Tyr Gly Ala Glu Thr Pro Lys Arg Val Ser Val Pro
290 295 300
Arg Ser His Ala Lys His Pro Thr Asp Thr Tyr Pro Asp Ala Ser Glu
305 310 315 320
Lys Ser Ile Gln Asn Ala Ile Asp Val Tyr Leu Pro Arg Phe Lys Asn
325 330 335
Lys Lys Lys Phe Asn Arg Thr Leu Cys Trp Cys Thr Asp Thr Ala Asp
340 345 350
Ala Ala Leu Leu Val Cys Glu His Pro Lys Trp Lys Asn Leu Ile Ile
355 360 365
Ala Ser Gly Asp Ser Gly His Ser Phe Lys Leu Leu Pro Asn Ile Gly
370 375 380
Lys His Val Val Glu Leu Met Glu Gly Arg Leu Ala Asp Asp Leu Lys
385 390 395 400
Asn Ala Trp Lys Trp Arg Pro Gly Gly Asp Ala Leu Lys Ser Thr Arg
405 410 415
Arg Gly His Ala Lys Asp Leu Ala Asp Met Pro Gly Trp Asn His Asp
420 425 430
Gly Glu Lys Ser Lys Met
435
<210> 3
<211> 1317
<212> DNA
<213>artificial sequence
<400> 3
atgtcttcgt cacgtaccac tacaaaagtc attgttgtcg gtggaggtgg cactataggg 60
tcatctacag cactgcatct tcttcgtgcg ggctacacac cctctaatat aacagttcta 120
gctccgtacc caatcccttc ggcacaatca gcaggaaatg atctcaataa gatcatgggt 180
atccgcgtac aaaacgatgt cgacgtacaa ctcagtttag aggccagaga tatgtggaag 240
aacgatgacg tattcaagca tttcttccac aacactggaa ggcttgattg tgcacattca 300
gaggaagagc ttaagtccct gaaaacggag tatcaaaagc tggttgatgc cggtcttgga 360
gaaactcatg aatggcttga taacgaagat gagattttga aaaaaatgcc attgctagag 420
cgagataaca taaaagggtg gaaagccata ttcagcgcag atggtggctg gttagcagcc 480
gcaaaagcga taaatgtaat cgggattata tcgaaagagc aaggcgttag atttggggtt 540
ggcagcgcag gatcattccg aaagcctcta tttgcaagcg acgacgtgac ttgcgtgggt 600
gttgagacgg tggacggaac gaagtactac ggcgataaag taatcatttc cgcaggcgcg 660
tggactccga cactggtaga cttggaagat cagtgttgtt caaaagcatg ggtttatgct 720
cacattcgtc taacaccaaa cgagattgct gagtatcaga atatgcccgt ggtatacaat 780
ggggatgttg gtttcttttt cgaacccgac gagtatggtg tgatgaaagt ctgcgatgaa 840
tttcccggtt tcacgagatt caaacagcat ctgccctacg gtgctgagac tcctaaacgg 900
gtatcagtgc ctcgctctca cgcgaaacat cccacagata cctaccccga tgcttctgaa 960
aaatcaatac agaatgccat tgacgtttac ctgccacgat tcaagaacaa gaagaagttc 1020
aatcgcacgc tatgctggtg tacagacaca gctgatgcag cgctgttggt ttgcgagcat 1080
ccgaaatgga agaatctcat tatcgcgtca ggtgacagcg gacactcctt caaacttctg 1140
ccgaatattg gaaaacatgt ggtggaactt atggagggtc ggttggctga cgatctaaaa 1200
aatgcatgga agtggagacc tggcggtgat gccctaaagt caacgcggcg aggccacgca 1260
aaagacctgg ctgatatgcc tggctggaac catgacgggg aaaaatcaaa aatgtag 1317
<210> 4
<211> 438
<212> PRT
<213>artificial sequence
<400> 4
Met Ser Ser Ser Arg Thr Thr Thr Lys Val Ile Val Val Gly Gly Gly
1 5 10 15
Gly Thr Ile Gly Ser Ser Thr Ala Leu His Leu Leu Arg Ala Gly Tyr
20 25 30
Thr Pro Ser Asn Ile Thr Val Leu Ala Pro Tyr Pro Ile Pro Ser Ala
35 40 45
Gln Ser Ala Gly Asn Asp Leu Asn Lys Ile Met Gly Ile Arg Val Gln
50 55 60
Asn Asp Val Asp Val Gln Leu Ser Leu Glu Ala Arg Asp Met Trp Lys
65 70 75 80
Asn Asp Asp Val Phe Lys His Phe Phe His Asn Thr Gly Arg Leu Asp
85 90 95
Cys Ala His Ser Glu Glu Glu Leu Lys Ser Leu Lys Thr Glu Tyr Gln
100 105 110
Lys Leu Val Asp Ala Gly Leu Gly Glu Thr His Glu Trp Leu Asp Asn
115 120 125
Glu Asp Glu Ile Leu Lys Lys Met Pro Leu Leu Glu Arg Asp Asn Ile
130 135 140
Lys Gly Trp Lys Ala Ile Phe Ser Ala Asp Gly Gly Trp Leu Ala Ala
145 150 155 160
Ala Lys Ala Ile Asn Val Ile Gly Ile Ile Ser Lys Glu Gln Gly Val
165 170 175
Arg Phe Gly Val Gly Ser Ala Gly Ser Phe Arg Lys Pro Leu Phe Ala
180 185 190
Ser Asp Asp Val Thr Cys Val Gly Val Glu Thr Val Asp Gly Thr Lys
195 200 205
Tyr Tyr Gly Asp Lys Val Ile Ile Ser Ala Gly Ala Trp Thr Pro Thr
210 215 220
Leu Val Asp Leu Glu Asp Gln Cys Cys Ser Lys Ala Trp Val Tyr Ala
225 230 235 240
His Ile Arg Leu Thr Pro Asn Glu Ile Ala Glu Tyr Gln Asn Met Pro
245 250 255
Val Val Tyr Asn Gly Asp Val Gly Phe Phe Phe Glu Pro Asp Glu Tyr
260 265 270
Gly Val Met Lys Val Cys Asp Glu Phe Pro Gly Phe Thr Arg Phe Lys
275 280 285
Gln His Leu Pro Tyr Gly Ala Glu Thr Pro Lys Arg Val Ser Val Pro
290 295 300
Arg Ser His Ala Lys His Pro Thr Asp Thr Tyr Pro Asp Ala Ser Glu
305 310 315 320
Lys Ser Ile Gln Asn Ala Ile Asp Val Tyr Leu Pro Arg Phe Lys Asn
325 330 335
Lys Lys Lys Phe Asn Arg Thr Leu Cys Trp Cys Thr Asp Thr Ala Asp
340 345 350
Ala Ala Leu Leu Val Cys Glu His Pro Lys Trp Lys Asn Leu Ile Ile
355 360 365
Ala Ser Gly Asp Ser Gly His Ser Phe Lys Leu Leu Pro Asn Ile Gly
370 375 380
Lys His Val Val Glu Leu Met Glu Gly Arg Leu Ala Asp Asp Leu Lys
385 390 395 400
Asn Ala Trp Lys Trp Arg Pro Gly Gly Asp Ala Leu Lys Ser Thr Arg
405 410 415
Arg Gly His Ala Lys Asp Leu Ala Asp Met Pro Gly Trp Asn His Asp
420 425 430
Gly Glu Lys Ser Lys Met
435
<210> 5
<211> 1317
<212> DNA
<213>artificial sequence
<400> 5
atgtcttcgt cacgtaccac tacaaaagtc attgttgtcg gtggaggtgg cactataggg 60
tcatctacag cactgcatct tcttcgtgcg ggctacacac cctctaatat aacagttcta 120
gatccgtacc caatcccttc ggcacaatca ggaggaaatg atctcaataa gatcatgggt 180
atccgcgtac aaaacgatgt cgacgtacaa ctcagtttag aggccagaga tatgtggaag 240
aacgatgacg tattcaagca tttcttccac aacactggaa ggcttgattg tgcacattca 300
gaggaagagc ttaagtccct gaaaacggag tatcaaaagc tggttgatgc cggtcttgga 360
gaaactcatg aatggcttga taacgaagat gagattttga aaaaaatgcc attgctagag 420
cgagataaca taaaagggtg gaaagccata ttcagcgcag atggtggctg gttagcagcc 480
gcaaaagcga taaatgtaat cgggattata tcgaaagagc aaggcgttag atttggggtt 540
ggcagcgcag gatcattccg aaagcctcta tttgcaagcg acgacgtgac ttgcgtgggt 600
gttgagacgg tggacggaac gaagtactac ggcgataaag taatcatttc cgcaggcgcg 660
tggactccga cactggtaga cttggaagat cagtgttgtt caaaagcatg ggtttatgct 720
cacattcgtc taacaccaaa cgagattgct gagtatcaga atatgcccgt ggtatacaat 780
ggggatgttg gtttcttttt cgaacccgac gagtatggtg tgatgaaagt ctgcgatgaa 840
tttcccggtt tcacgagatt caaacagcat ctgccctacg gtgctgagac tcctaaacgg 900
gtatcagtgc ctcgctctca cgcgaaacat cccacagata cctaccccga tgcttctgaa 960
aaatcaatac agaatgccat tgacgtttac ctgccacgat tcaagaacaa gaagaagttc 1020
aatcgcacgc tatgctggtg tacagacaca gctgatgcag cgctgttggt ttgcgagcat 1080
ccgaaatgga agaatctcat tatcgcgtca ggtgacagcg gacactcctt caaacttctg 1140
ccgaatattg gaaaacatgt ggtggaactt atggagggtc ggttggctga cgatctaaaa 1200
aatgcatgga agtggagacc tggcggtgat gccctaaagt caacgcggcg aggccacgca 1260
aaagacctgg ctgatatgcc tggctggaac catgacgggg aaaaatcaaa aatgtag 1317
<210> 6
<211> 438
<212> PRT
<213>artificial sequence
<400> 6
Met Ser Ser Ser Arg Thr Thr Thr Lys Val Ile Val Val Gly Gly Gly
1 5 10 15
Gly Thr Ile Gly Ser Ser Thr Ala Leu His Leu Leu Arg Ala Gly Tyr
20 25 30
Thr Pro Ser Asn Ile Thr Val Leu Asp Pro Tyr Pro Ile Pro Ser Ala
35 40 45
Gln Ser Gly Gly Asn Asp Leu Asn Lys Ile Met Gly Ile Arg Val Gln
50 55 60
Asn Asp Val Asp Val Gln Leu Ser Leu Glu Ala Arg Asp Met Trp Lys
65 70 75 80
Asn Asp Asp Val Phe Lys His Phe Phe His Asn Thr Gly Arg Leu Asp
85 90 95
Cys Ala His Ser Glu Glu Glu Leu Lys Ser Leu Lys Thr Glu Tyr Gln
100 105 110
Lys Leu Val Asp Ala Gly Leu Gly Glu Thr His Glu Trp Leu Asp Asn
115 120 125
Glu Asp Glu Ile Leu Lys Lys Met Pro Leu Leu Glu Arg Asp Asn Ile
130 135 140
Lys Gly Trp Lys Ala Ile Phe Ser Ala Asp Gly Gly Trp Leu Ala Ala
145 150 155 160
Ala Lys Ala Ile Asn Val Ile Gly Ile Ile Ser Lys Glu Gln Gly Val
165 170 175
Arg Phe Gly Val Gly Ser Ala Gly Ser Phe Arg Lys Pro Leu Phe Ala
180 185 190
Ser Asp Asp Val Thr Cys Val Gly Val Glu Thr Val Asp Gly Thr Lys
195 200 205
Tyr Tyr Gly Asp Lys Val Ile Ile Ser Ala Gly Ala Trp Thr Pro Thr
210 215 220
Leu Val Asp Leu Glu Asp Gln Cys Cys Ser Lys Ala Trp Val Tyr Ala
225 230 235 240
His Ile Arg Leu Thr Pro Asn Glu Ile Ala Glu Tyr Gln Asn Met Pro
245 250 255
Val Val Tyr Asn Gly Asp Val Gly Phe Phe Phe Glu Pro Asp Glu Tyr
260 265 270
Gly Val Met Lys Val Cys Asp Glu Phe Pro Gly Phe Thr Arg Phe Lys
275 280 285
Gln His Leu Pro Tyr Gly Ala Glu Thr Pro Lys Arg Val Ser Val Pro
290 295 300
Arg Ser His Ala Lys His Pro Thr Asp Thr Tyr Pro Asp Ala Ser Glu
305 310 315 320
Lys Ser Ile Gln Asn Ala Ile Asp Val Tyr Leu Pro Arg Phe Lys Asn
325 330 335
Lys Lys Lys Phe Asn Arg Thr Leu Cys Trp Cys Thr Asp Thr Ala Asp
340 345 350
Ala Ala Leu Leu Val Cys Glu His Pro Lys Trp Lys Asn Leu Ile Ile
355 360 365
Ala Ser Gly Asp Ser Gly His Ser Phe Lys Leu Leu Pro Asn Ile Gly
370 375 380
Lys His Val Val Glu Leu Met Glu Gly Arg Leu Ala Asp Asp Leu Lys
385 390 395 400
Asn Ala Trp Lys Trp Arg Pro Gly Gly Asp Ala Leu Lys Ser Thr Arg
405 410 415
Arg Gly His Ala Lys Asp Leu Ala Asp Met Pro Gly Trp Asn His Asp
420 425 430
Gly Glu Lys Ser Lys Met
435
<210> 7
<211> 1317
<212> DNA
<213>artificial sequence
<400> 7
atgtcttcgt cacgtaccac tacaaaagtc attgttgtcg gtggaggtgg cactataggg 60
tcatctacag cactgcatct tcttcgtgcg ggctacacac cctctaatat aacagttcta 120
gatccgtacc caatcccttc ggcacaatca gcaggaaatg atctcaataa gatcatgggt 180
atcaaggtac aaaacgatgt cgacgtacaa ctcagtttag aggccagaga tatgtggaag 240
aacgatgacg tattcaagca tttcttccac aacactggaa ggcttgattg tgcacattca 300
gaggaagagc ttaagtccct gaaaacggag tatcaaaagc tggttgatgc cggtcttgga 360
gaaactcatg aatggcttga taacgaagat gagattttga aaaaaatgcc attgctagag 420
cgagataaca taaaagggtg gaaagccata ttcagcgcag atggtggctg gttagcagcc 480
gcaaaagcga taaatgtaat cgggattata tcgaaagagc aaggcgttag atttggggtt 540
ggcagcgcag gatcattccg aaagcctcta tttgcaagcg acgacgtgac ttgcgtgggt 600
gttgagacgg tggacggaac gaagtactac ggcgataaag taatcatttc cgcaggcgcg 660
tggactccga cactggtaga cttggaagat cagtgttgtt caaaagcatg ggtttatgct 720
cacattcgtc taacaccaaa cgagattgct gagtatcaga atatgcccgt ggtatacaat 780
ggggatgttg gtttcttttt cgaacccgac gagtatggtg tgatgaaagt ctgcgatgaa 840
tttcccggtt tcacgagatt caaacagcat ctgccctacg gtgctgagac tcctaaacgg 900
gtatcagtgc ctcgctctca cgcgaaacat cccacagata cctaccccga tgcttctgaa 960
aaatcaatac agaatgccat tgacgtttac ctgccacgat tcaagaacaa gaagaagttc 1020
aatcgcacgc tatgctggtg tacagacaca gctgatgcag cgctgttggt ttgcgagcat 1080
ccgaaatgga agaatctcat tatcgcgtca ggtgacagcg gacactcctt caaacttctg 1140
ccgaatattg gaaaacatgt ggtggaactt atggagggtc ggttggctga cgatctaaaa 1200
aatgcatgga agtggagacc tggcggtgat gccctaaagt caacgcggcg aggccacgca 1260
aaagacctgg ctgatatgcc tggctggaac catgacgggg aaaaatcaaa aatgtag 1317
<210> 8
<211> 438
<212> PRT
<213>artificial sequence
<400> 8
Met Ser Ser Ser Arg Thr Thr Thr Lys Val Ile Val Val Gly Gly Gly
1 5 10 15
Gly Thr Ile Gly Ser Ser Thr Ala Leu His Leu Leu Arg Ala Gly Tyr
20 25 30
Thr Pro Ser Asn Ile Thr Val Leu Asp Pro Tyr Pro Ile Pro Ser Ala
35 40 45
Gln Ser Ala Gly Asn Asp Leu Asn Lys Ile Met Gly Ile Lys Val Gln
50 55 60
Asn Asp Val Asp Val Gln Leu Ser Leu Glu Ala Arg Asp Met Trp Lys
65 70 75 80
Asn Asp Asp Val Phe Lys His Phe Phe His Asn Thr Gly Arg Leu Asp
85 90 95
Cys Ala His Ser Glu Glu Glu Leu Lys Ser Leu Lys Thr Glu Tyr Gln
100 105 110
Lys Leu Val Asp Ala Gly Leu Gly Glu Thr His Glu Trp Leu Asp Asn
115 120 125
Glu Asp Glu Ile Leu Lys Lys Met Pro Leu Leu Glu Arg Asp Asn Ile
130 135 140
Lys Gly Trp Lys Ala Ile Phe Ser Ala Asp Gly Gly Trp Leu Ala Ala
145 150 155 160
Ala Lys Ala Ile Asn Val Ile Gly Ile Ile Ser Lys Glu Gln Gly Val
165 170 175
Arg Phe Gly Val Gly Ser Ala Gly Ser Phe Arg Lys Pro Leu Phe Ala
180 185 190
Ser Asp Asp Val Thr Cys Val Gly Val Glu Thr Val Asp Gly Thr Lys
195 200 205
Tyr Tyr Gly Asp Lys Val Ile Ile Ser Ala Gly Ala Trp Thr Pro Thr
210 215 220
Leu Val Asp Leu Glu Asp Gln Cys Cys Ser Lys Ala Trp Val Tyr Ala
225 230 235 240
His Ile Arg Leu Thr Pro Asn Glu Ile Ala Glu Tyr Gln Asn Met Pro
245 250 255
Val Val Tyr Asn Gly Asp Val Gly Phe Phe Phe Glu Pro Asp Glu Tyr
260 265 270
Gly Val Met Lys Val Cys Asp Glu Phe Pro Gly Phe Thr Arg Phe Lys
275 280 285
Gln His Leu Pro Tyr Gly Ala Glu Thr Pro Lys Arg Val Ser Val Pro
290 295 300
Arg Ser His Ala Lys His Pro Thr Asp Thr Tyr Pro Asp Ala Ser Glu
305 310 315 320
Lys Ser Ile Gln Asn Ala Ile Asp Val Tyr Leu Pro Arg Phe Lys Asn
325 330 335
Lys Lys Lys Phe Asn Arg Thr Leu Cys Trp Cys Thr Asp Thr Ala Asp
340 345 350
Ala Ala Leu Leu Val Cys Glu His Pro Lys Trp Lys Asn Leu Ile Ile
355 360 365
Ala Ser Gly Asp Ser Gly His Ser Phe Lys Leu Leu Pro Asn Ile Gly
370 375 380
Lys His Val Val Glu Leu Met Glu Gly Arg Leu Ala Asp Asp Leu Lys
385 390 395 400
Asn Ala Trp Lys Trp Arg Pro Gly Gly Asp Ala Leu Lys Ser Thr Arg
405 410 415
Arg Gly His Ala Lys Asp Leu Ala Asp Met Pro Gly Trp Asn His Asp
420 425 430
Gly Glu Lys Ser Lys Met
435
<210> 9
<211> 1317
<212> DNA
<213>artificial sequence
<400> 9
atgtcttcgt cacgtaccac tacaaaagtc attgttgtcg gtggaggtgg cactataggg 60
tcatctacag cactgcatct tcttcgtgcg ggctacacac cctctaatat aacagttcta 120
gatccgtacc caatcccttc ggcacaatca gcaggaaatg atctcaataa gatcatgggt 180
atccgcgtac aaaacgatgt cgacgtacaa ctcagtttag aggccagaga tatgtggaag 240
aacgatgacg tattcaagca tttcttccac aacactggaa ggcttgattg tgcacattca 300
gaggaagagc ttaagtccct gaaaacggag tatcaaaagc tggttgatgc cggtcttgga 360
gaaactcatg aatggcttga taacgaagat gagattttga aaaaaatgcc attgctagag 420
cgagataaca taaaagggtg gaaagccata ttcagcgcag atggtggctg gttagcagcc 480
gcaaaagcga taaatgtaat cgggattata tcgaaagagc aaggcgttag atttggggtt 540
ggcagcgcag gatcattccg aaagcctcta tttgcaagcg acgacgtgac ttgcgtgggt 600
gttgagacgg tggacggaac gaagtactac ggcgataaag taatcatttc cgcaggcgcg 660
tggactccga cactggtaga cttggaagat cagtgttgtt caaaagcatg ggtttatgct 720
cacattcgtc taacaccaaa cgagattgct gagtatcaga atatgcccgt ggtatacaat 780
ggggatgttg gtttcttttt cgaacccgac gagtatggtg tgatgaaagt ctgcgatgaa 840
tttcccggtt tcacgagatt caaacagcat ctgccctacg gtgctgagac tcctaaacgg 900
gtatcagtgc ctcgctctca cgcgaaacat cccacagata cctaccccga tgcttctgaa 960
aaatcaatac agaatgccat tgacgtttac ctgccacgat tcaagaacaa gaagaagttc 1020
aatcgcacgc tatgctggtg tacagacaca gctgatgcag cgctgttggt ttgcgagcat 1080
ccgaaatgga agaatctcat tatcgggtca ggtgacagcg gacactcctt caaacttctg 1140
ccgaatattg gaaaacatgt ggtggaactt atggagggtc ggttggctga cgatctaaaa 1200
aatgcatgga agtggagacc tggcggtgat gccctaaagt caacgcggcg aggccacgca 1260
aaagacctgg ctgatatgcc tggctggaac catgacgggg aaaaatcaaa aatgtag 1317
<210> 10
<211> 438
<212> PRT
<213>artificial sequence
<400> 10
Met Ser Ser Ser Arg Thr Thr Thr Lys Val Ile Val Val Gly Gly Gly
1 5 10 15
Gly Thr Ile Gly Ser Ser Thr Ala Leu His Leu Leu Arg Ala Gly Tyr
20 25 30
Thr Pro Ser Asn Ile Thr Val Leu Asp Pro Tyr Pro Ile Pro Ser Ala
35 40 45
Gln Ser Ala Gly Asn Asp Leu Asn Lys Ile Met Gly Ile Arg Val Gln
50 55 60
Asn Asp Val Asp Val Gln Leu Ser Leu Glu Ala Arg Asp Met Trp Lys
65 70 75 80
Asn Asp Asp Val Phe Lys His Phe Phe His Asn Thr Gly Arg Leu Asp
85 90 95
Cys Ala His Ser Glu Glu Glu Leu Lys Ser Leu Lys Thr Glu Tyr Gln
100 105 110
Lys Leu Val Asp Ala Gly Leu Gly Glu Thr His Glu Trp Leu Asp Asn
115 120 125
Glu Asp Glu Ile Leu Lys Lys Met Pro Leu Leu Glu Arg Asp Asn Ile
130 135 140
Lys Gly Trp Lys Ala Ile Phe Ser Ala Asp Gly Gly Trp Leu Ala Ala
145 150 155 160
Ala Lys Ala Ile Asn Val Ile Gly Ile Ile Ser Lys Glu Gln Gly Val
165 170 175
Arg Phe Gly Val Gly Ser Ala Gly Ser Phe Arg Lys Pro Leu Phe Ala
180 185 190
Ser Asp Asp Val Thr Cys Val Gly Val Glu Thr Val Asp Gly Thr Lys
195 200 205
Tyr Tyr Gly Asp Lys Val Ile Ile Ser Ala Gly Ala Trp Thr Pro Thr
210 215 220
Leu Val Asp Leu Glu Asp Gln Cys Cys Ser Lys Ala Trp Val Tyr Ala
225 230 235 240
His Ile Arg Leu Thr Pro Asn Glu Ile Ala Glu Tyr Gln Asn Met Pro
245 250 255
Val Val Tyr Asn Gly Asp Val Gly Phe Phe Phe Glu Pro Asp Glu Tyr
260 265 270
Gly Val Met Lys Val Cys Asp Glu Phe Pro Gly Phe Thr Arg Phe Lys
275 280 285
Gln His Leu Pro Tyr Gly Ala Glu Thr Pro Lys Arg Val Ser Val Pro
290 295 300
Arg Ser His Ala Lys His Pro Thr Asp Thr Tyr Pro Asp Ala Ser Glu
305 310 315 320
Lys Ser Ile Gln Asn Ala Ile Asp Val Tyr Leu Pro Arg Phe Lys Asn
325 330 335
Lys Lys Lys Phe Asn Arg Thr Leu Cys Trp Cys Thr Asp Thr Ala Asp
340 345 350
Ala Ala Leu Leu Val Cys Glu His Pro Lys Trp Lys Asn Leu Ile Ile
355 360 365
Gly Ser Gly Asp Ser Gly His Ser Phe Lys Leu Leu Pro Asn Ile Gly
370 375 380
Lys His Val Val Glu Leu Met Glu Gly Arg Leu Ala Asp Asp Leu Lys
385 390 395 400
Asn Ala Trp Lys Trp Arg Pro Gly Gly Asp Ala Leu Lys Ser Thr Arg
405 410 415
Arg Gly His Ala Lys Asp Leu Ala Asp Met Pro Gly Trp Asn His Asp
420 425 430
Gly Glu Lys Ser Lys Met
435
<210> 11
<211> 28
<212> DNA
<213>artificial sequence
<400> 11
ttagaattcg atgtcttcgt cacgtacc 28
<210> 12
<211> 31
<212> DNA
<213>artificial sequence
<400> 12
atactcgagc taatgatgat gatgatgatg c 31
<210> 13
<211> 32
<212> DNA
<213>artificial sequence
<400> 13
gttctagctc cgtacccaat cccttcggca ca 32
<210> 14
<211> 31
<212> DNA
<213>artificial sequence
<400> 14
gtacggagct agaactgtta tattagaggg t 31
<210> 15
<211> 33
<212> DNA
<213>artificial sequence
<400> 15
caatcaggag gaaatgatct caataagatc atg 33
<210> 16
<211> 33
<212> DNA
<213>artificial sequence
<400> 16
atttcctcct gattgtgccg aagggattgg gta 33
<210> 17
<211> 32
<212> DNA
<213>artificial sequence
<400> 17
ggtatcaagg tacaaaacga tgtcgacgta ca 32
<210> 18
<211> 31
<212> DNA
<213>artificial sequence
<400> 18
gttttgtacc ttgataccca tgatcttatt g 31
<210> 19
<211> 31
<212> DNA
<213>artificial sequence
<400> 19
attatcgggt caggtgacag cggacactcc t 31
<210> 20
<211> 33
<212> DNA
<213>artificial sequence
<400> 20
cacctgaccc gataatgaga ttcttccatt tcg 33
Claims (10)
1. a kind of Fructoamino-acid-oxidase mutant, which is characterized in that the Fructoamino-acid-oxidase mutant by
Fructoamino-acid-oxidase parent mutant with amino acid sequence shown in SEQ ID NO:2 obtains, specifically, the fruit
Glycosyl amino-acid oxidase enzyme mutant:
It is amino acid sequence the 41st as shown in SEQ ID NO:2 day with amino acid sequence shown in SEQ ID NO:4
Aspartic acid (D) sports alanine (A) and obtains；
It is the third of amino acid sequence the 51st as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:6
Propylhomoserin (A) sports glycine (G) and obtains；
It is amino acid sequence the 62nd smart ammonia as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:8
Sour (R) sports lysine (K) and obtains；
It is amino acid sequence the 369th as shown in SEQ ID NO:2 with amino acid sequence shown in SEQ ID NO:10
Alanine (A) sports glycine (G) and obtains.
2. Fructoamino-acid-oxidase mutant according to claim 1, which is characterized in that the Fructoamino-acid
Enzyme mutant is aoxidized compared with the Fructoamino-acid-oxidase parent with amino acid sequence shown in SEQ ID NO:2, it is right
The catalytic activity of fructosyl lysine improves.
3. Fructoamino-acid-oxidase mutant according to claim 2, which is characterized in that described to rely ammonia to fructosyl
The catalytic activity of acid rises to raising at least 63%.
4. a kind of isolated polynucleotides, which is characterized in that the isolated polynucleotide encoding is appointed in -3 according to claim 1
Fructoamino-acid-oxidase mutant described in one；
Preferably, the isolated polynucleotides:
With nucleotide sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:4 is encoded；
With nucleotide sequence shown in SEQ ID NO:5, amino acid sequence shown in SEQ ID NO:6 is encoded；
With nucleotide sequence shown in SEQ ID NO:7, amino acid sequence shown in SEQ ID NO:8 is encoded；
With nucleotide sequence shown in SEQ ID NO:9, amino acid sequence shown in SEQ ID NO:10 is encoded.
5. a kind of recombinant DNA, which is characterized in that the recombinant DNA includes carrier and connect with the carrier according to claim 4
The isolated polynucleotides.
6. a kind of host cell, which is characterized in that the host cell is converted with recombinant DNA according to claim 5.
7. host cell according to claim 6, which is characterized in that the host cell is Bacillus coli cells.
8. a kind of method for expressing Fructoamino-acid-oxidase mutant according to any one of claim 1-3,
It is characterized in that, the expression includes making host cell according to claim 7 in LB liquid medium at 37 DEG C
After lower culture a period of time, IPTG inducing expression is added.
9. a kind of for measuring the detection kit of the glycosylated protein comprising fructosyl lysine, which is characterized in that described
Detection kit includes Fructoamino-acid-oxidase mutant according to any one of claim 1-3.
10. a kind of detection method for nondiagnostic glycosylated protein of the measurement comprising fructosyl lysine, feature exist
In the detection method includes: to make the sample of the glycosylated protein with described in comprising fructosyl lysine and according to right
It is required that Fructoamino-acid-oxidase mutant described in any one of 1-3 contacts, and measure by the Fructoamino-acid oxygen
Change the amount for the hydrogen peroxide that enzyme mutant catalysis generates.
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Cited By (3)
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| CN111676236A (en) * | 2020-03-12 | 2020-09-18 | 北京达成生物科技有限公司 | Escherichia coli expression method of recombinant FLOD protein |
| CN114875002A (en) * | 2021-07-01 | 2022-08-09 | 温州医科大学 | Arginine mutated DNA/RNA ligase |
| CN115725526A (en) * | 2022-08-15 | 2023-03-03 | 南京立顶医疗科技有限公司 | Fructosyl lysine oxidase mutant FODt and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676236A (en) * | 2020-03-12 | 2020-09-18 | 北京达成生物科技有限公司 | Escherichia coli expression method of recombinant FLOD protein |
| CN114875002A (en) * | 2021-07-01 | 2022-08-09 | 温州医科大学 | Arginine mutated DNA/RNA ligase |
| CN115725526A (en) * | 2022-08-15 | 2023-03-03 | 南京立顶医疗科技有限公司 | Fructosyl lysine oxidase mutant FODt and preparation method and application thereof |
| CN115725526B (en) * | 2022-08-15 | 2023-10-27 | 南京立顶医疗科技有限公司 | Fructose lysine oxidase mutant FODt and preparation method and application thereof |
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