WO2015096621A1 - 果糖氨基酸氧化酶、制备方法及含该酶的糖化白蛋白检测试剂盒 - Google Patents

果糖氨基酸氧化酶、制备方法及含该酶的糖化白蛋白检测试剂盒 Download PDF

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WO2015096621A1
WO2015096621A1 PCT/CN2014/093580 CN2014093580W WO2015096621A1 WO 2015096621 A1 WO2015096621 A1 WO 2015096621A1 CN 2014093580 W CN2014093580 W CN 2014093580W WO 2015096621 A1 WO2015096621 A1 WO 2015096621A1
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amino acid
fructose
oxidase
acid oxidase
acid sequence
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PCT/CN2014/093580
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English (en)
French (fr)
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邹炳德
邹继华
汪屹
贾江花
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宁波美康生物科技股份有限公司
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Priority to US15/108,320 priority Critical patent/US9988609B2/en
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/03—Oxidoreductases acting on the CH-NH group of donors (1.5) with oxygen as acceptor (1.5.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/90—Developer
    • C12Q2326/96—4-Amino-antipyrine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
    • G01N2333/90605—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4)
    • G01N2333/90633—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3) in general
    • G01N2333/90638—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3) in general with a definite EC number (1.4.3.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
    • G01N2333/9065—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5)
    • G01N2333/90672—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3) in general

Definitions

  • the present invention relates to the field of in vitro diagnostics, in particular to a fructose lysine oxidase mutant having high thermostability, a method for determining glycated albumin, and a kit for measuring glycated albumin.
  • the present invention can be used for clinical tests and is capable of accurately measuring glycated albumin.
  • Diabetes is a type of metabolic disease that occurs in people with high blood sugar, which can cause serious damage to most systems, especially nerves and blood vessels.
  • World Health Organization the number of people with diabetes in China has ranked first in the world. Diabetes will be the most serious public health problem in China in the next 50 years.
  • Glycated albumin is a product of non-enzymatic glycation of glucose and albumin N-terminus in human serum, 90% of which react with lysine ⁇ -NH2 residues in the albumin chain.
  • the reaction principle is that the two first form an unstable Glycosylamine or Schiff Base, which is then subjected to an irreversible rearrangement of glucosamine (Amadori) to form a stable aminoketone (ketoamine). ).
  • Amadori glucosamine
  • ketoamine stable aminoketone
  • albumin detection can be used to detect average blood glucose levels over the past 2-3 weeks.
  • glycated albumin has become an indispensable test for diabetic patients, and it is more suitable as an indicator for assessing the risk of hospitalization and death in diabetic dialysis patients than glycated hemoglobin.
  • the reaction principle is to first digest the glycated protein into a low molecular weight glycated polypeptide using a protease, and then use the fructose amino acid enzyme to catalyze the oxidation reaction of the glycated polypeptide to produce a polypeptide (or amino acid), glucurophenone and H 2 O 2 .
  • the released H 2 O 2 was determined by endpoint reaction colorimetry, and its absorbance at 600 nm was proportional to the concentration of glycated albumin.
  • the specific reaction process is as follows:
  • fructose amino acid oxidase is a key enzyme in the determination of glycated albumin, and fructose amino acid oxidase becomes the key to accurately determine glycated albumin in human serum.
  • Fructose amino acid oxidase can also be called fructose amino acid enzyme, amadoriase, ketamine oxidase and the like.
  • fructose amino acid oxidases have been found in many bacteria, yeasts and fungi, for example from Aspergillus, Penicillium, Fusarium, Pichia, cones. Coniochaeta, Eupencilum, Corynebacterium, etc. (Non-patent document 1Lin, Z. and J. Zheng (2010). "Occurrence, characteristics, and Applications of fructosyl amine oxidases (amadoriases).
  • the present invention is directed to the above-mentioned deficiencies of the prior art, and provides a fructose amino acid oxidase having high heat stability.
  • Non-Patent Document 4 Mennella, C., RC Borrelli, et al. (2005). Substrate specificity of amadoriase I from Aspergillus fumigatus.
  • the present invention uses nucleotide sequence 2 as a template to design primer sequence 3 (5'-atggcgcctt caattttgag cactg-3') and primer sequence 4 (5'-ttacggacct ctgctctctc caatc-3') for error-prone PCR, through reasonable control
  • primer sequence 3 (5'-atggcgcctt caattttgag cactg-3')
  • primer sequence 4 (5'-ttacggacct ctgctctctc caatc-3') for error-prone PCR, through reasonable control
  • concentration of manganese ions in the PCR reaction system controls the mutation frequency to 1 to 2 nucleotide mutations per 1 Kb nucleotide.
  • Non-Patent Document 7 Miyazaki, K. (2003) .Creating random mutagenesis libraries by megaprimer PCR of whole plasmid (MEGAWHOP). Directed Evolution Library Creation, Springer: 23-28), the resulting product was digested with restriction endonuclease DpnI to remove the template plasmid. Thereafter, TOP10 competent cells were transformed and plated on an LB solid plate containing ampicillin, and the obtained clones were about 10,000, so that the library capacity obtained by the random mutation was about 10,000.
  • the resulting clone was mixed with a coating bar, collected in a centrifuge tube, and the plasmid was extracted, and the resulting plasmid was transformed into BL21 (ED3) for further screening.
  • the above transformants were inoculated into a 96-well plate in a medium of 150 ul LB per well containing an ampicillin antibiotic, and the well plate was used as a retention plate.
  • the next day the cells were transferred to another 96-well plate in the same order.
  • the medium used was 150 ul LB/well, and induced with ampicillin and IPTG.
  • the cells were cultured at 37 ° C for 6 hours, centrifuged at 3800 rpm, and the medium was removed. The plate is used as an analysis plate.
  • lysate 100 mM Tris, pH 8.0; 0.4 mg/ml sodium deoxycholate; 0.8 mg/ml CTAB; 20 mM KCl; 80 mM MgSO4
  • lysate 100 mM Tris, pH 8.0; 0.4 mg/ml sodium deoxycholate; 0.8 mg/ml CTAB; 20 mM KCl; 80 mM MgSO4
  • centrifugation was carried out at 3800 rpm for 15 minutes, and 50 ul of the lysate was taken in a new 96-well plate.
  • Another 50 ul of the lysate was placed in a 96-well PCR plate, placed in a 96-well PCR machine, heat-treated at 50 ° C for 15 minutes, and transferred to another 96-well plate.
  • the mutation of the above six sites is improved in thermal stability, if the above mutation is mutated to other amino acids, the thermal stability is also higher than that of the wild type. Thus, the present invention has largely attempted other mutations at the above sites. Meanwhile, since it is considered that the thermal stability can be further improved if a single mutation site is superimposed, a combination of various mutation sites is also tried.
  • mutant strain was transformed into BL21 (DE3), cultured, induced, expressed, purified, and analyzed by enzymatic properties, and it was found that the thermal stability of these mutant strains was further improved.
  • mutants were mutated to the following mutants by site-directed mutagenesis: 59E ⁇ F, 98E ⁇ C, 225G ⁇ F; 59E ⁇ F, 225G ⁇ D, 277K ⁇ N; 59E ⁇ F,277K ⁇ N,285E ⁇ I;98E ⁇ P,225G ⁇ N,285E ⁇ Q;225G ⁇ L,285E ⁇ I,355D ⁇ T; transforming the constructed mutant strain into BL21(DE3) for culture , induction, expression, purification, and enzymatic properties analysis revealed that the thermal stability of these mutant strains was further improved.
  • mutant strain was transformed into BL21 (DE3), cultured, induced, expressed, purified, and analyzed by enzymatic properties, and it was found that the thermal stability of these mutant strains was further improved.
  • mutant strain was transformed into BL21 (DE3), cultured, induced, expressed, purified, and analyzed by enzymatic properties, and it was found that the thermal stability of these mutant strains was further improved.
  • the structure and function are often similar between two sequences with high homology, and the beneficial effect of mutation of one sequence is in another sequence with high homology.
  • the same mutation is carried out, generally with a homology of 80% or more, preferably 90% or more, more preferably 96% or more, and most preferably 99% or more, and similarly advantageous effects are often obtained.
  • a large number of mutations and combinatorial mutation studies were carried out on sequences having amino acid sequence homology of sequence 2 equal to or higher than 80%.
  • Fructose amino acid oxidase from the following strains was selected as the test object to illustrate the enzyme having more than 80% homology with sequence 1, and the mutation at the corresponding site will also lead to an increase in the thermal stability of the enzyme.
  • These strains are: Aspergillus niger CBS 513.88, Aspergillus clavatus NRRL 1, Neosartorya fischeri NRRL 181, Aspergillus fumigatus Af293; corresponding to sequence 1
  • the homology was 80%, 83%, 96%, and 99%, respectively.
  • Aspergillus niger CBS 513.88 has a homology of 80% with sequence 1, and its corresponding mutation sites are 59E, 98E, 225G, 277K, 285E, 355D, by selectively selecting several single amino acid mutations. As well as a combination of mutations at several different sites, it was found that the thermal stability of the enzyme was also increased to varying degrees. For example, the thermal stability of the following mutant strains is particularly obvious: 59E ⁇ F, 98E ⁇ C, 225G ⁇ F, 277K ⁇ N, 285E ⁇ F, 355D ⁇ L.
  • Aspergillus clavatus NRRL 1 has a homology of 83% with sequence 1, and its corresponding mutation sites are 59E, 98E, 225G, 277N, 285E, 355D, by selectively selecting several single amino acid mutations. As well as a combination of mutations at several different sites, it was found that the thermal stability of the enzyme was also increased to varying degrees. For example, the thermal stability of the following mutant strains is particularly obvious: 59E ⁇ F, 98E ⁇ C, 225G ⁇ F, 285E ⁇ F, 355D ⁇ L.
  • Neosartoya fischeri NRRL 181 has 96% homology with sequence 1, and its corresponding mutation sites are 59E, 98E, 225G, 277K, 285E, 355D, by selectively selecting several single amino acids. Mutations, as well as combinations of mutations at several different sites, have been found to increase the thermal stability of the enzyme to varying degrees. For example, the thermal stability of the following mutant strains is particularly obvious: 59E ⁇ F, 98E ⁇ C, 225G ⁇ F, 277K ⁇ N, 285E ⁇ F, 355D ⁇ L.
  • Aspergillus fumigatus Af293 has 99% homology with sequence 1, and its corresponding mutation sites are 59E, 98E, 225G, 277K, 285E, 355D, by selective selection of several individual Amino acid mutations, as well as combinations of mutations at several different sites, have been found to increase the thermal stability of the enzyme to varying degrees.
  • the thermal stability of the following mutant strains is particularly obvious: 59E ⁇ F, 98E ⁇ C, 225G ⁇ F, 277K ⁇ N, 285E ⁇ F, 355D ⁇ L.
  • the mutation of the amino acid in the present invention is replaced by an abbreviation character, which is common knowledge, and the specific meanings are as follows: glycine G, alanine A, leucine L, isoleucine I, proline V, proline P, Phenylalanine F, methionine M, tryptophan W, serine S, glutamine Q, threonine T, cysteine C, asparagine N, tyrosine Y, aspartate D, glutamic acid E, lysine K, arginine R, histidine H.
  • 285E ⁇ F represents that the glutamic acid at position 285 is mutated to phenylalanine, and the rest are similar.
  • the vector used in this study is pET-22b and the host (host cell) is BL21 (DE3).
  • any simple carrier replacement such as pET-20b, pET-28b, pET-32a, pQE30, pTrc99a, etc., or host bacterial replacement, such as Rosetta, Origami, M15, etc., should be considered identical.
  • Technical effects will be considered to fall within the scope of the claims.
  • Any addition of a purification tag at the N-terminus or C-terminus of the mutant enzyme, the behavior of the signal peptide should also be considered to fall within the scope of the claims.
  • the preparation of fructose amino acid oxidase can be carried out using an expression purification technique of an already known enzyme.
  • the corresponding mutant strain is inoculated, and when the OD 600 is grown to 0.5 to 1, isopropylthiogalactoside (IPTG) is induced, and at this time, in order to facilitate the expression thereof, low temperature induction may be selected, such as 25 ° C. , 20 ° C, 16 ° C, etc., according to the induction temperature and expression, determine the optimal induction time to obtain the best expression.
  • IPTG isopropylthiogalactoside
  • centrifugation, sonication, SDS-PAGE, and the like can be carried out by a conventional method.
  • the corresponding method can be selected using the label carried by the recombinase. For example, if the recombinant plasmid contains a His-tag, it can be affinity-purified using a nickel column.
  • the above-mentioned fructose amino acid oxidase having good thermal stability can be used for the development of a glycated albumin detection method, and a glycated albumin detection kit can be further developed.
  • the main principle is that, under the action of protease, glycated albumin is degraded by the action of protease, and the fructose amino acid or fructose polypeptide is oxidized to glucose under the action of fructose amino acid oxidase. Amino acid or polypeptide, hydrogen peroxide, while consuming a certain amount of oxygen. The amount of glycated albumin in the sample is then calculated by measuring the production of the product or the amount of substrate consumed.
  • any sample containing glycated albumin, glycated polypeptide, or glycated amino acid can be used as a sample for testing. Since this method is mainly used for clinical tests, the common sample is serum. In theory, hemolysis should be avoided. After the sample is collected, it should be kept at 2 ⁇ 8 °C and measured in time.
  • Buffer Any buffer of appropriate concentration and pH can be used here as long as it is active against the enzyme in the assay There should be no inhibition and no interference with the detection process.
  • Typical buffers are: trishydroxymethylaminomethane hydrochloride buffer, acetic acid-sodium acetate buffer, phthalic acid-hydrochloric acid buffer or glycine-hydrochloric acid buffer.
  • the concentration of the buffer can be determined depending on the case, and is preferably 20 to 200 mmol/L, and more preferably 50 to 100 mmol/L.
  • protease capable of degrading glycated albumin can be used herein, preferably trypsin, azeotropic protease, protease A, proteinase K, and the like.
  • the added protease should promote the full degradation of glycated albumin, and the corresponding concentration should be 10 to 100 KU/L, preferably 40 KU/L, depending on its source.
  • Product formation There are three main products, namely glucose, amino acids and hydrogen peroxide.
  • the content of glycated albumin can be calculated by measuring any of these three products.
  • the determination of glucose can be determined by the hexokinase method.
  • the principle is that phosphorylation of glucose and ATP is catalyzed by hexokinase to produce glucose-6-phosphate (G-6-P) and ADP.
  • the former dehydrogenates under the catalysis of glucose-6-phosphate dehydrogenase (G6PD) to produce 6-phosphogluconate (6-GP), and simultaneously reduces NADP to NADPH.
  • the rate of NADPH formation is proportional to the glucose concentration at 340 nm. The rate of increase in absorbance was monitored and the glucose concentration was calculated.
  • Amino acids can be determined by reacting with a fluorescent amine to determine the fluorescence.
  • Hydrogen peroxide can be detected by coupling peroxidase to H 2 O 2 and then Trinder reaction to generate color.
  • the chromogen here may be N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), N-ethyl-N-(3-sulfopropyl)-3-methyl Sodium aniline (TOPS), sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS).
  • TODB N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt
  • TOPS N-ethyl-N-(3-sulfopropyl)-3-methyl Sodium aniline
  • TOOS sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline
  • the consumption of the substrate mainly refers to O 2 , and the consumption of O 2 can be detected by the oxygen electrode.
  • a corresponding glycated albumin detection kit kit can be developed.
  • the main principle is to select the appropriate buffer. Under the action of protease, glycated albumin is degraded into fructose amino acid or fructose polypeptide. At this time, fructose amino acid or fructose polypeptide is oxidized to glucose and amino acid under the action of fructose amino acid oxidase. Or peptide, hydrogen peroxide, while consuming a certain amount of oxygen. The amount of glycated albumin in the sample is then calculated by measuring the production of the product or the amount of substrate consumed.
  • the most common method is to detect hydrogen peroxide in the product, that is, to detect by Trinder reaction, which is also the coupling mode used in most kits at present, and the embodiments in this specification focus on this.
  • Trinder reaction which is also the coupling mode used in most kits at present, and the embodiments in this specification focus on this.
  • the glycated albumin detection kit kit comprises reagent 1 and reagent 2, wherein reagent 1 comprises a buffer, a protease, a peroxidase, a 4-aminoantipyrine, a preservative.
  • Reagent 2 contains a buffer, a fructose amino acid oxidase mutant, a chromogen, and a preservative.
  • Buffer Any buffer of appropriate concentration and pH can be used here as long as it is active against the enzyme in the assay There should be no inhibition and no interference with the detection process.
  • Typical buffers are: trishydroxymethylaminomethane hydrochloride buffer, acetic acid-sodium acetate buffer, phthalic acid-hydrochloric acid buffer or glycine-hydrochloric acid buffer.
  • the concentration of the buffer can be determined depending on the case, and is preferably 20 to 200 mmol/L, and more preferably 50 to 100 mmol/L.
  • protease capable of degrading glycated albumin can be used herein, preferably trypsin, azeotropic protease, protease A, proteinase K, and the like.
  • the added protease should promote the full degradation of glycated albumin, and the corresponding concentration should be 10 to 100 KU/L, preferably 40 KU/L, depending on its source.
  • This enzyme acts as a coupling, and may be added at a concentration of 10 to 100 KU/L, preferably 30 KU/L, as needed.
  • the purpose of the preservative is to prevent the growth of bacteria in the kit, and to ensure that the sensitivity of the kit for long-term storage is not affected.
  • the preservatives are 2-methyl-4-isothiazolin-3-one, proclin300 and the like.
  • the amount of the preservative added should be from 0.01 to 0.05%, preferably 0.02%.
  • the fructose amino acid oxidase refers to the high heat stability fructose amino acid oxidase of the present invention, and the enzyme should be added in an amount sufficient to sufficiently react the substrate fructose amino acid or fructose polypeptide, and the amount added should be 10 to 100 KU. /L, preferably 20 to 50 KU/L, and most preferably 28 KU/L.
  • the chromogen of the present invention may be N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), N-ethyl-N-(3-sulfopropyl)-3 -Methylaniline sodium salt (TOPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS,) chromogen in the kit can be 1 to 10 mmol/L, preferably 2 mmol/L.
  • TODB N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt
  • TOPS N-ethyl-N-(3-sulfopropyl)-3 -Methylaniline sodium salt
  • TOOS N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt
  • the kit for determining glycated albumin according to the above invention comprises reagent 1 and reagent 2, and the concentration of each component in the reagent is:
  • Preservative 0.01 to 0.05% Preservative 0.01 to 0.05%.
  • the percentage of glycated albumin can be calculated by dividing the concentration of glycated albumin by the concentration of albumin.
  • the kit Because its performance is better than the previous fructose amino acid oxidase, the kit meets the requirements in terms of sensitivity and anti-interference, and its performance in all aspects after long-term maintenance has not decreased significantly.
  • the invention provides a stable, simple, rapid, sensitive and accurate quantitative detection reagent, which has good dilution linearity, accuracy and precision, and has good consistency with the HPLC detection method.
  • the invention uses the glycated albumin assay kit of the invention to determine the glycated albumin by enzymatic method, and can be used in the clinical laboratory for the determination of large-scale samples instead of HPLC.
  • the operation is simple and the linear range is wide. Low cost and high application value.
  • Figure 1 shows the linear range of glycated albumin detection.
  • Figure 2 is a correlation diagram of the present invention and HPLC detection.
  • SEQ ID NO: 2 The sequence shown in SEQ ID NO: 2 was synthesized by whole-gene synthesis and cloned into the pET-22b vector, and the cleavage sites used were NdeI and XhoI.
  • the plasmid pET-Ama thus obtained was the template for the following error-prone PCR and WHOP-PCR.
  • reaction conditions are:
  • the above amount of MnCl 2 can be appropriately adjusted to obtain a suitable mutation frequency, that is, a 1-2 nucleotide mutation/1 Kb nucleotide.
  • the fragment obtained by error-prone PCR which was about 1.4 Kb in size, was recovered as a primer for the next round of WHOP-PCR.
  • the product of 50 ul of WHOP-PCR was digested with 1 ul of DpnI and reacted at 37 ° C for 2 hours to completely remove the template DNA.
  • the above-mentioned digested product was transformed into TOP10 competent cells in 5 tubes, and the library capacity of 10000 or more in total was obtained the next day.
  • the resulting clone was mixed with a coating bar, collected in a centrifuge tube, and the plasmid was extracted, and the resulting plasmid was transformed into BL21 (ED3) for further screening.
  • the above transformants were inoculated into a 96-well plate, and the last well was inoculated with a wild type strain as a control, and the medium used was 150 ul LB per well, containing ampic antibiotic, and the well plate was used as a retention plate.
  • the next day, the cells were transferred to another 96-well plate in the same order.
  • the medium used was 150 ul LB/well, and induced with ampicillin and IPTG.
  • the cells were cultured at 37 ° C for 6 hours, centrifuged at 3800 rpm, and the medium was removed. The plate is used as an analysis plate.
  • lysate 100 mM Tris, pH 8.0; 0.4 mg/ml sodium deoxycholate; 0.8 mg/ml CTAB; 20 mM KCl; 80 mM MgSO4
  • lysate 100 mM Tris, pH 8.0; 0.4 mg/ml sodium deoxycholate; 0.8 mg/ml CTAB; 20 mM KCl; 80 mM MgSO4
  • centrifugation was carried out at 3800 rpm for 15 minutes, and 50 ul of the lysate was taken in a new 96-well plate.
  • Another 50 ul of the lysate was placed in a 96-well PCR plate, placed in a 96-well PCR machine, heat-treated at 50 ° C for 15 minutes, and transferred to another 96-well plate.
  • the purified fructose amino acid oxidase was diluted to about 10 ug/ml in a buffer of 100 mM Tris, pH 8.0.
  • the heat-treated 50 ul of fructose amino acid oxidase was transferred to a 96-well plate while 50 ⁇ l of unheated fructose amino acid oxidase was taken on the same 96-well plate. Incubate at 37 ° C for 10 min.
  • a coloring solution (Tris, 100 mM, pH 8.0; TOOS solution, 15 mM, previously incubated to 37 ° C; 4-APP, 0.5 mM; POD, 40 U/ml; fructose lysine, 15 mM), reacted at 37 ° C for 30 min.
  • the absorbance at 555 nm was recorded using a microplate reader.
  • the absorbance of the heat-treated fructose amino acid oxidase is divided by the absorbance of the unheated fructose amino acid oxidase, and the obtained value is the residual enzyme activity ratio at this temperature.
  • 1,59 is the introduction of point mutations of amino acids
  • primer sequence 5 5'-tgctagttat tgctcagcgg-3'
  • sequence 6 5'-acaagatta tgttacacag cgagctg-3'
  • a fragment of interest containing a point mutation was obtained.
  • the PCR system is 50ul:
  • the PCR reaction conditions are:
  • the obtained target fragment was purified and used in the next round of WHOP-PCR primers.
  • the specific reaction system is shown in Table 5.
  • the PCR reaction conditions are:
  • the obtained mutant strain was inoculated overnight, and transferred to a fresh ampicillin-containing LB medium at a ratio of 1% the next day.
  • an IPTG of about 0.4 mM was added to induce about 5 mM.
  • the fructose amino acid oxidase obtained by the purification was analyzed for its thermal stability as described above. The specific results are shown in the table below.
  • the amino acid sequence of the fructose amino acid oxidase derived from Aspergillus niger CBS 513.88 has 80% homology with the amino acid sequence of fructose amino acid oxidase derived from Aspergillus fumigatus.
  • the amino acid sequence is shown in SEQ ID NO: 68 and the nucleotide sequence is shown in SEQ ID NO: 69.
  • Sequence 69 was synthesized by whole gene synthesis and cloned into the NdeI and XhoI cleavage sites of the pET-22b vector.
  • fructosyl amino acid oxidase of Aspergillus niger CBS 513.88 were subjected to single base mutation or a combination of different sites according to the above point mutation method.
  • the obtained mutant strain was inoculated overnight, and transferred to a fresh ampicillin-containing LB medium at a ratio of 1% the next day.
  • an IPTG of about 0.4 mM was added to induce about 5 mM.
  • the fructose amino acid oxidase obtained by the purification was analyzed for its thermal stability as described above. The specific results are shown in the table below.
  • the amino acid sequence of the fructose amino acid oxidase derived from Aspergillus clavatus NRRL 1 has an amino acid sequence of 83% from the amino acid sequence of fructose amino acid oxidase derived from Aspergillus fumigatus.
  • the amino acid sequence is shown in SEQ ID NO: 70
  • the nucleotide sequence is shown in SEQ ID NO: 71.
  • Sequence 71 was synthesized by whole gene synthesis and cloned into the NdeI and XhoI cleavage sites of the pET-22b vector.
  • the amino acid sequence of the fructose amino acid oxidase derived from Neosartoya fischeri NRRL 181 has 96% homology with the amino acid sequence of fructose amino acid oxidase derived from Aspergillus fumigatus.
  • the amino acid sequence is shown in SEQ ID NO: 72
  • the nucleotide sequence is shown in SEQ ID NO: 73. Sequence 73 was synthesized by whole gene synthesis and cloned into the NdeI and XhoI cleavage sites of the pET-22b vector.
  • the amino acid sequence of fructose amino acid oxidase derived from Aspergillus fumigatus Af293 has 99% homology with the amino acid sequence of fructose amino acid oxidase derived from Aspergillus fumigatus.
  • the amino acid sequence is shown in SEQ ID NO: 74
  • the nucleotide sequence is shown in SEQ ID NO: 75. Sequence 75 was synthesized by whole gene synthesis and cloned into the NdeI and XhoI cleavage sites of the pET-22b vector.
  • Ammonium sulfate precipitation the supernatant obtained in the step (1) is subjected to fractional precipitation using an ammonium sulfate solution, and the finally collected precipitate is dissolved in buffer A to obtain a crude extract;
  • Affinity chromatography balance the nickel column with buffer A, and then adsorb the crude extract obtained in step (2) on the nickel column. After the adsorption is completed, the gradient elution with the imidazole solution is performed to collect the eluate;
  • Dialysis The eluate in step (3) was placed in a dialysis bag, placed in a dialyzate at 4 ° C, and magnetically stirred overnight for dialysis.
  • the buffer A in the step (2) is: 50 mM potassium phosphate buffer, pH 8.0, 500 mM NaCl;
  • the imidazole solution in the step (3) is: 50 mM potassium phosphate buffer, pH 8.0, 500 mM NaCl, 20 mM to 1000 mM imidazole;
  • the dialysate in the step (4) is: 50 mM potassium phosphate buffer, pH 8.0, 500 mM NaCl.
  • the fructose amino acid oxidase mutant used in the present embodiment is specifically a mutant strain M76. In fact, other mutant strains can also be used.
  • Samples tested should use fresh serum and hemolysis should be avoided. If the serum is not checked in time after collection, it should be stored at 4 ° C, theoretically should not exceed 2 weeks.
  • Detection conditions a dominant wavelength of 600 nm and a sub-wavelength of 700 nm.
  • GA high-value serum was taken and diluted with physiological saline to different gradients, and the measurements were performed separately.
  • the linear range of glycated albumin was detected by this method from 21.0 to 1200 umol/L, and r 2 >0.990.
  • a high- and low-value quality control and two sample sera were tested 20 times, with mean values of 204umol/L, 751umol/L, 251umol/L, 373umol/L, and CV of 1.1%, 0.7%, and 0.8%, respectively. , 0.6%.
  • a high-value and low-value quality control and two sample sera were continuously measured for 10 days, with mean values of 204umol/L, 751umol/L, 251umol/L, 373umol/L, and CV of 1.2%, 0.7%, and 1.3, respectively. %, 1.0%.
  • the stability of the reagents on the Hitachi Biochemical Analyzer can last at least 4 weeks.
  • the detection of glycated albumin, combined with the detection of albumin, can be obtained by dividing the glycated albumin concentration by the albumin concentration to calculate the percentage of glycated albumin.
  • Albumin detection reagents are as follows
  • Detection conditions a dominant wavelength of 600 nm and a sub-wavelength of 700 nm.
  • Reaction method endpoint method.
  • the reference value of glycated albumin is 11-16%, and the increase of glycated albumin value is common in diabetes. This index can be used as a reference indicator for mid-term glycemic control in diabetic patients.
  • fructose amino acid oxidase mutants involved in the present invention are numerous, it is impossible to verify one by one in the glycated albumin detection kit, so only a few typical fructose amino acid oxidase mutants are selected for glycated albumin detection. Kits and their performance were evaluated to demonstrate that the fructose amino acid oxidase mutants of the present invention are suitable for glycated albumin assay kits and are not intended to limit the invention.
  • the detection methods of the kits involved, the linear range, the intra-assay precision, the inter-assay precision, the opening stability, the correlation with HPLC, and the anti-interference verification methods are the same as described in Examples 20 to 25.
  • a glycated albumin detection kit comprising reagent 1 and reagent 2, wherein:
  • the fructose amino acid oxidase mutant strain is tested by the following representatives: M 1, M 13, M28, M35, M50, M56, M64, M73, M79, M80, M81, M90 prepared in the above examples. , M 100, M 109, M 120.

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Abstract

提供一种果糖氨基酸氧化酶，具有SEQID.No.1所示的氨基酸序列或与该氨基酸序列有80%以上同源性的果糖氨基酸氧化酶中，其选自以下（a）至（f）中的氨基酸对应的位置上具有1个或更多的氨基酸残基进行取代，所得的果糖氨基酸氧化酶具有更高的热稳定性：（a）59位的谷氨酸，（b）98位的谷氨酸，（c）225位的甘氨酸，（d）277位的赖氨酸，（e）285位的谷氨酸，（f）355位的天冬氨酸。还提供了上述酶的制备方法和含有该酶的测定糖化白蛋白的试剂盒。

Description

果糖氨基酸氧化酶、制备方法及含该酶的糖化白蛋白检测试剂盒 技术领域
本发明涉及体外诊断领域，具体说是提供一种热稳定性高的果糖赖氨酸氧化酶突变体，一种用于测定糖化白蛋白的方法，一种测定糖化白蛋白的试剂盒。本发明可用于临床检验，能够精确地测定糖化白蛋白。
背景技术
糖尿病是发生在高血糖人群中的一类代谢型疾病，可导致机体多数系统受到严重的损害，尤其是神经及血管。根据世界卫生组织预计，中国糖尿病人数已居世界第一位。糖尿病将成为未来50年中国最严重的公共卫生问题。
糖化白蛋白(Glycated albumin，GA)是指人血清中葡萄糖与白蛋白N末端发生非酶促糖化反应而形成的产物，其中90％与白蛋白链内的赖氨酸ε-NH2残基发生反应，其反应原理为两者首先形成不稳定的葡基胺(Glycosylamine)或Schiff碱基(Schiff Base)，后者再经不可逆的葡糖胺(Amadori)重排反应形成稳定的氨基酮(酮胺)。因白蛋白的半衰期大约是20天，所以糖化白蛋白检测可被用于检测过去2-3周平均血糖水平。目前，糖化白蛋白已成为一项糖尿病患者必不可少的检测项目，与糖化血红蛋白相比，其更适合作为评估糖尿病透析患者住院治疗及死亡风险的指标。
所以如何能精确地测定出人血清中的糖化白蛋白量成为临床检测糖化白蛋白的关键。目前市场主要采用酶法用于检测人血清中的糖化白蛋白。其反应原理为首先使用蛋白酶将糖化蛋白消化成低分子量的糖化多肽，随后使用果糖氨基酸酶催化糖化多肽发生氧化反应生成多肽(或氨基酸)，葡萄糖醛酮和H2O2。释放的H2O2通过终点反应比色法来测定，其在600nm处的吸收值与糖化白蛋白的浓度成比例。具体反应过程如下：
Figure PCTCN2014093580-appb-000001
Figure PCTCN2014093580-appb-000002
Figure PCTCN2014093580-appb-000003
由上述反应原理和反应步骤可知，果糖氨基酸氧化酶为糖化白蛋白测定中的关键酶，果糖氨基酸氧化酶成为能否精确地测定人血清中糖化白蛋白的关键。
果糖氨基酸氧化酶又可以称为果糖氨基酸酶，阿马道里酶，酮氨氧化酶等。已经在许多细菌、酵母和真菌中发现了这类果糖氨基酸氧化酶，例如有来自曲霉属(Aspergillus)、青霉属(Penicillium)、镰孢属(Fusarium)、毕赤酵母属(Pichia)、锥毛壳属(Coniochaeta)、正青霉属(Eupencillum)、棒杆菌属(Corynebacterium)等(非-专利文件1Lin,Z.and J.Zheng(2010)."Occurrence,characteristics,and  applications of fructosyl amine oxidases(amadoriases)."Appl Microbiol Biotechnol86(6):1613-1619.)。上述果糖氨基酸氧化酶均有可能应用于糖化白蛋白检测试剂盒。但同时，由于在实际应用中诊断试剂盒需要一定时间的保存，对稳定性有较高的要求，因而要求所使用的原料—酶—也应该稳定。而上述公开资料所显示的酶的热稳定性均不太理想。来自土曲霉(Aspergillus terreus)GP1的果糖氨基酸氧化酶在45℃热处理10分钟显示残留酶活为约40％(非-专利文件2:Yoshida,N.,Y.Sakai,et al.(1996)."Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins."European Journal of Biochemistry 242(3):499-505.)。来自尖孢镰孢(Fusarium oxysporum)S-1F4的果糖氨基酸氧化酶的45℃热处理5分钟显示残留酶活为约10％(非-专利文件3:Sakai,Y.,N.Yoshida,et al.(1995)."Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4."Bioscience,biotechnology,and biochemistry 59(3):487.)。来自Coniochaetidium savoryi ATCC36547的果糖氨基酸氧化酶在37℃热处理30分钟显示残留酶活为80％(日本专利申请公开号2004-275013；)。正是由于现有的果糖氨基酸氧化酶的热稳定性较差，使得目前市场上的糖化白蛋白检测试剂盒稳定性都不能满足要求，很多情况下，都是在试剂盒中添加大量的稳定剂，以提高试剂盒的稳定性(国际专利申请公开号WO2002061119A1)，这就导致了试剂的粘度较高，以及其它一系列问题，不利于在生化仪上的应用。
所以急需开发一种热稳定性高的果糖氨基酸氧化酶，并利用该果糖氨基酸氧化酶研发出一种既具有高灵敏度，又能长期稳定的糖化白蛋白检测试剂盒。
发明内容
本发明针对现有技术的上述不足，提供一种具有高热稳定性的果糖氨基酸氧化酶。
由于来自于烟曲霉(Aspergillus fumigatus)的Amadoriase I(其氨基酸序列为序列1即SEQ ID.No.1所示序列，其核苷酸序列为序列2即SEQ ID.No.2所示序列)具有对底物果糖赖氨酸的活性远远大于果糖缬氨酸的特点(非-专利文件4:Mennella,C.,R.C.Borrelli,et al.(2005)."Substrate specificity of amadoriase I from Aspergillus fumigatus."Ann N Y Acad Sci 1043:837-844.)，该特点使得其特别适合于糖化白蛋白检测试剂盒，因为糖化白蛋白的糖基化位点正是赖氨酸残基，与糖化血红蛋白的缬氨酸残基相区别，有利于在检测中降低其它因素的干扰。因此以此为出发，对该酶进行改造，以提高其热稳定性。由于Amadorise I的序列与先前报道的FAOX-TE和FPOX-CE的序列同源性很低(非-专利文件5：Sakaue,R.and N.Kajiyama(2003)."Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution."Applied and environmental microbiology 69(1):139-145.非-专利文 件6：Hirokawa,K.,A.Ichiyanagi,et al.(2008)."Enhancement of thermostability of fungal deglycating enzymes by directed evolution."Appl Microbiol Biotechnol78(5):775-781.)，因此无法通过简单的序列比对获得相关信息，而必须通过大量的创造性的研究来对该酶改造的相关信息。
具体而言是利用随机突变的方法，提高Amadorise I的热稳定性，方法如下：
(1)以Amadorise I的果糖氨基酸氧化酶基因序列为模板，进行易错PCR扩增，建立果糖氨基酸氧化酶的突变文库；
(2)将所述突变文库转入大肠杆菌，利用96孔板进行培养，并诱导表达；
(3)将细菌进行原位裂解，并利用醌法测定酶活性，筛选出热稳定性高的果糖赖氨酸氧化酶。
本发明以核苷酸序列2为模板，设计引物序列3(5’-atggcgcctt caattttgag cactg-3’)和引物序列4(5’-ttacggacct ctgctctctc caatc-3’)，进行易错PCR，通过合理控制PCR反应体系中锰离子的浓度，将突变频率控制在1～2个核苷酸突变/1Kb核苷酸。将所获得的含突变核苷酸的片段纯化后，以含核苷酸序列2的pET-22b质粒为模板进行全质粒PCR(WHOP-PCR)(非-专利文件7:Miyazaki,K.(2003).Creating random mutagenesis libraries by megaprimer PCR of whole plasmid(MEGAWHOP).Directed Evolution Library Creation,Springer:23-28)，所得产物用限制性内切酶DpnI进行酶切消化，去除模板质粒。之后，转化TOP10感受态细胞，涂布于含氨苄抗生素的LB固体平板上，所得克隆约为10000个，故该随机突变所获得的库容量约为10000个。
将所得克隆用涂布棒混匀后，收集于离心管中，抽质粒，所得质粒转化BL21(ED3)，以备下一步筛选用。接着，将上述转化子接种于96孔板中，所用培养基为150ul LB/每孔，含氨苄抗生素，该孔板作为保留板。次日按照相同顺序转接至另一块96孔板中，所用培养基为150ul LB/每孔，同时添加氨苄抗生素和IPTG进行诱导，于37℃下培养6小时，3800rpm离心收菌，去掉培养基，该孔板作为分析板。
在分析板中加入150ul裂解液(100mM Tris，pH8.0；0.4mg/ml脱氧胆酸钠；0.8mg/ml CTAB；20mM KCl；80mM MgSO4)。于室温下裂解半小时后，3800rpm离心15分钟，取50ul裂解液于一块新的96孔板中。另取50ul裂解液于96孔PCR板中，置于96孔PCR仪中，在50℃下加热处理15分钟，转移至另一块96孔板中。在上述两块中分别加入100ul的显色液(Tris，100mM，pH8.0；TOOS溶液，15mM；4-APP，0.5mM；POD，40U/ml；果糖赖氨酸，15mM)。通过30分钟的显色，利 用酶标仪记录其吸光值。通过计算两者吸光度的比值，判断热处理后的粗酶液的残留酶活比例。对于热稳定性提高了的突变株，其理论上的酶活残留比例将较野生型高。
本发明通过对10000多个突变株的筛选后，总共筛选获得了6个热稳定性有所提高的突变株。经测序分析，发现这6个突变株均为单碱基突变，其核苷酸序列突变分别为177G→T，293A→C，764G→C，830A→C，853G→C，1063G→C，相应的氨基酸序列突变分别为59E→N，98E→A，225G→A，277K→S，285E→Q，355D→H。
由于上述6个位点的突变提高的其热稳定性，故而如果上述突变成其他氨基酸，其热稳定性较野生型高的可能性也非常高。因而,本发明又大量尝试了上述位点的其他突变。同时，因为考虑到若将单一突变位点进行叠加，可以进一步提高热稳定性，所以也尝试了各种不同突变位点的组合。
对于59号位点的Glu，通过定点突变的方法，分别替代为其他19种氨基酸，构建19种突变质粒，对这19种突变质粒分别转化表达菌株BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现绝大部分突变株的热稳定性有所提高，其中以下突变体的热稳定性提高较为明显，分别是L，I，V，F，M，W，T，C，N，Y，D，H。
对于98号位点的Glu，通过定点突变的方法，分别替代为其他19种氨基酸，构建19种突变质粒，对这19种突变质粒分别转化表达菌株BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现绝大部分突变株的热稳定性有所提高，其中以下突变体的热稳定性提高较为明显，分别是A，L，I，V，P，F，S，T，C，N，Y，D，H。
对于225号位点的Gly，通过定点突变的方法，分别替代为其他19种氨基酸，构建19种突变质粒，对这19种突变质粒分别转化表达菌株BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现绝大部分突变株的热稳定性有所提高，其中以下突变体的热稳定性提高较为明显，分别是A，L，F，M，W，S，N，D。
对于277号位点的Lys，通过定点突变的方法，分别替代为其他19种氨基酸，构建19种突变质粒，对这19种突变质粒分别转化表达菌株BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现绝大部分突变株的热稳定性有所提高，其中以下突变体的热稳定性提高较为明显，分别是A，L，I，F，S，T，N，Y，R，H。
对于285号位点的Glu，通过定点突变的方法，分别替代为其他19种氨基酸，构建19种突变质粒，对这19种突变质粒分别转化表达菌株BL21(DE3)，进行培养， 诱导，表达，纯化，酶学性质分析，结果发现绝大部分突变株的热稳定性有所提高，其中以下突变体的热稳定性提高较为明显，分别是A，L，I，F，M，W，Q，N。
对于355号位点的Asp，通过定点突变的方法，分别替代为其他19种氨基酸，构建19种突变质粒，对这19种突变质粒分别转化表达菌株BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现绝大部分突变株的热稳定性有所提高，其中以下突变体的热稳定性提高较为明显，分别是L，I，V，F，M，W，T，C，Y，R，H。
通过对以上位点的具体分析，发现这些位点确实都对该酶的热稳定性有较大贡献。为此，考虑到若将不同的突变位点进行组合，极有可能会进一步提高酶的热稳定性。所以又对不同的突变位点进行了组合，比如2个位点组合，3个位点组合，4个位点组合，5个位点组合，6个位点组合。但是由于每个位点都有十个甚至更多的氨基酸突变可以选择，其不同位点的组合将是一个天文数字，不可能每一个都进行研究。故而只能选取其中较有代表性的突变组合，以此来说明上述位点的组合是有效的。
对于2个突变位点的组合，通过定点突变的方法，将其突变为以下几种突变体：59E→F，98E→C；59E→F，225G→D；59E→F，277K→N；59E→F，285E→F；98E→P，225G→N；98E→P，277K→S；225G→L，277K→A；225G→L，285E→I；277K→Y，355D→T；285E→I，355D→W；将构建好的突变株转化至BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现这些突变株的热稳定性进一步提高。
对于3个突变位点的组合，通过定点突变的方法，将其突变为以下几种突变体：59E→F，98E→C，225G→F；59E→F，225G→D，277K→N；59E→F，277K→N，285E→I；98E→P，225G→N，285E→Q；225G→L，285E→I，355D→T；将构建好的突变株转化至BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现这些突变株的热稳定性进一步提高。
对于4个突变位点的组合，通过定点突变的方法，将其突变为以下几种突变体：59E→F，98E→C，225G→F，277K→N；59E→F，225G→D，277K→N，285E→F；，将构建好的突变株转化至BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现这些突变株的热稳定性进一步提高。
对于5个突变位点的组合，通过定点突变的方法，将其突变为以下几种突变体：59E→F，98E→C，225G→F，277K→N，285E→F；将构建好的突变株转化至BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现这些突变株的热稳定性进一步提高。
对于6个突变位点的组合，通过定点突变的方法，将其突变为以下几种突变体：59E→F，98E→C，225G→F，277K→N，285E→F，355D→L；将构建好的突变株转化至BL21(DE3)，进行培养，诱导，表达，纯化，酶学性质分析，结果发现这些突变株的热稳定性进一步提高。
同时，作为一般惯用手段，在同源性很高的两条序列之间，其结构和功能往往很相似，对其中一个序列进行突变获得的有益效果，在另外一条同源性很高的序列之上进行同样的突变，一般来说同源性80％以上，优选地90％以上，更优选地96％以上，最为优选地99％以上，往往也会获得相似的有益效果。因而本发明在此基础上，对与序列2的氨基酸序列同源性等于或高于80％的序列进行了大量突变及其组合突变研究。
来自于以下几个菌株的果糖氨基酸氧化酶被选作试验对象，用以说明与序列1具有80％以上的同源性的酶，其相应位点的突变也将导致该酶热稳定性的增高。这几个菌株分别为：黑曲霉(Aspergillus niger)CBS 513.88，棒曲霉(Aspergillus clavatus)NRRL 1，新萨托菌(Neosartorya fischeri)NRRL 181，烟曲霉(Aspergillus fumigatus)Af293；相应地与序列1的同源性分别为80％，83％，96％，99％。
黑曲霉(Aspergillus niger)CBS 513.88与序列1的同源性为80％，其相应的突变位点为59E，98E，225G，277K，285E，355D，通过有选择性的挑选几种单个氨基酸突变，以及几种不同位点的组合突变，发现的确该酶的热稳定性也有不同程度的提高。比如以下的突变株热稳定性提高尤为明显：59E→F，98E→C，225G→F，277K→N，285E→F，355D→L。
棒曲霉(Aspergillus clavatus)NRRL 1与序列1的同源性为83％，其相应的突变位点为59E，98E，225G，277N，285E，355D，通过有选择性的挑选几种单个氨基酸突变，以及几种不同位点的组合突变，发现的确该酶的热稳定性也有不同程度的提高。比如以下的突变株热稳定性提高尤为明显：59E→F，98E→C，225G→F，285E→F，355D→L。
新萨托菌(Neosartorya fischeri)NRRL 181与序列1的同源性为96％，其相应的突变位点为59E，98E，225G，277K，285E，355D，通过有选择性的挑选几种单个氨基酸突变，以及几种不同位点的组合突变，发现的确该酶的热稳定性也有不同程度的提高。比如以下的突变株热稳定性提高尤为明显：59E→F，98E→C，225G→F，277K→N，285E→F，355D→L。
烟曲霉(Aspergillus fumigatus)Af293与序列1的同源性为99％，其相应的突变位点为59E，98E，225G，277K，285E，355D，通过有选择性的挑选几种单个 氨基酸突变，以及几种不同位点的组合突变，发现的确该酶的热稳定性也有不同程度的提高。比如以下的突变株热稳定性提高尤为明显：59E→F，98E→C，225G→F，277K→N，285E→F，355D→L。
本发明中氨基酸的突变均用缩写字符代替，其为公知常识，具体含义如下：甘氨酸G，丙氨酸A，亮氨酸L，异亮氨酸I，缬氨酸V，脯氨酸P，苯丙氨酸F，甲硫氨酸M，色氨酸W，丝氨酸S，谷氨酰胺Q，苏氨酸T，半胱氨酸C，天冬酰胺N，酪氨酸Y，天冬氨酸D，谷氨酸E，赖氨酸K，精氨酸R，组氨酸H。本发明中285E→F，代表第285位的谷氨酸突变为苯丙氨酸，其余亦类似。
这里值得说明的是，本研究所使用的载体为pET-22b，宿主菌(宿主细胞)为BL21(DE3)。根据一般常识，任何简单的进行载体更换，如pET-20b，pET-28b，pET-32a，pQE30，pTrc99a等，或者进行宿主菌更换，如Rosetta，Origami，M15等，都应当被认为具有相同的技术效果，都将被认为落入权利要求保护范围之内。任何在突变酶的N端或C端添加纯化标签，已经信号肽的行为，也应当被认为落入权利要求保护范围之内。
作为一般地，可以利用已经公知的酶的表达纯化技术来进行果糖氨基酸氧化酶的制备。例如，接种相应的突变株菌种，待OD600生长至0.5～1时，进行异丙基硫代半乳糖苷(IPTG)诱导，此时为了有利于其表达，可以选择低温诱导，如25℃，20℃，16℃等，根据诱导温度及表达情况，确定最适的诱导时间，以获得最佳的表达量。表达之后可以利用常规方法进行离心，超声破碎，SDS-PAGE检测表达量等。至于纯化时，可以利用重组酶所带的标签选择相应的方法。例如，若重组质粒含有His-tag，可以利用镍柱进行亲和纯化。
可以利用上述具有良好热稳定性的果糖氨基酸氧化酶，进行糖化白蛋白检测方法的研发，并进一步开发出糖化白蛋白检测试剂盒。
其主要原理是，选择适当的缓冲液，在蛋白酶的作用下，糖化白蛋白被降解成果糖氨基酸或者果糖多肽，此时在果糖氨基酸氧化酶的作用下，将果糖氨基酸或者果糖多肽氧化成葡萄糖、氨基酸或者多肽、过氧化氢，同时消耗一定的氧气。然后通过测定产物的生产或者是底物的消耗量，来计算出样本中糖化白蛋白的含量。
样本要求：理论上只要是含有糖化白蛋白、糖化多肽、糖化氨基酸的样本都可以作为检测用的样本。由于本方法主要应用于临床检验，故常见的样本为血清。理论上应当避免溶血，样本采集后应当在2～8℃保持并及时测定。
缓冲液：任何适当浓度及pH的缓冲液都能在此使用，只要其对检测过程的酶活反 应无抑制作用，不干扰检测过程即可。典型的缓冲液有：三羟甲基氨基甲烷盐酸盐缓冲液、乙酸-乙酸钠缓冲液、邻苯二甲酸-盐酸缓冲液或甘氨酸-盐酸缓冲液等。缓冲液的浓度可根据情况而确定，优选20～200mmol/L，更为优选地为50～100mmol/L。
蛋白酶：任何能够降解糖化白蛋白的蛋白酶都可以用于此，优选地有胰蛋白酶、嗜碱性蛋白酶、蛋白酶A、蛋白酶K等。所添加的蛋白酶应当促使糖化白蛋白能够充分降解，根据其不同来源，相应的浓度应为10～100KU/L，优选40KU/L。
产物的生成：产物的生成主要有三种，即葡萄糖、氨基酸、过氧化氢。可以通过测定这三种产物中的任何一种，来计算确定糖化白蛋白的含量。
葡萄糖的测定可以通过己糖激酶法测定，其原理是在己糖激酶催化下，葡萄糖和ATP发生磷酸化反应，生成葡萄糖-6-磷酸(G-6-P)与ADP。前者在葡萄糖-6-磷酸脱氢酶(G6PD)催化下脱氢，生成6-磷酸葡萄糖酸(6-GP)，同时使NADP还原成NADPH，NADPH的生成速率与葡萄糖浓度呈正比，在波长340nm监测吸光度升高速率，计算葡萄糖浓度。
氨基酸可以通过与荧光胺反应，最终测定荧光来测定。
过氧化氢则可以通过偶联过氧化物酶生成H2O2，再通过Trinder反应，生成颜色来检测。这里的色原可以是N,N-二(4-磺丁基)-3-甲基苯胺二钠盐(TODB)，N-乙基-N-(3-磺丙基)-3-甲基苯胺钠盐(TOPS)，N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺钠盐(TOOS)。
底物的消耗，底物主要是指O2，O2的消耗可以通过氧电极来检测。
利用上述方法，可以开发出相应的糖化白蛋白检测试剂盒试剂盒。主要原理是，选择适当的缓冲液，在蛋白酶的作用下，糖化白蛋白被降解成果糖氨基酸或者果糖多肽，此时在果糖氨基酸氧化酶的作用下，将果糖氨基酸或者果糖多肽氧化成葡萄糖、氨基酸或者多肽、过氧化氢，同时消耗一定的氧气。然后通过测定产物的生产或者是底物的消耗量，来计算出样本中糖化白蛋白的含量。在实际应用中，最常用的方法是检测产物中的过氧化氢，即通过Trinder反应来检测，这也是目前绝大多数试剂盒使用的偶联方式，本说明书中的实施例着重以此来进行实验，借以说明，但并不排除试剂盒中可以用检测其它物质来实现对糖化白蛋白的检测。
该糖化白蛋白检测试剂盒试剂盒包含试剂1和试剂2，其中试剂1包含缓冲液，蛋白酶，过氧化物酶，4-氨基安替比林，防腐剂。试剂2包含缓冲液，果糖氨基酸氧化酶突变株，色原，防腐剂。
缓冲液：任何适当浓度及pH的缓冲液都能在此使用，只要其对检测过程的酶活反 应无抑制作用，不干扰检测过程即可。典型的缓冲液有：三羟甲基氨基甲烷盐酸盐缓冲液、乙酸-乙酸钠缓冲液、邻苯二甲酸-盐酸缓冲液或甘氨酸-盐酸缓冲液等。缓冲液的浓度可根据情况而确定，优选20～200mmol/L，更为优选地为50～100mmol/L。
蛋白酶：任何能够降解糖化白蛋白的蛋白酶都可以用于此，优选地有胰蛋白酶、嗜碱性蛋白酶、蛋白酶A、蛋白酶K等。所添加的蛋白酶应当促使糖化白蛋白能够充分降解，根据其不同来源，相应的浓度应为10～100KU/L，优选40KU/L。
过氧化物酶：该酶起到偶联作用，根据需要可添加浓度为10～100KU/L，优选地为30KU/L。
防腐剂的目的是防止细菌在试剂盒中的滋生，保证试剂盒长期保存的灵敏度等不受影响。所述的防腐剂有2-甲基-4-异噻唑啉-3-酮、proclin300等。为了有效达到这种目的，防腐剂的添加量应为0.01～0.05％，优选0.02％。
所述的果糖氨基酸氧化酶是指本发明的高热稳定性果糖氨基酸氧化酶，该酶的添加量应当以能够使底物果糖氨基酸或者果糖多肽充分反应为准，所添加的量应为10～100KU/L，优选地为20～50KU/L，最优选地为28KU/L。
本发明所述的色原可以是N,N-二(4-磺丁基)-3-甲基苯胺二钠盐(TODB)，N-乙基-N-(3-磺丙基)-3-甲基苯胺钠盐(TOPS)，N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺钠盐(TOOS，)色原在试剂盒中的浓度可以为1～10mmol/L，优选地为2mmol/L。
具体的，本发明上述的测定糖化白蛋白的试剂盒，该试剂盒包括试剂1和试剂2，试剂中各组分浓度为：
试剂1：
缓冲液  20～200mmol/L
蛋白酶  10～100KU/L
过氧化物酶  10～100KU/L
4-氨基安替比林  5～50mM/L
防腐剂  0.01～0.05％
试剂2：
缓冲液  20～200mmol/L
上述六个位点中的1个或更多氨基酸取代后所得的果糖氨基酸氧化酶10～ 100KU/L
色原    1～10mmol/L
防腐剂  0.01～0.05％。
若将本发明的糖化白蛋白的检测，与白蛋白的检测相结合，可以得出糖化白蛋白浓度除以白蛋白浓度算出糖化白蛋白的百分比值。
由于其性能优于以前的果糖氨基酸氧化酶，使得试剂盒在灵敏度、抗干扰等方面满足要求之外，其长期保持后的各方面性能均无明显下降。
本发明提供了一种稳定、简单、快速、灵敏、准确定量的检测试剂，其检测具有良好的稀释直线性、准确度、精密度，并与HPLC检测法有良好的一致性。本发明同现技术相比，采用本发明的糖化白蛋白测定试剂盒，酶法测定糖化白蛋白，完全可以替代HPLC应用于临床实验室进行大批量样本的测定，其操作简便，线性范围宽，成本低，具有很高的应用价值。
附图说明
图1糖化白蛋白检测线性范围图。
图2本发明与HPLC检测的相关性图。
具体实施方式
下面结合具体实施例进一步阐述本发明，但并不限于下述实施例。
实施例1
突变文库的构建
1、对于序列2所示的的序列，利用全基因合成的方法进行合成，并克隆至pET-22b载体上，所用酶切位点为NdeI和XhoI。以此得到的质粒pET-Ama为以下易错PCR和WHOP-PCR的模板。
2、易错PCR反应体系及条件
表1 反应体系为100ul：
名称 体积(ul)
dNTPMixture(各2.5mM) 8
dTTP(100mM) 0.8
dCTP(100mM) 0.8
10*PCRBuffer 10
上游引物(5mM)，序列3 20
下游引物(5mM)，序列4 20
MnCl2(5mM) 10
Mg2+(25mM) 14
Taq酶(5U/ul) 1
模板(10ng/ul) 5
水 12
反应条件为：
95℃5min；94℃30sec；55℃30sec；72℃2min；30个循环；72℃10min；4℃保存。
上述的MnCl2用量可以适当进行调整，以便获得合适的突变频率，即1～2核苷酸突变/1Kb核苷酸。
3、WHOP-PCR反应体系及条件
将易错PCR获得的片段，大小约1.4Kb进行胶回收后，用作下一轮WHOP-PCR的引物。
表2 WHOP-PCR反应体系，50ul：
Figure PCTCN2014093580-appb-000004
WHOP-PCR反应条件：
98℃30sec；98℃10sec；60℃10sec；72℃2.5min；24个循环；72℃5min；4℃保存。
4、酶切及转化
在50ul的WHOP-PCR的产物加入1ul的DpnI进行酶切，于37℃下反应2小时，以彻底除去模板DNA。
将上述酶切后的产物分5管转化TOP10感受态细胞，次日得到总共10000个单克隆以上的库容量。
实施例2
突变文库的筛选
1、培养、诱导、表达
将所得克隆用涂布棒混匀后，收集于离心管中，抽质粒，所得质粒转化BL21(ED3)，以备下一步筛选用。接着，将上述转化子接种于96孔板中，其中最后一个孔接种野生型菌株以作为对照，所用培养基为150ul LB/每孔，含氨苄抗生素，该孔板作为保留板。次日按照相同顺序转接至另一块96孔板中，所用培养基为150ul LB/每孔，同时添加氨苄抗生素和IPTG进行诱导，于37℃下培养6小时，3800rpm离心收菌，去掉培养基，该孔板作为分析板。
2、筛选
在分析板中加入150ul裂解液(100mM Tris，pH8.0；0.4mg/ml脱氧胆酸钠；0.8mg/ml CTAB；20mM KCl；80mM MgSO4)。于室温下裂解半小时后，3800rpm离心15分钟，取50ul裂解液于一块新的96孔板中。另取50ul裂解液于96孔PCR板中，置于96孔PCR仪中，在50℃下加热处理15分钟，转移至另一块96孔板中。在上述两块中分别加入100ul的显色液(Tris，100mM，pH8.0；TOOS溶液，15mM；4-APP，0.5mM；POD，40U/ml；果糖赖氨酸，15mM)。通过30分钟的显色，利用酶标仪记录其吸光值。通过计算两者吸光度的比值，判断热处理后的粗酶液的残留酶活比例。对于热稳定性提高了的突变株，其理论上的酶活残留比例将较野生型高。
本发明通过对10000多个突变株的筛选后，总共筛选获得了6个热稳定性有所提高的突变株。经测序分析，发现这6个突变株均为单碱基突变，其核苷酸序列突变分别为177G→T，293A→C，764G→C，830A→C，853G→C，1063G→C，相应的氨基酸序列突变分别为59E→N，98E→A，225G→A，277K→S，285E→Q，355D→H。
实施例3
果糖氨基酸氧化酶的酶活测定及热稳定性分析
经纯化后的果糖氨基酸氧化酶，在100mM Tris，pH8.0的缓冲液中稀释成约10ug/ml。
取50ul该果糖氨基酸氧化酶于96孔PCR中，于50℃下热处理15min，4℃保存。
将经热处理过的50ul果糖氨基酸氧化酶转移至96孔板上，同时取未加热过的果糖氨基酸氧化酶50ul于同一块96孔板上。于37℃保温10min。
加入事先已孵育至37℃的显色液(Tris，100mM，pH8.0；TOOS溶液，15mM； 4-APP，0.5mM；POD，40U/ml；果糖赖氨酸，15mM)，于37℃下反应30min。
利用酶标仪记录其在555nm处的吸光度。
将热处理过的果糖氨基酸氧化酶的吸光值除以未热处理过的果糖氨基酸氧化酶的吸光值，所得数值即为在该温度下的残留酶活比例。
对上述随机突变获得的6个突变株及野生型果糖氨基酸氧化酶进行热稳定性分析，其结果见表3
表3 随机突变获得的突变株的热稳定性分析
Figure PCTCN2014093580-appb-000005
实施例4
59位氨基酸的取代的热稳定性分析
1、59为氨基酸的点突变的引入
第一轮PCR以pET-Ama为模板，分别用含有点突变的引物序列5(5’-tgctagttat tgctcagcgg-3’)、序列6(5’-acaagatta tgttacacag cgagctg-3’)作为上游引物扩增，获得含有点突变的目的片段。PCR体系为50ul：
表4 PCR体系
Figure PCTCN2014093580-appb-000006
Figure PCTCN2014093580-appb-000007
PCR反应条件为：
98℃30sec；98℃10sec；60℃10sec；72℃30sec；30个循环；72℃5min；4℃保存。
所得目的片段经纯化后，用于下一轮WHOP-PCR的引物，其具体反应体系见表5
表5 WHOP-PCR反应体系
Figure PCTCN2014093580-appb-000008
PCR反应条件为：
98℃30sec；98℃10sec；60℃10sec；72℃2.5min；24个循环；72℃5min；4℃保存。
在50ul的WHOP-PCR的产物中加入1ul的DpnI进行酶切，于37℃下反应2小时，以彻底除去模板DNA。然后转化BL21(DE3)，经测序正确后，用于热稳定性分析。
2.59位氨基酸取代后的热稳定性
将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表6 59位氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000009
实施例5
98位氨基酸取代的热稳定性分析
按照上述方法，在98位氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表7 98位氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000010
Figure PCTCN2014093580-appb-000011
实施例6
225位氨基酸取代的热稳定性分析
按照上述方法，在225位氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表8 225位氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000012
实施例7
277位氨基酸取代的热稳定性分析
按照上述方法，在277位氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表9 277位氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000013
Figure PCTCN2014093580-appb-000014
实施例8
285位氨基酸取代的热稳定性分析
按照上述方法，在285位氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表10 285位氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000015
Figure PCTCN2014093580-appb-000016
实施例9
355位氨基酸取代的热稳定性分析
按照上述方法，在355位氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表11 355位氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000017
Figure PCTCN2014093580-appb-000018
实施例10
两个不同突变位点氨基酸取代的结合的热稳定性分析
按照上述方法，在两个不同位点氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表12 两个不同突变位点为氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000019
Figure PCTCN2014093580-appb-000020
实施例11
三个不同突变位点氨基酸取代的结合的热稳定性分析
按照上述方法，在三个不同位点氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表13 三个不同突变位点为氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000021
实施例12
四个不同突变位点氨基酸取代的结合的热稳定性分析
按照上述方法，在四个不同位点氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表14 四个不同突变位点为氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000022
实施例13
五个不同突变位点氨基酸取代的结合的热稳定性分析
按照上述方法，在五个不同位点氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表15 五个不同突变位点为氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000023
Figure PCTCN2014093580-appb-000024
实施例14
六个不同突变位点氨基酸取代的结合的热稳定性分析
按照上述方法，在六个不同位点氨基酸上引入不同的点突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表16 六个不同突变位点为氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000025
实施例15
来自黑曲霉(Aspergillus niger)CBS 513.88的果糖氨基酸氧化酶的突变及其组合的热稳定性分析
通过Blast可知，来自于黑曲霉(Aspergillus niger)CBS 513.88的果糖氨基酸氧化酶的氨基酸序列与来自烟曲霉(Aspergillus fumigatus)的果糖氨基酸氧化酶的氨基酸序列的同源性为80％。其氨基酸序列见序列68，核苷酸序列见序列69，利用全基因合成的方法，将序列69合成，并克隆于pET-22b载体的NdeI和XhoI酶切位点中。
经序列分析发现，相应于Amadoriase I的59，98，225，277，285，355位点的氨基酸，在黑曲霉(Aspergillus niger)CBS 513.88的果糖氨基酸氧化酶上的相应位点为59E，98E，225G，277K，285E，355D。
按照上述点突变的方法，将黑曲霉(Aspergillus niger)CBS 513.88的果糖氨 基酸氧化酶的这些位点进行单碱基突变或不同位点的组合突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表17 黑曲霉(Aspergillus niger)CBS 513.88的果糖氨基酸氧化酶的氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000026
实施例16
来自棒曲霉(Aspergillus clavatus)NRRL 1的果糖氨基酸氧化酶的突变及其组合的热稳定性分析
通过Blast可知，来自于棒曲霉(Aspergillus clavatus)NRRL 1的果糖氨基酸氧化酶的氨基酸序列与来自烟曲霉(Aspergillus fumigatus)的果糖氨基酸氧化酶的氨基酸序列的同源性为83％。其氨基酸序列见序列70，核苷酸序列见序列71，利用全基因合成的方法，将序列71合成，并克隆于pET-22b载体的NdeI和XhoI酶切位点中。
经序列分析发现，相应于Amadoriase I的59，98，225，277，285，355位 点的氨基酸，在棒曲霉(Aspergillus clavatus)NRRL 1的果糖氨基酸氧化酶上的相应位点为59E，98E，225G，277N，285E，355D。
按照上述点突变的方法，将棒曲霉(Aspergillus clavatus)NRRL 1的果糖氨基酸氧化酶的这些位点进行单碱基突变或不同位点的组合突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表18 棒曲霉(Aspergillus clavatus)NRRL 1的果糖氨基酸氧化酶的氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000027
实施例17
来自新萨托菌(Neosartorya fischeri)NRRL 181的果糖氨基酸氧化酶的突变及其组合的热稳定性分析
通过Blast可知，来自于新萨托菌(Neosartorya fischeri)NRRL 181的果糖氨基酸氧化酶的氨基酸序列与来自烟曲霉(Aspergillus fumigatus)的果糖氨基酸氧化酶的氨基酸序列的同源性为96％。其氨基酸序列见序列72，核苷酸序列见序列73，利用全基因合成的方法，将序列73合成，并克隆于pET-22b载体的NdeI和XhoI酶切位点中。
经序列分析发现，相应于Amadoriase I的59，98，225，277，285，355位点的氨基酸，在新萨托菌(Neosartorya fischeri)NRRL 181的果糖氨基酸氧化酶上 的相应位点为59E，98E，225G，277K，285E，355D。
按照上述点突变的方法，将新萨托菌(Neosartorya fischeri)NRRL 181的果糖氨基酸氧化酶的这些位点进行单碱基突变或不同位点的组合突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表19 新萨托菌(Neosartorya fischeri)NRRL 181的果糖氨基酸氧化酶的氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000028
实施例18
来自烟曲霉(Aspergillus fumigatus)Af293的果糖氨基酸的突变及其组合的热稳定性分析
通过Blast可知，来自于烟曲霉(Aspergillus fumigatus)Af293的果糖氨基酸氧化酶的氨基酸序列与来自烟曲霉(Aspergillus fumigatus)的果糖氨基酸氧化酶的氨基酸序列的同源性为99％。其氨基酸序列见序列74，核苷酸序列见序列75，利用全基因合成的方法，将序列75合成，并克隆于pET-22b载体的NdeI和XhoI酶切位点中。
经序列分析发现，相应于Amadoriase I的59，98，225，277，285，355位点的氨基酸，在烟曲霉(Aspergillus fumigatus)CBS 513.88的果糖氨基酸氧化酶上的相应位点为59E，98E，225G，277K，285E，355D。
按照上述点突变的方法，将烟曲霉(Aspergillus fumigatus)Af293的果糖氨基酸氧化酶的这些位点进行单碱基突变或不同位点的组合突变。将所获得的突变株，接种过夜，次日按照1％的比例转接至新鲜的含氨苄的LB培养基中，待OD600长至0.8左右时，加入终浓度为0.4mM的IPTG诱导约5个小时，离心收菌，破碎，纯化。纯化所得的果糖氨基酸氧化酶按照上述方法分析其热稳定性。具体结果见下表。
表20 烟曲霉(Aspergillus fumigatus)Af293的果糖氨基酸氧化酶的氨基酸取代的热稳定性分析
Figure PCTCN2014093580-appb-000029
实施例19
制备方法
1、菌体的获得：培养含有不同突变基因的菌种培养至OD600约0.8时，加入IPTG诱导5小时，离心收集菌体，收集的菌体用缓冲液重新悬浮，超声破碎，离心收集上清；
2、硫酸铵沉淀：使用硫酸铵溶液对步骤(1)所得上清液进行分级沉淀，将最后收集得到的沉淀溶于buffer A中，得到粗提液；
3、亲和层析：用buffer A平衡镍柱，再将步骤(2)得到的粗提液上镍柱吸附，吸附结束后，用咪唑溶液进行梯度洗脱，收集洗脱液；
4、透析：将步骤(3)中的洗脱液装至透析袋中，置于4℃透析液中，并磁力搅拌透析过夜。
所述步骤(2)中buffer A为：50mM磷酸钾缓冲液，pH 8.0，500mM NaCl；
所述步骤(3)中咪唑溶液为：50mM磷酸钾缓冲液，pH 8.0，500mM NaCl，20mM～1000mM咪唑；
所述步骤(4)中透析液为：50mM磷酸钾缓冲液，pH 8.0，500mM NaCl。
实施例20
糖化白蛋白检测试剂盒的线性范围
包括试剂1和试剂2，其中：
试剂1：
Figure PCTCN2014093580-appb-000030
试剂2：
Figure PCTCN2014093580-appb-000031
本实施例所用果糖氨基酸氧化酶突变体具体为M76号突变株，事实上，其他突变株亦可以使用。
测试的样本应当使用新鲜的血清，应当避免溶血。血清收集后若不及时进行检查，则应到存储于4℃，理论上应不超过2周。
检测条件：主波长600nm，副波长700nm。
检测步骤：见表21
表21：检测步骤
Figure PCTCN2014093580-appb-000032
Figure PCTCN2014093580-appb-000033
线性范围，见图1：
取GA高值血清，用生理盐水稀释成不同梯度，分别进行测定。
用该方法检测糖化白蛋白的线性范围21.0～1200umol/L，r2>0.990。
实施例21
糖化白蛋白检测试剂盒的精密度试验
对一高值和低值质控和两份样本血清分别进行20次测试，均值分别为204umol/L，751umol/L，251umol/L，373umol/L，CV分别为1.1％，0.7％，0.8％，0.6％。
表22 批内精密度试验
Figure PCTCN2014093580-appb-000034
对一高值和低值质控和两份样本血清分别进行连续测定10天，均值分别为204umol/L，751umol/L，251umol/L，373umol/L，CV分别为1.2％，0.7％，1.3％，1.0％。
表23 批间精密度试验
Figure PCTCN2014093580-appb-000035
实施例22
糖化白蛋白检测试剂盒的开瓶稳定性试验
试剂在日立生化仪上开瓶稳定性至少可持续4周。
表24 开瓶稳定性试验
GA 0天 7天 14天 21天 28天
质控1：198umol/LGA 203 202 206 202 206
质控2：750umol/LGA 753 740 738 736 745
N109657270umol/L 274 271 274 269 272
实施例23
糖化白蛋白检测试剂盒与HPLC相关性
通过将本发明的糖化白蛋白检测试剂盒同经典的HPLC检测方法测定同样的样本，进行比较，发现其相关性良好，R2>0.995。
实施例24
糖化白蛋白检测试剂盒抗干扰试验
发现当维生素C的浓度低于0.2mmol/L，血红蛋白低于1.6g/L，结合胆红素低于0.32mmol/L，非结合胆红素低于0.32mmol/L时，甘油三脂低于20mmol/L，血糖低于20g/L，尿酸低于36g/L时，其对糖化白蛋白测定值的干扰小于10％。
表25 干扰试验
Figure PCTCN2014093580-appb-000036
Figure PCTCN2014093580-appb-000037
实施例25
糖化白蛋白百分比值的测定
将糖化白蛋白的检测，与白蛋白的检测相结合，可以得出糖化白蛋白浓度除以白蛋白浓度算出糖化白蛋白的百分比值。
白蛋白检测试剂如下
试剂3：
琥珀酸缓冲液            0.05mol/L
聚氧乙烯(23)十二烷基醚  2.4g/L
溴甲酚绿                1.8×10-4mol/L
检测条件：主波长600nm，副波长700nm。
反应方法：终点法。
检测步骤：见表26
表26：检测步骤
Figure PCTCN2014093580-appb-000038
利用上述方法，对20份血清进行测定，并计算糖化白蛋白的百分比，见表27。
表27 20份血清糖化白蛋白的百分比
Figure PCTCN2014093580-appb-000039
Figure PCTCN2014093580-appb-000040
通常情况下，糖化白蛋白的参考值为11～16％，糖化白蛋白值的升高常见于糖尿病，该指标可以作为糖尿病患者中期血糖控制的参考指标。
实施例26
利用其它果糖氨基酸氧化酶突变体配制的糖化白蛋白检测试剂盒的性能
由于本发明所涉及的果糖氨基酸氧化酶突变体众多，不可能一一在糖化白蛋白检测试剂盒中逐个验证，故而只选取几个最典型的果糖氨基酸氧化酶突变体，用于糖化白蛋白检测试剂盒，并对它们的性能进行评估，用以说明本发明所涉及的果糖氨基酸氧化酶突变体适合于糖化白蛋白检测试剂盒，不能用于限制本发明。所涉及的试剂盒的检测方法，线性范围，批内精密度，批间精密度，开口稳定性，与HPLC的相关性，抗干扰等的验证方法均于实施例20～25所述相同。
糖化白蛋白检测试剂盒，包括试剂1和试剂2，其中：
试剂1：
Figure PCTCN2014093580-appb-000041
Figure PCTCN2014093580-appb-000042
试剂2：
Figure PCTCN2014093580-appb-000043
本实施例中，果糖氨基酸氧化酶突变株选用以下几种代表进行试验：即上述实施例制备的M 1，M 13，M28，M35，M50，M56，M64，M73，M79，M80，M81，M90，M 100，M 109，M 120。
上述果糖氨基酸氧化酶突变体及其相应的糖化白蛋白检测试剂盒的性能具体见下表。
表28 利用不同果糖氨基酸氧化酶突变株制备的糖化白蛋白检测试剂盒的性能
Figure PCTCN2014093580-appb-000044
Figure PCTCN2014093580-appb-000045
本发明的上述实施例是对本发明的说明而不能用于限制本发明，与本发明的权利要求书相当的含义和范围内的任何改变，都应认为是包括在权利要求书的范围内。

Claims (20)

  1. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(a)至(f)中的氨基酸对应的位置上具有1个或更多的氨基酸残基进行取代，所得的果糖氨基酸氧化酶；与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (a)59位的谷氨酸，
    (b)98位的谷氨酸，
    (c)225位的甘氨酸，
    (d)277位的赖氨酸，
    (e)285位的谷氨酸，
    (f)355位的天冬氨酸。
  2. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(g)至(l)中的氨基酸对应的位置被取代为以下(g)至(l)中记载的取代后的氨酸酸残基，所得的果糖氨基酸氧化酶；与与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (g)59位的谷氨酸被取代为L、I、V、F、M、W、T、C、N、Y、D或H；
    (h)98位的谷氨酸酸被取代为A、L、I、V、P、F、S、T、C、N、Y、D或H；
    (i)225位的甘氨酸被取代为A、L、F、M、W、S、N或D；
    (j)277位的赖氨酸被取代为A、L、I、F、S、T、N、Y、R或H；
    (k)285位的谷氨酸被取代为A、L、I、F、M、W、Q或N；
    (l)355位的天冬氨酸被取代为L、I、V、F、M、W、T、C、Y、R或H。
  3. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(m)至(v)中的氨基酸被取代为以下(m)至(v)中记载的取代后的氨酸酸残基，所得的果糖氨基酸氧化酶；与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (m)59E→F，98E→C；
    (n)59E→F，225G→D；
    (o)59E→F，277K→N；
    (p)59E→F，285E→F；
    (q)98E→P，225G→N；
    (r)98E→P，277K→S；
    (s)225G→L，277K→A；
    (t)225G→L，285E→I；
    (u)277K→Y，355D→T；
    (v)285E→I，355D→W。
  4. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(w)至(aa)中的氨基酸被取代为以下(w)至(aa)中记载的取代后的氨酸酸残基，所得的果糖氨基酸氧化酶；与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (w)59E→F，98E→C，225G→F
    (x)59E→F，225G→D，277K→N
    (y)59E→F，277K→N，285E→I
    (z)98E→P，225G→N，285E→Q
    (aa)225G→L，285E→I，355D→T。
  5. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(ab)至(ac)中的氨基酸被取代为以下(ab)至(ac)中记载的取代后的氨酸酸残基，所得的果糖氨基酸氧化酶；与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (ab)59E→F，98E→C，225G→F，277K→N；
    (ac)59E→F，225G→D，277K→N，285E→F。
  6. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(ad)中的氨基酸被取代为(ad)中记载的取代后的氨酸酸残基，所得的果糖氨基酸氧化酶；与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (ad)59E→F，98E→C，225G→F，277K→N，285E→F。
  7. 一种果糖氨基酸氧化酶，其特征在于，具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶中，其选自以下(ae)中的氨基酸被取代为(ae)中记载的取代后的氨酸酸残基，所得的果糖氨基酸氧化酶；与取代前的具有SEQ ID.No.1所示的氨基酸序列或与该氨基酸序列有80％以上同源性的果糖氨基酸氧化酶相比较，具有更高的热稳定性：
    (ae)59E→F，98E→C，225G→F，277K→N，285E→F，355D→L。
  8. 一种果糖氨基酸氧化酶，其特征在于，所述的与具有SEQID.No.1所示的氨基酸序列有80％以上同源性的果糖氨基酸氧化酶来自于黑曲霉(Aspergillus niger)CBS513.88，棒曲霉(Aspergillus clavatus)NRRL1，新萨托菌(Neosartorya fischeri)NRRL 181或烟曲霉(Aspergillus fumigatus)Af293。
  9. 一种编码上述权利要求1～8任何一项所述的果糖氨基酸氧化酶的基因。
  10. 一种包含权利要求9所述的果糖氨基酸氧化酶基因的重组载体。
  11. 一种包含权利要求10所述重组载体的宿主细胞。
  12. 一种制备果糖氨基酸氧化酶的方法，其特征在于包括以下步骤：
    (1)菌体的获得：培养含有不同突变基因的菌种培养至OD600生长至0.5～1时，加入异丙基硫代半乳糖苷诱导5小时，离心收集菌体，收集的菌体用缓冲液重新悬浮，超声破碎，离心收集上清液；
    (2)硫酸铵沉淀：使用硫酸铵溶液对步骤(1)所得上清液进行分级沉淀，将最后收集得到的沉淀溶于buffer A中，得到粗提液；
    (3)亲和层析：用buffer A平衡镍柱，再将步骤(2)得到的粗提液上镍柱吸附， 吸附结束后，用咪唑溶液进行梯度洗脱，收集洗脱液；
    (4)透析：将步骤(3)中的洗脱液装至透析袋中，置于4℃透析液中，并磁力搅拌透析过夜；
    所述步骤(2)中buffer A为：50mM磷酸钾缓冲液，pH8.0，500mM NaCl；
    所述步骤(3)中咪唑溶液为：50mM磷酸钾缓冲液，pH8.0，500mM NaCl，20mM～1000mM咪唑；
    所述步骤(4)中透析液为：50mM磷酸钾缓冲液，pH8.0，500mM NaCl。
  13. 一种测定糖化白蛋白的试剂盒，其特征在于：该试剂盒包括试剂1和试剂2，试剂中各组分浓度为：
    试剂1：
    缓冲液 20～200mmol/L，
    蛋白酶 10～100KU/L，
    过氧化物酶 10～100KU/L，
    4-氨基安替比林 5～50mM/L，
    防腐剂 0.01～0.05％；
    试剂2：
    缓冲液 20～200mmol/L，
    权利要求1～8任一权利所述的果糖氨基酸氧化酶 10～100KU/L，
    色原 1～10mmol/L，
    防腐剂 0.01～0.05％。
  14. 根据权利要求13所述的测定糖化白蛋白的试剂盒，其特征在于：所述的缓冲液为三羟甲基氨基甲烷盐酸盐缓冲液、乙酸-乙酸钠缓冲液、邻苯二甲酸-盐酸缓冲液或甘氨酸-盐酸缓冲液。
  15. 根据权利要求13所述的测定糖化白蛋白的试剂盒，其特征在于：所述的蛋白酶为胰蛋白酶、嗜碱性蛋白酶、蛋白酶A或蛋白酶K。
  16. 根据权利要求13所述的测定糖化白蛋白的试剂盒，其特征在于：所述的色原为N,N-二(4-磺丁基)-3-甲基苯胺二钠盐、N-乙基-N-(3-磺丙基)-3-甲基苯胺钠盐或N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺钠盐。
  17. 根据权利要求13所述的测定糖化白蛋白的试剂盒，其特征在于：所述的防腐 剂为2-甲基-4异噻唑啉-3-酮、proclin300。
  18. 一种用于测定糖化白蛋白的方法，其特征在于：
    (1)利用蛋白酶处理样本，使之降解生成糖化氨基酸或者糖化多肽；
    (2)利用上述权利要求1～8中的任何一项果糖氨基酸氧化酶，将糖化氨基酸或者糖化多肽氧化；
    (3)通过检测产物的生成或底物的消耗来检测样本中糖化白蛋白的含量。
  19. 根据权利要求18所述的用于测定糖化白蛋白的方法，其特征在于，步骤(1)所述的蛋白酶是胰蛋白酶、嗜碱性蛋白酶、蛋白酶A或蛋白酶K。
  20. 根据权利要求18所述的用于测定糖化白蛋白的方法，其特征在于，步骤(3)所述的产物生成是通过偶联过氧化物酶生成H2O2，再通过Trinder反应，生成颜色来检测。
PCT/CN2014/093580 2013-12-27 2014-12-11 果糖氨基酸氧化酶、制备方法及含该酶的糖化白蛋白检测试剂盒 WO2015096621A1 (zh)

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