Fructose amino acid oxidase, preparation method and glycatedalbumin detection kit comprising oxidase
A fructose amino acid and glycosylated albumin technology, applied in biochemical equipment and methods, redox enzymes, botanical equipment and methods, etc., can solve the problems of unsatisfactory stability, poor thermal stability, unfavorable application of biochemical instruments, etc.
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Embodiment 1
[0078] Mutation library construction
[0079]1. The sequence shown in Sequence 2 is synthesized by the method of total gene synthesis, and cloned into the pET-22b vector, and the restriction sites used are NdeI and XhoI. The thus obtained plasmid pET-Ama was used as a template for the following error-prone PCR and WHOP-PCR.
[0080] 2. Error-prone PCR reaction system and conditions
[0081] The reaction system in Table 1 is 100ul:
[0082] name
Volume (ul)
dNTP Mixture (2.5mM each)
8
dTTP (100mM)
0.8
dCTP (100mM)
0.8
10*PCRBuffer
10
Upstream primer (5mM), Sequence 3
20
Downstream primer (5mM), sequence 4
20
[0083] MnCl 2 (5mM)
10
Mg 2+ (25mM)
14
Taq enzyme (5U / ul)
1
Template (10ng / ul)
5
water
12
[0084] The reaction conditions are:
[0085] 95°C for 5min; 94°C for 30sec; 55°C for 30sec; 72°C for 2min; 30 cycles; 72°C ...
Embodiment 2
[0097] Screening of mutant libraries
[0098] 1. Culture, induction and expression
[0099] After mixing the obtained clones with a coating stick, they were collected in a centrifuge tube, and the plasmid was extracted, and the obtained plasmid was transformed into BL21 (ED3) for the next step of screening. Next, the above-mentioned transformant was inoculated in a 96-well plate, wherein the last well was inoculated with a wild-type strain as a control, and the medium used was 150ulLB / well containing ampicillin antibiotic, and the orifice plate was used as a retention plate. The next day, transfer to another 96-well plate in the same order. The medium used is 150ulLB / well, and ampicillin antibiotics and IPTG are added for induction at the same time. Cultivate at 37°C for 6 hours, collect bacteria by centrifugation at 3800rpm, and remove the medium. This orifice plate serves as the assay plate.
[0100] 2. Screening
[0101] Add 150ul of lysis solution (100mM Tris, pH8.0; 0....
Embodiment 3
[0104] Enzyme Activity Determination and Thermal Stability Analysis of Fructose Amino Acid Oxidase
[0105] The purified fructose amino acid oxidase was diluted to about 10 ug / ml in 100 mM Tris, pH 8.0 buffer.
[0106] Take 50ul of the fructose amino acid oxidase in a 96-well PCR, heat-treat at 50°C for 15min, and store at 4°C.
[0107] Transfer 50ul of heat-treated fructose amino acid oxidase to a 96-well plate, and take 50ul of unheated fructose amino acid oxidase on the same 96-well plate. Incubate at 37°C for 10 minutes.
[0108] Add the chromogenic solution (Tris, 100mM, pH8.0; TOOS solution, 15mM; 4-APP, 0.5mM; POD, 40U / ml; fructoselysine, 15mM) that has been incubated to 37°C beforehand, and incubate at 37°C React for 30 minutes.
[0109] The absorbance at 555 nm was recorded using a microplate reader.
[0110] Divide the absorbance value of the heat-treated fructose amino acid oxidase by the absorbance value of the non-heat-treated fructose amino acid oxidase, and ...
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