Fructose amino acid oxidase, preparation method and enzyme-containing kit for detecting glycated albumin

Abstract

Fructosyl amino acid oxidase is provided, which has an amino acid sequence as shown in SEQID. No. 1 or fructosyl amino acid oxidase having a homology of more than 80% with this amino acid sequence, on a corresponding site of an amino acid selected from following (a) to (f), having one or more amino acid residues conducting a substitution, obtained fructosyl amino acid oxidase having a higher thermostability: (a) 59-site glutamic acid, (b) 98-site glutamic acid, (c) 225-site glycine, (d) 277-site lysine, (e) 285-site glutamic acid, and (f) 355-site aspartic acid. The method for preparing the above oxidase and the test kit containing the enzyme for determining glycated albumin are also provided.

Claims

1. A fructosyl amino acid oxidase comprising the amino acid sequence of SEQ ID No: 1, wherein glutamic acid at position 59 of SEQ ID No: 1 is substituted with an amino acid selected from the group consisting of L, I, V, F, M, W, T, C, N, Y, D, and H, and wherein the fructosyl amino acid exhibits a higher thermostability compared to the fructosyl amino acid oxidase of SEQ ID No: 1.

2. A fructosyl amino acid oxidase comprising the amino acid sequence of SEQ ID No: 1, wherein glutamic acid at position 59 of SEQ ID No: 1 is substituted with phenylalanine (F), wherein the fructosyl amino acid oxidase further comprises an additional substitution to SEQ ID No: 1 selected from the group consisting of substitutions (a) to (k), wherein the fructosyl amino acid exhibits a higher thermostability compared to the fructosyl amino acid oxidase of SEQ ID No: 1, and wherein the substitutions (a) to (k) are as follows: (a) E98C (b) G225D (c) K227N (d) E285F (e) E98C and G225F (f) G225D and K227N (g) K227N and E285I (h) E98C, G225F, and K277N (i) G225D, K277N, and E285F (j) E98C, G225F, K277N, and E285F (k) E98C, G225F, K227N, E285F, and D355L.

3. A polynucleotide encoding the fructosyl amino acid oxidase of claim 1.

4. A test kit for determining glycated albumin, the kit comprising a reagent 1 and reagent 2, wherein reagent 1 comprises 20-200 mM buffer, 10-100 KU/L protease, 10-100 KU/L peroxidase, 5-50 mM 4-amino-antipyrine, and 0.01 to 0.05% preservative, and wherein reagent 2 comprises 20-200 mM buffer, 10-100 KU/L of the fructosyl amino acid oxidase of claim 1, 1-10 mM chromogen, and 0.01 to 0.05% preservative.

5. The test kit of claim 4, wherein the buffer is selected from the group consisting of tris-hydrochloride buffer, acetic acid-sodium acetate buffer, phthalic-acid-hydrochloric acid buffer, and glycine-hydrochloric acid buffer.

6. The test kit of claim 4, wherein the protease is selected from the group consisting of trypsin, basophilic protease, proteinase A, and proteinase K.

7. The test kit of claim 4, wherein chromogen is selected from the group consisting of N, N-di(4-sulfobutyl)-3-methylaniline disodium salt, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt.

8. The test kit of claim 4, wherein the preservative is 2-methyl-4-isothiazolin-3-one.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a glycated albumin detecting linear range diagram.

(2) FIG. 2 is a diagram of correlation of the present invention with HPLC detection.

DETAILED DESCRIPTION

(3) The present invention is further illustrated below with reference to Embodiments. However, the present invention is not limited to the following Embodiments.

(4) Embodiment One

(5) Construction of mutagenesis libraries

(6) 1. For the sequence shown in Sequence 2, using whole gene synthesis method to perform the synthesis, and it is cloned into pET-22b vector. Restriction sites NdeI and XhoI are used. Thereby obtained plasmid pET-Ama is the following error-prone PCR and WHOP-PCR template.

(7) 2. Error-Prone PCR Reaction System and Conditions

(8) TABLE-US-00001 TABLE 1 Reaction system is 100 ul: Name Volume (ul) dNTP Mixture (2.5 mM for each) 8 dTTP (100 mM) 0.8 dCTP (100 mM) 0.8 10 * PCR Buffer 10 Upstream primer (5 mM), sequence 3 20 Downstream primer (5 mM), 20 sequence 4 MnCl.sub.2 (5 mM) 10 Mg.sup.2+ (25 mM) 14 Taq Enzyme (5 U/ul) 1 Template (10 ng/ul) 5 Water 12

(9) Reaction conditions are:

(10) 95 C. 5 min; 94 C. 30 sec; 55 C. 30 sec; 72 C. 2 min; 30 cycles; 72 C. 10 min; 4 C. preservation.

(11) The amount of the above MnCl.sub.2 can be adjusted as appropriate, in order to obtain a suitable mutagenesis frequency, i.e., 1 to 2 nucleotide mutageneses/1 Kb nucleotides.

(12) 3. WHOP-PCR reaction system and conditions

(13) The error-prone PCR fragment with the size of about 1.4 Kb is recycled using gel, and is used as primers in the next round WHOP-PCR.

(14) TABLE-US-00002 TABLE 2 WHOP-PCR reaction system, 50 ul: Name Volume (ul) dNTP Mixture (2.5 mM for each) 4 5 * HF Buffer 10 error-prone PCR fragments (25 ng/ul) 20 Phusion Hot Start II High-Fidelity 0.5 DNA Polymerase (5 U/ul) Template (20 ng/ul) 5 Water 10.5

(15) WHOP-PCR reaction conditions:

(16) 98 C. 30 sec; 98 C. 10 sec; 60 C. 10 sec; 72 C. 2.5 min; 24 cycles; 72 C. 5 min; 4 C. preservation.

(17) 4. Digestion and transformation

(18) 1 ul of DpnI is added into 50 ul of WHOP-PCR product, to perform the enzyme digestion. At 37 C., the reaction lasts for 2 hours to completely remove the template DNA.

(19) The digested product described above is divided into five tubes and transformed into TOP10 competent cells. The next day, a total library capacity of more than 10000 monoclone is obtained.

(20) Embodiment Two

(21) Mutagenesis libraries screening

(22) 1. Cultivating, inducing, and expressing

(23) The obtained clones, after mixing by a coating stick, are collected in a centrifuge tube. Plasmids are extracted. The obtained plasmids are transformed into BL21 (ED3), to be ready for the next screening use. Next, the transformants are inoculated in a 96-well plate, wherein the last well is inoculated with a wild-type strain as a control. The cultivation medium is 150 ul LB/per well, containing the antibiotic ampicillin. This well plate is used as a retention plate. The next day, in the same order, it is transferred to another 96-well plate. The cultivation medium is 150 ul LB/per well. Meanwhile, the antibiotic ampicillin and IPTG are added for induction. It is cultivated at 37 (C for 6 hours. Bacteria is harvested by a centrifugation of 3800 rpm. The medium is removed. This plate is used as an assay plate.

(24) 2. Screening

(25) 150 ul lysis solution (100 mM Tris, pH 8.0; 0.4 mg/ml sodium deoxycholate; 0.8 mg/ml CTAB; 20 m MKCl; 80 mM MgSO4) is added to the assay plate. After lysis at room temperature for half an hour, it is centrifuged at 3800 rpm for 15 minutes. Another 50 ul lysate is added to a new 96-well plate. Another 50 ul of lysis solution is taken in a 96-well PCR plate, which is placed in a 96-well PCR instrument. It is heat-treated at 50 C. for 15 minutes. It is transferred to another 96-well plate. In the above-mentioned two plates, 100 ul of chromogenic solution (Tris, 100 mM, pH8.0; TOOS solution, 15 mM; 4-APP, 0.5 mM; POD, 40 U/ml; fructose lysine, 15 mM) is added respectively. By the chromogenic treatment for 30 minutes, the absorbance value is recorded using a microplate reader. By calculating a ratio of both absorbance, the activity proportion of residual enzyme of crude enzyme solution after the heat treatment is determined. For mutant strains having improved thermostability, their theoretical activity residual rate will be higher than that of the wild type.

(26) In the present invention, by screening more than 10000 mutant strains, six mutant strains with enhanced thermostability are obtained in total by screening. After the sequencing analysis, it is found that these six mutant strains are all single-base mutagenesis. Their nucleotide sequence mutageneses are 177G.fwdarw.T, 293A.fwdarw.C, 764G.fwdarw.C, 830A.fwdarw.C, 853G.fwdarw.C, 1063G.fwdarw.C, respectively. Corresponding amino acid sequence mutagenises are 59E.fwdarw.N, 98E.fwdarw.A, 225G.fwdarw.A, 277K.fwdarw.S, 285E.fwdarw.Q, and 355D.fwdarw.H, respectively.

(27) Embodiment Three

(28) Determination of fructosyl amino acid oxidases activity and thermostability analysis

(29) In a buffer of 100 mM Tris, pH 8.0, purified fructosyl amino acid oxidase is diluted to approximately 10 ug/ml.

(30) 50 ul of fructosyl amino acid oxidase is taken into a 96-well PCR. It is heat-treated at 50 C. for 15 min. It is stored under 4 C.

(31) 50 ul of heat-treated fructosyl amino acid oxidase is transferred to a 96-well plate. Meanwhile, unheated 50 ul fructosyl amino acid oxidase is taken to a 96-well plate. It is incubated at 37 C. for 10 min.

(32) Color developing solution which has previously been incubated to 37 C. (Tris, 100 mM, pH 8.0; TOOS solution, 15 mM; 4-APP, 0.5 mM; POD, 40 U/ml; fructose lysine, 15 mM) is added. The reaction is conducted at 37 C. for 30 min.

(33) The absorbance at 555 nm is recorded using a microplate reader.

(34) The absorbance value of heat-treated fructosyl amino acid oxidase is divided by the absorbance value of non-heat-treated fructosyl amino acid oxidase. The resulting value is the ratio of residual activity at this temperature.

(35) Thermostability analysis is performed on these six mutant strains obtained from random mutageneses and wild-type fructosyl amino acid oxidases. The results are shown in Table 3.

(36) TABLE-US-00003 TABLE 3 Thermostability analysis of mutant strains obtained from random mutageneses Amino 50 C., 15 min, Activity ratio of Plasmid mutagenesis residual enzyme pET-Ama None 5% pET-Ama-59N E.fwdarw.N 15% pET-Ama-98A E.fwdarw.A 39% pET-Ama- G.fwdarw.A 31% 225A pET-Ama- K.fwdarw.S 34% 277S pET-Ama- E.fwdarw.Q 38% 285Q pET-Ama- D.fwdarw.H 28% 355H

(37) Embodiment Four

(38) Thermostability analysis of 59-site amino acid substitution

(39) 1. Introducing 59-site amino acid site-directed mutagenesis

(40) In the first round of the PCR, pET-Ama is used as a template, primer sequence 5 (5-tgctagttattgctcagcgg-3), Sequence 6 (5-acaagattatgttacacagcgagctg-3) containing the site-directed mutagenesis are used as upstream primers respectively. Fragment containing site-directed mutagenesis is obtained. PCR system is 50 ul:

(41) TABLE-US-00004 TABLE 4 PCR system Name Volume (ul) dNTP Mixture (2.5 mM for each) 4 5 * HF Buffer 10 Upstream primer, sequence 6 10 (5 pm/ul) T7-Ter, sequence 5 (5 pm/ul) 10 Phusion Hot Start II High- 0.5 Fidelity DNA Polymerase (5 U/ul) Template (1 ng/ul) 5 Water 10.5

(42) PCR reaction conditions are:

(43) 98 C. 30 sec; 98 C. 10 sec; 60 C. 10 sec; 72 C. 30 sec; 30 cycles; 72 C. 5 min; 4 C. preservation.

(44) After the obtained target fragment is purified, it is used as a primer in the next round WHOP-PCR. Specific reaction system is shown in Table 5

(45) TABLE-US-00005 TABLE 5 WHOP-PCR reaction system Name Volume (ul) dNTP Mixture (2.5 mM for each) 4 5 * HF Buffer 10 Target PCR production (25 ng/ul) 20 Phusion Hot Start II High- 0.5 Fidelity DNA Polymerase (5 U/ul) TemplatepET-Ama (20 ng/ul) 5 Water 10.5

(46) PCR reaction conditions are:

(47) 98 C. 30 sec; 98 C. 10 sec; 60 C. 10 sec; 72 C. 2.5 min; 24 cycles; 72 C. 5 min; 4 C. preservation.

(48) 1 ul of DpnI is added into 50 ul of WHOP-PCR product to perform enzyme digestion. The reaction last for 2 hours under 37 C., so that the template DNA is removed completely. Then, BL21 (DE3) is transformed. After sequencing correctly, it is used for thermostability analysis.

(49) Thermostability after 2.59-site amino acid substitution

(50) The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to a fresh LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG with a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(51) TABLE-US-00006 TABLE 6 Thermostability analysis of 59-site amino acid substitution 50 C., 15 min, Activity Number ratio of of mutant Amino acid Primers residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M1 pET-Ama-59L E.fwdarw.L pET-Ama 5, 6 32% M2 pET-Ama-59I E.fwdarw.I pET-Ama 5, 7 50% M3 pET-Ama-59V E.fwdarw.V pET-Ama 5, 8 20% M4 pET-Ama-59F E.fwdarw.F pET-Ama 5, 9 60% M5 pET-Ama- E.fwdarw.M pET-Ama 5, 10 42% 59M M6 pET-Ama- E.fwdarw.W pET-Ama 5, 11 55% 59W M7 pET-Ama-59T E.fwdarw.T pET-Ama 5, 12 38% M8 pET-Ama-59C E.fwdarw.C pET-Ama 5, 13 25% M9 pET-Ama-59N E.fwdarw.N pET-Ama 5, 14 15% M10 pET-Ama-59Y E.fwdarw.Y pET-Ama 5, 15 58% M11 pET-Ama-59D E.fwdarw.D pET-Ama 5, 16 18% M12 pET-Ama-59H E.fwdarw.H pET-Ama 5, 17 30%

(52) Embodiment Five

(53) Thermostability analysis of 98-site amino acid substitution

(54) According to the above method, different site-directed mutageneses are introduced in the 98-site amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to a fresh LB medium containing ampicillin. After OD.sub.600 grown to about 0.8, IPTG with a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(55) TABLE-US-00007 TABLE 7 Thermostability analysis of 98-site amino acid substitution 50 C., 15 min, Activity Number ratio of Amino of mutant acid Primers residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M13 pET-Ama- E.fwdarw.A pET-Ama 5, 18 39% 98A M14 pET-Ama-98L E.fwdarw.L pET-Ama 5, 19 22% M15 pET-Ama-98I E.fwdarw.I pET-Ama 5, 20 40% M16 pET-Ama- E.fwdarw.V pET-Ama 5, 21 55% 98V M17 pET-Ama-98P E.fwdarw.P pET-Ama 5, 22 61% M18 pET-Ama-98F E.fwdarw.F pET-Ama 5, 23 33% M19 pET-Ama-98S E.fwdarw.S pET-Ama 5, 24 60% M20 pET-Ama-98T E.fwdarw.T pET-Ama 5, 25 20% M21 pET-Ama-98C E.fwdarw.C pET-Ama 5, 26 44% M22 pET-Ama- E.fwdarw.N pET-Ama 5, 27 42% 98N M23 pET-Ama- E.fwdarw.Y pET-Ama 5, 28 25% 98Y M24 pET-Ama- E.fwdarw.D pET-Ama 5, 29 45% 98D M25 pET-Ama- E.fwdarw.H pET-Ama 5, 30 18% 98H

(56) Embodiment Six

(57) Thermostability analysis of 225-site amino acid substitution

(58) According to the above method, different site-directed mutageneses are introduced in 225-site amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to a fresh LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG with a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(59) TABLE-US-00008 TABLE 8 Thermostability analysis of 225-site amino acid substitution 50 C., Number 15 min, of Amino Activity ratio mutant acid Primers of residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M26 pET-Ama- G.fwdarw.A pET-Ama 5, 31 31% 225A M27 pET-Ama- G.fwdarw.L pET-Ama 5, 32 28% 225L M28 pET-Ama- G.fwdarw.F pET-Ama 5, 33 44% 225F M29 pET-Ama- G.fwdarw.M pET-Ama 5, 34 14% 225M M30 pET-Ama- G.fwdarw.W pET-Ama 5, 35 18% 225W M31 pET-Ama- G.fwdarw.S pET-Ama 5, 36 22% 225S M32 pET-Ama- G.fwdarw.N pET-Ama 5, 37 16% 225N M33 pET-Ama- G.fwdarw.D pET-Ama 5, 38 28% 225D

(60) Embodiment Seven

(61) Thermostability analysis of 277-site amino acid substitution

(62) According to the above method, different site-directed mutageneses are introduced in 277-site amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(63) TABLE-US-00009 TABLE 9 Thermostability analysis of 277-site amino acid substitution 50 C., 15 min, Number Amino Activity of acid ratio mutant muta- Primers of residual enzyme Plasmid geneses Template sequence enzyme WT1 pET-Ama / / / 5% M34 pET-Ama-277A K.fwdarw.A pET-Ama 5, 39 36% M35 pET-Ama-277L K.fwdarw.L pET-Ama 5, 40 59% M36 pET-Ama-277I K.fwdarw.I pET-Ama 5, 41 28% M37 pET-Ama-277F K.fwdarw.F pET-Ama 5, 42 47% M38 pET-Ama-277S K.fwdarw.S pET-Ama 5, 43 34% M39 pET-Ama-277T K.fwdarw.T pET-Ama 5, 44 44% M40 pET-Ama-277N K.fwdarw.N pET-Ama 5, 45 48% M41 pET-Ama-277Y K.fwdarw.Y pET-Ama 5, 46 49% M42 pET-Ama-277R K.fwdarw.R pET-Ama 5, 47 23% M43 pET-Ama-277H K.fwdarw.H pET-Ama 5, 48 59%

(64) Embodiment Eight

(65) Thermostability analysis of 285-site amino acid substitution

(66) According to the above method, different site-directed mutageneses are introduced in 285-site amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(67) TABLE-US-00010 TABLE 10 Thermostability analysis of 285-site amino acid substitution 50 C., 15 min, Number Amino Activity of acid ratio mutant muta- Primers of residual enzyme Plasmid geneses Template sequence enzyme WT1 pET-Ama / / / 5% M44 pET-Ama-285A E.fwdarw.A pET-Ama 5, 49 20% M45 pET-Ama-285L E.fwdarw.L pET-Ama 5, 50 46% M46 pET-Ama-285I E.fwdarw.I pET-Ama 5, 51 43% M47 pET-Ama-285F E.fwdarw.F pET-Ama 5, 52 52% M48 pET-Ama-285M E.fwdarw.M pET-Ama 5, 53 21% M49 pET-Ama-285W E.fwdarw.W pET-Ama 5, 54 13% M50 pET-Ama-285Q E.fwdarw.Q pET-Ama 5, 55 38% M51 pET-Ama-285N E.fwdarw.N pET-Ama 5, 56 29%

(68) Embodiment Nine

(69) Thermostability analysis of 355-site amino acid substitution

(70) According to the above method, different site-directed mutageneses are introduced in the 355-site amino acids. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(71) TABLE-US-00011 TABLE 11 Thermostability analysis of 355-site amino acid substitution 50 C., 15 min, Activity Number ratio of of mutant Amino acid Primers residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M52 pET-Ama- D.fwdarw.L pET- 5, 57 55% 355L Ama M53 pET-Ama-355I D.fwdarw.I pET- 5, 58 31% Ama M54 pET-Ama- D.fwdarw.V pET- 5, 59 36% 355V Ama M55 pET-Ama- D.fwdarw.F pET- 5, 60 34% 355F Ama M56 pET-Ama- D.fwdarw.M pET- 5, 61 43% 355M Ama M57 pET-Ama- D.fwdarw.W pET- 5, 62 26% 355W Ama M58 pET-Ama- D.fwdarw.T pET- 5, 63 41% 355T Ama M59 pET-Ama- D.fwdarw.C pET- 5, 64 39% 355C Ama M60 pET-Ama- D.fwdarw.Y pET- 5, 65 35% 355Y Ama M61 pET-Ama- D.fwdarw.R pET- 5, 66 20% 355R Ama M62 pET-Ama- D.fwdarw.H pET- 5, 67 78% 355H Ama

(72) Embodiment Ten

(73) Thermostability analysis of a combination of amino acid substitutions on two different mutagenesis sites

(74) According to the above method, different site-directed mutageneses are introduced on two different sites of the amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(75) TABLE-US-00012 TABLE 12 Thermostability analysis of amino acid substitutions on two different mutagenesis sites 50 C., Number 15 min, of Activity ratio mutant Amino acid Primers of residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M63 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 26 74% 59F98C 98E.fwdarw.C 59F M64 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 38 72% 59F225D 225G.fwdarw.D 59F M65 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 45 76% 59F277N 277K.fwdarw.N 59F M66 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 52 77% 59F285F 285E.fwdarw.F, 59F M67 pET-Ama- 98E.fwdarw.P, pET-Ama- 5, 56 69% 98P225N 225G.fwdarw.N 98P M68 pET-Ama- 98E.fwdarw.P, pET-Ama- 5, 43 70% 98P277S 277K.fwdarw.S 98P M69 pET-Ama- 225G.fwdarw.L, pET-Ama- 5, 39 51% 225L277A 277K.fwdarw.A 225L M70 pET-Ama- 225G.fwdarw.L, pET-Ama- 5, 51 55% 225L285I 285E.fwdarw.I 225L M71 pET-Ama- 277K.fwdarw.Y, pET-Ama- 5, 63 59% 277Y355T 355D.fwdarw.T 277Y M72 pET-Ama- 285E.fwdarw.I, pET-Ama- 5, 62 62% 285I355W 355D.fwdarw.W 285I

(76) Embodiment Eleven

(77) Thermostability analysis of a combination of amino acid substitutions on three different mutagenesis sites

(78) According to the above method, different site-directed mutageneses are introduced on three different sites of the amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(79) TABLE-US-00013 TABLE 13 Thermostability analysis of a combination of amino acid substitutions on three different mutagenesis sites 50 C., 15 min, Number Activity of ratio of mutant Amino acid Primers residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M73 pET-Ama- 59E.fwdarw.F, pET- 5, 33 85% 59F98C225F 98E.fwdarw.C, Ama- 225G.fwdarw.F 59F98C M74 pET-Ama- 59E.fwdarw.F pET- 5, 45 83% 59F225D277N 225G.fwdarw.D, Ama- 277 K.fwdarw.N 59F225D M75 pET-Ama- 59E.fwdarw.F, pET- 5, 51 83% 59F277N285I 277K.fwdarw.N, Ama- 285E.fwdarw.I 59F277N M76 pET-Ama- 98E.fwdarw.P, pET- 5, 55 79% 98P225N285Q 225G.fwdarw.N, Ama- 285E.fwdarw.Q 98P225N M77 pET-Ama- 225G.fwdarw.L, pET- 5, 63 75% 225L285I355T 285E.fwdarw.I, Ama- 355D.fwdarw.T 225L285I

(80) Embodiment Twelve

(81) Thermostability analysis of a combination of amino acid substitutions on four different mutagenesis sites

(82) According to the above method, different site-directed mutageneses are introduced on four different sites of the amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(83) TABLE-US-00014 TABLE 14 Thermostability analysis of amino acid substitutions on four different mutagenesis sites Number 50 C., 15 min, of Activity ratio of mutant Amino acid Primers residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M78 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 45 92% 59F98C225F277N 98E.fwdarw.C, 59F98C225F 225G.fwdarw.F, 277K.fwdarw.N M79 pET-Ama- 59E.fwdarw.F, pET-Ama-59F225D277N 5, 52 93% 59F225D277N285F 225G.fwdarw.D, 277 K.fwdarw.N, 285E.fwdarw.F

(84) Embodiment Thirteen

(85) Thermostability analysis of a combination of amino acid substitutions on five different mutagenesis sites

(86) According to the above method, different site-directed mutageneses are introduced on five different sites of amino acids. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(87) TABLE-US-00015 TABLE 15 Thermostability analysis of amino acid substitutions on five different mutagenesis sites 50 C., 15 min, Number Activity of ratio of mutant Amino acid Primers residual enzyme Plasmid mutageneses Template sequence enzyme WT1 pET-Ama / / / 5% M80 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 52 >96% 59F98C225F277N285F 98E.fwdarw.C, 59F98C225F277N 225G.fwdarw.F, 277K.fwdarw.N, 285E.fwdarw.F

(88) Embodiment Fourteen

(89) Thermostability analysis of a combination of amino acid substitutions on six different mutagenesis sites

(90) According to the above method, different site-directed mutageneses are introduced on six different sites of amino acid. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(91) TABLE-US-00016 TABLE 16 Thermostability analysis of amino acid substitutions on six different mutagenesis sites Number of 50 C., 15 min, mutant Amino acid Primers Activity ratio of enzyme Plasmid mutageneses Template sequence residual enzyme WT1 pET-Ama / / / 5% M81 pET-Ama- 59E.fwdarw.F, pET-Ama- 5, 57 >98% 59F98C225F277N285F355L 98E.fwdarw.C, 59F98C225F277N283F 225G.fwdarw.F, 277K.fwdarw.N, 285E.fwdarw.F, 355D.fwdarw.L

(92) Embodiment Fifteen

(93) Thermostability analysis of mutageneses of fructosyl amino acid oxidases from Aspergillus niger CBS513.88 and the combination thereof

(94) As is known from Blast, the amino acid sequence of fructosyl amino acid oxidases from Aspergillus niger CBS513.88 and the amino acid sequence of fructosyl amino acid oxidases from Aspergillus fumigatus have a homology of 80%. Its amino acid sequence is shown a sequence 68. The nucleotide sequence of the sequence 69 is shown. Use whole gene synthesis method, sequences 69 are synthesized, and cloned into the NdeI and XhoI restriction sites of pET-22b vector.

(95) After the sequence analysis, it is found that, corresponding to amino acids of sites 59, 98, 225, 277, 285, and 355 of Amadoriase I, corresponding sites on fructosyl amino acid oxidases of Aspergillus niger CBS513.88 are 59E, 98E, 225G, 277K, 285E, and 355D.

(96) According to the above method of site-directed mutagenesis, these sites of fructosyl amino acid oxidases of Aspergillus niger CBS513.88 is single-base mutated or mutated on a combination of different sites. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(97) TABLE-US-00017 TABLE 17 Thermostability analysis of amino acid substitutions of fructosyl amino acid oxidases of Aspergillus niger CBS513.88 Number of 50 C., 15 min, mutant Activity ratio of enzyme Amino acid mutageneses residual enzyme WT2 / <5% M82 59E.fwdarw.F 20% M83 98E.fwdarw.C 22% M84 225G.fwdarw.F 30% M85 277K.fwdarw.N 38% M86 285E.fwdarw.F 32% M87 355D.fwdarw.L 26% M88 59E.fwdarw.F, 98E.fwdarw.C, 37% M89 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 51% M90 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, 75% M91 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, 93% 285E.fwdarw.F, M92 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, >98% 285E.fwdarw.F, 355D.fwdarw.L

(98) Embodiment Sixteen

(99) Thermostability analysis of mutageneses of fructosyl amino acid oxidases from Aspergillus clavatus NRRL1 and the combination thereof

(100) As is known from Blast, the amino acid sequence of fructosyl amino acid oxidases from Aspergillus clavatus NRRL1 and the amino acid sequence of fructosyl amino acid oxidases from Aspergillus fumigatus have a homology of 83%. Its amino acid sequence is shown in sequence 70. The nucleotide sequence is shown in sequence 71. Using whole gene synthesis method, sequences 71 is synthesized and cloned into the NdeI and XhoI restriction sites of pET-22b vector.

(101) After the sequence analysis, it is found that, corresponding to amino acids of sites 59, 98, 225, 277, 285, and 355 of Amadoriase I, corresponding sites on fructosyl amino acid oxidases of Aspergillus clavatus NRRL1 are 59E, 98E, 225G, 277N, 285E, and 355D.

(102) According to the above method of site-directed mutagenesis, these sites of fructosyl amino acid oxidases of Aspergillus clavatus NRRL1 are single-base mutagenesis or mutated on a combination of different sites. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(103) TABLE-US-00018 TABLE 18 Thermostability of amino acid substitutions of fructose amino acid oxidase of Aspergillus clavatus NRRL1 Number 50 C., 15 min, of mutant Activity ratio of enzyme Amino acid mutageneses residual enzyme WT3 / <5% M93 59E.fwdarw.F 30% M94 98E.fwdarw.C 26% M95 225G.fwdarw.F 37% M96 285E.fwdarw.F 36% M97 355D.fwdarw.L 46% M98 59E.fwdarw.F, 98E.fwdarw.C, 47% M99 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 65% M100 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 90% 285E.fwdarw.F, M101 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, >95% 285E.fwdarw.F, 355D.fwdarw.L

(104) Embodiment Seventeen

(105) Thermostability analysis of mutageneses of fructosyl amino acid oxidases from Neosartorya fischeri NRRL181 and the combination thereof

(106) As is known from Blast, the amino acid sequence of fructosyl amino acid oxidases derived from Neosartorya fischeri NRRL181 and the amino acid sequence of fructosyl amino acid oxidases from Aspergillus fumigatus have a homology of 96%. Its amino acid sequence is shown in sequence 72. The nucleotide sequence is shown in sequence 73. Using whole gene synthesis method, sequences 73 is synthesized and cloned into the NdeI and XhoI restriction sites of vector pET-22b.

(107) After the sequence analysis, it is found that, corresponding to amino acids of sites 59, 98, 225, 277, 285, and 355 of Amadoriase I, corresponding sites on fructosyl amino acid oxidases of Neosartorya fischeri NRRL181 are 59E, 98E, 225G, 277K, 285E, and 355D.

(108) According to the above method of site-directed mutagenesis, these sites of fructosyl amino acid oxidases of Neosartorya fischeri NRRL181 are single-base mutated or mutated on a combination of different sites. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8, IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(109) TABLE-US-00019 TABLE 19 Thermostability analysis of amino acid substitutions of fructosyl amino acid oxidases of Neosartorya fischeri NRRL181 Number of 50 C., 15 min, mutant Activity ratio of enzyme Amino acid mutageneses residual enzyme WT4 / <5% M102 59E.fwdarw.F 33% M103 98E.fwdarw.C 42% M104 225G.fwdarw.F 29% M105 277K.fwdarw.N 35% M106 285E.fwdarw.F 55% M107 355D.fwdarw.L 44% M108 59E.fwdarw.F, 98E.fwdarw.C, 61% M109 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 72% M110 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, 88% M111 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, 94% 285E.fwdarw.F, M112 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, >98% 285E.fwdarw.F, 355D.fwdarw.L

(110) Embodiment Eighteen

(111) Thermostability analysis of mutageneses of the fructose amino acid from Aspergillus fumigatus Af293 and the combination thereof

(112) As is known from Blast, the amino acid sequence of fructosyl amino acid oxidases from the amino acid sequence of Aspergillus fumigatus A1293 and fructosyl amino acid oxidases from Aspergillus fumigatus have a homology of 99%. Its amino acid sequence is shown in sequence 74. The nucleotide sequence is shown in sequence 75. Using whole gene synthesis method, sequences 75 is synthesized, and is cloned into the NdeI and XhoI restriction sites of pET-22b vector.

(113) After the sequence analysis, it is found that, corresponding to amino acids of sites 59, 98, 225, 277, 285, and 355 of Amadoriase I, corresponding sites on fructosyl amino acid oxidases of the Aspergillus fumigatus CBS513.88 are 59E, 98E 225G, 277K, 285E, and 355D.

(114) According to the above method of site-directed mutagenesis, these sites of fructosyl amino acid oxidases of Aspergillus fumigatus Af293 are single-base mutated or mutated on a combination of different sites. The obtained mutant is inoculated overnight. The next day, in a proportion of 1%, it is transferred to LB medium containing ampicillin. After OD.sub.600 grows to about 0.8. IPTG having a final concentration of 0.4 mM is added. It is induced for about five hours. Bacteria are harvested by centrifugation, broken, and purified. Thermostability analysis as described above is performed on purified fructosyl amino acid oxidases. Detailed results are shown in the table below.

(115) TABLE-US-00020 TABLE 20 Thermostability analysis of amino acid substitutions of fructosyl amino acid oxidases of Aspergillus fumigatus Af293 Number 50 C., 15 min, of mutant Activity ratio of enzyme Amino acid mutageneses residual enzyme WT5 / <5% M113 59E.fwdarw.F 50% M114 98E.fwdarw.C 42% M115 225G.fwdarw.F 40% M116 277K.fwdarw.N 41% M117 285E.fwdarw.F 55% M118 355D.fwdarw.L 48% M119 59E.fwdarw.F, 98E.fwdarw.C, 67% M120 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 82% M121 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, 277K.fwdarw.N, 90% M123 59E.fwdarw.F, 98E.fwdarw.C, 225G.fwdarw.F, >98% 277K.fwdarw.N, 285E.fwdarw.F, 355D.fwdarw.L

(116) Embodiment Nineteen

(117) Preparation Method

(118) 1. Obtaining the strain: when the strain containing different mutated genes has been cultivated to OD.sub.600 of about 0.8, IPTG is added. It is induced for 5 hours. The strain is harvested by centrifugation. The collected strain is re-suspended with the buffer, and is ultrasonically disrupted. The supernatant is collected by centrifugation.

(119) 2. Ammonium sulfate precipitation: using an ammonium sulfate solution, the supernatant obtained in step (1) is subjected to fractional precipitation. The final collected and obtained precipitate is dissolved in buffer A, so as to obtain a crude extracted solution.

(120) Affinity chromatography: using buffer A balanced nickel column, the crude extract solution obtained in step (2) is adsorbed on a nickel column. After the absorption finishes, gradient elution is performed with an imidazole solution. The eluent is collected.

(121) 4. Dialysis: the eluate in step (3) is put into a dialysis bag. It is put in a dialysate at 4 C., and is dialyzed overnight with magnetic stirring.

(122) Buffer A in step (2) is: 50 mM potassium phosphate buffer, pH 8.0, 500 mM NaCl.

(123) Imidazole solution in step (3) is: 50 mM potassium phosphate buffer, pH 8.0, 500 mM NaCl, 20 mM1000 mM imidazole.

(124) Dialyzate in step (4) is: 50 mM potassium phosphate buffer, pH 8.0, 500 mM NaCl.

(125) Embodiment Twenty

(126) The linear range of glycated albumin test kit

(127) Including reagent 1 and reagent 2, wherein:

(128) Reagent 1:

(129) Tris hydrochloride buffer 50 mmol/L

(130) Protease K40 KU/L

(131) Peroxidase 30 KU/L

(132) 4-amino-antipyrine 10 mmol/L

(133) Methyl-4-isothiazolin-3-one 0.02%

(134) Reagent 2:

(135) Tris hydrochloride buffer 50 mmol/L

(136) Mutants of fructosyl amino acid oxidases 28 KU/L

(137) N. N-bis(4-sulfobutyl)-3-methylaniline (TODB) 2 mmol/L

(138) Methyl-4-isothiazolin-3-one 0.02%

(139) The mutant of fructosyl amino acid oxidase used in the present embodiment particularly is No. M76 mutant. In fact, other mutants also can be used.

(140) Fresh serum samples should be used for the test. The hemolysis should be avoided. After the serum is collected, if not promptly tested, it should be stored at 4 CC. Theoretically, it should not be stored for more than two weeks.

(141) Detection conditions: Main wavelength of 600 nm, sub-wavelength of 700 nm.

(142) Detection steps: shown in Table 21

(143) TABLE-US-00021 TABLE 21 Detection steps Sample 20 ul Reagent 1 200 ul mixing uniformly, incubating for 5 min at 37 C. Reagent 2 50 ul mixing uniformly, determining the absorbance value A1 within 20 s, incubating for 5 min at 37 C., determining the absorbance A2, calculating A = A2 A1

(144) The linear range, shown in FIG. 1:

(145) The serum having high GA value is taken, and is diluted into different gradients using normal saline, and will be measured respectively.

(146) The linear range of glycated albumin detected by this method is 21.01200 umol/L, r2>0.990.

(147) Embodiment Twenty-one

(148) Precision test of glycated albumin test kit

(149) A high and low quality control and two serum samples are tested 20 times respectively. Mean values are 204 umol/L, 751 umol/L, 251 umol/L, 373 umol/L respectively. CVs are 1.1%, 0.7%, 0.8%, 0.6% respectively.

(150) TABLE-US-00022 TABLE 22 Intra-batch precision test Quality control Quality control Serum 2: 1: 2: Serum 1: 375 umol/ 198 umol/L 750 umol/L 241 umol/L L GA GA GA GA Testing 20 20 20 20 times Mean values 204 751 251 373 SD 2.15 4.91 1.94 2.41 CV % 1.1 0.7 0.8 0.6

(151) A high and low value quality control and two serum samples are continuously measured for 10 days respectively. Mean values are 204 umol/L, 751 umol/L, 251 umol/L, 373 umol/L, respectively. CVs are 1.2%, 0.7%, 1.3%, 1.0%, respectively.

(152) TABLE-US-00023 TABLE 23 Inter-batch precision test Quality control Quality control Serum 1: Serum 2: 1: 2: 241 umol/ 375 umol/ 198 umol/L 750 umol/L L L GA GA GA GA Testing times 20 20 20 20 Mean values 204 751 251 373 SD 2.39 9.59 5.18 6.75 CV % 1.2 1.3 2.1 1.8

(153) Embodiment Twenty-two

(154) Open-bottle stability test of glycated albumin test kit

(155) On Hitachi biochemical analyzer, the open-bottle stability of the reagent can sustain for at least 4 weeks.

(156) TABLE-US-00024 TABLE 24 Open-bottle stability test GA 0 days 7 days 14 days 21 days 28 days Quality control 203 202 206 202 206 1: 198 umol/L GA Quality control 753 740 738 736 745 2: 750 umol/L GA N109657 270 umol/L 274 271 274 269 272

(157) Embodiment Twenty-three

(158) Glycated albumin test kit and HPLC correlation

(159) The glycated albumin detection test kit of the present invention and classical HPLC detection methods are used in the determination of the same sample, after comparison, it is found that the correlation is good, R2>0.995.

(160) Embodiment Twenty-four

(161) Anti-interference test of the glycated albumin test kit

(162) It is found that, when the concentration of vitamin C is less than 0.2 mmol/L, hemoglobin less than 1.6 g/L, bilirubin less than 0.32 mmol/L, uncoupling bilirubin less than 0.32 mmol/L, triglyceride less than 20 mmol/L, blood glucose less than 20 g/L, uric acid less than 36 g/L, their interference of the measured value of glycated albumin is less than 10%.

(163) TABLE-US-00025 TABLE 25 Interference test Interference Interferences concentration Deviation Vitamin C 0.2 mmol/L 2.1% Hemoglobin 1.6 g/L 3.1% Bilirubin 0.32 mmol/L 1.9% Uncoupling bilirubin 0.32 mmol/L 2.8% Triglyceride 20 mmol/L 6.6% Blood glucose 20 g/L 1.5% Uric acid 36 g/L 5.9%

(164) Embodiment Twenty-five

(165) Determination of the percentage of glycated albumin

(166) The detection of glycated albumin and the detection of albumin are combined, so as to obtain a percentage value of the glycated albumin through calculating the glycated albumin concentration divided by the albumin concentration.

(167) The Albumin detection reagent is as follows

(168) Reagent 3:

(169) Succinate buffer 0.05 mol/L

(170) Polyoxyethytene (23) lauryl ether 2.4 g/L

(171) Bromocresol green 1.810.sup.4 mol/L

(172) Test conditions: main wavelength of 600 nm, sub-wavelength of 700 nm.

(173) Reaction method: end-point method.

(174) Detection steps: shown in Table 26

(175) TABLE-US-00026 TABLE 26 Detection steps Sample 3 ul Reagent 3 300 ul mixing uniformly, 37 C. incubating for 1 min, reading the absorbance A

(176) Using the above method, 20 samples of serum are detected. The percentage of glycated albumin is calculated, as shown in Table 27.

(177) TABLE-US-00027 TABLE 27 Percentages of glycated albumin of 20 samples of serum Percentage Glycated albumin (Glycated Sample number (umol/L) Albumin (g/dL) albumin/Albumin) 1 120 2.33 12.3% 2 135 2.06 15.6% 3 255 4.21 14.2% 4 267 4.11 15.2% 5 186 4.03 10.9% 6 155 2.72 13.5% 7 198 3.15 14.8% 8 290 3.97 17.1% 9 112 1.82 14.8% 10 156 2.76 13.4% 11 178 2.73 15.4% 12 134 2.51 12.7% 13 125 2.58 11.6% 14 301 4.19 16.8% 15 148 2.79 12.6% 16 198 3.15 14.8% 17 205 3.68 13.1% 18 175 3.48 11.9% 19 246 4.62 12.5% 20 278 4.14 15.7%

(178) Typically, the reference value of glycated albumin is from 11% to 16%. An increased glycated albumin value is common in diabetes. The index can be used as a reference indicator of medium-term glucose control of diabetic patients.

(179) Embodiment Twenty-six

(180) Performance of glycated albumin test kit formulated using mutants of other fructosyl amino acid oxidases

(181) Since mutants of fructosyl amino acid oxidases involved in the present invention are so many, it is impossible to verify them in the glycated albumin test kit one by one. Therefore, only a few of the most typical mutants of fructosyl amino acid oxidases are selected for the glycated albumin detection test kit. Their performances are evaluated. Mutants of fructosyl amino acid oxidases involved in the present invention that are suitable for the glycated albumin test kits are used to illustrate the present invention, and cannot limit the present invention. The detection method of the involved test kit, verification methods of linear range, the intra-batch precision, the precision inter-batch, the open-bottle stability, the correlation with HPLC, the anti-interference, and etc. are the same as those of Embodiments twenty to twenty-five.

(182) Glycated albumin test kit includes reagent 1 and reagent 2, wherein:

(183) Reagent 1:

(184) Tris hydrochloride buffer 50 mmol/L

(185) Protease K 40 KU/L

(186) Peroxidase 30 KU/L

(187) 4-amino-antipyrine 10 mmol/L

(188) Methyl-4-isothiazolin-3-one 0.02%

(189) Reagent 2:

(190) Tris hydrochloride buffer 50 mmol/L

(191) Fructosyl amino acid oxidase mutant 28 KU/L

(192) N, N-bis(4-sulfobutyl)-3-methylaniline (TODB) 2 mmol/L

(193) Methyl-4-isothiazolin-3-one 0.02%

(194) In this embodiment, the following representatives are selected from mutant strains of fructosyl amino acid oxidases to be tested: i.e., those prepared in the above embodiments, M1, M13, M28, M35, M50, M56, M64, M73, M79, M80, M81, M90, M100, M109, and M120.

(195) Performances of the above mutants of fructosyl amino acid oxidases and their corresponding glycated albumin test kit are shown in the table below in details.

(196) TABLE-US-00028 TABLE 28 Performances of glycated albumin test kit prepared using different mutant strains of amino acid oxidase mutant fructose Percentage Glycated albumin (Glycated Sample number (umol/L) Albumin (g/dL) albumin/Albumin) 1 120 2.33 12.3% 2 135 2.06 15.6% 3 255 4.21 14.2% 4 267 4.11 15.2% 5 186 4.03 10.9% 6 155 2.72 13.5% 7 198 3.15 14.8% 8 290 3.97 17.1% 9 112 1.82 14.8% 10 156 2.76 13.4% 11 178 2.73 15.4% 12 134 2.51 12.7% 13 125 2.58 11.6% 14 301 4.19 16.8% 15 148 2.79 12.6% 16 198 3.15 14.8% 17 205 3.68 13.1% 18 175 3.48 11.9% 19 246 4.62 12.5% 20 278 4.14 15.7%

(197) The above embodiments of the present invention are descriptions of the present invention, but not for limiting the present invention. Any change equivalent to the concept and scope of claims of the present invention should all be construed as falling within the scope of the claims.