Homocysteine detection using fusion enzymes
A homocysteine, fusion enzyme technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as difficult control, restricting catalytic efficiency, etc., to improve accuracy and stability, and improve detection. horizontal effect
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Embodiment 1
[0025] Expression and purification of embodiment 1 fusion enzyme protein
[0026]The fusion enzyme protein uses a flexible linker peptide (GGGGS) 2 , applied to the fusion of homocysteine S-methyltransferase (EC2.1.1.10) and adenosylhomocysteinase (EC3.3.1.1), and adenosylhomocysteinase (EC3.3.1.1) and methionine adenosyltransferase fusion (EC2.5.1.6), the construction method of the fusion gene is consistent:
[0027] Design the upstream primer with the EcoR I restriction site for the first enzyme gene, and the downstream primer with GGGGS and the 5' end partial sequence of the second enzyme gene; design the 3' end partial sequence with GGGGS and the first enzyme gene An upstream primer, and a downstream primer with a Xhol I restriction site for the second enzyme gene fragment. Using the American ABI gradient PCR instrument (Veriti TM 96), the amplification system and reaction conditions of the two are the same (total volume 100ul, 10ul of 10*PCR buffer, 10ul of 100umo / L ...
Embodiment 2
[0039] Embodiment 2 Utilizes the comparison of the detection accuracy of the reagent prepared by fusion enzyme and common enzyme cycle reagent
[0040] Reagents prepared using fusion enzymes contain the following components:
[0041] Reagent components
Dosage per liter
Phosphate buffer, pH7.6, 37°C
100mM
TCEP
26.5mM
Sodium azide
7.7mM
ATP
2mM
adenosine deaminase
3ku
H-G-Aase
10ku
A-E-Mase
10ku
bovine serum albumin
0.5%
alpha-ketoglutarate
7.5mM
Mannitol
60mM
Methionine
0.5mM
Glycerin
100g
reduced coenzyme NADH
0.29mM
glutamate dehydrogenase
2ku
[0042] The contrast reagent is a commercially available general enzyme cycle reagent, which is calibrated with a prepared homocysteine standard solution with a concentration of 15.1 μM. The detection sample is a prepared linear sample, and the concentrations of h...
Embodiment 3
[0054] Embodiment 3 utilizes the comparison of the thermal stability of the reagent prepared by fusion enzyme and common enzyme cycle reagent
[0055] The liquid dual-reagent reagent prepared by fusion enzyme contains the following components:
[0056] Reagent 1:
Dosage per liter
Tris buffer, pH9.1, 20.0°C
100mM
TCEP
26.5mM
Sodium azide
15.0mM
Methionine
0.5mM
alpha-ketoglutarate
7.5mM
Triton X-100
2.0g
bovine serum albumin
2.0g
reduced coenzyme NADH
0.29mM
adenosine deaminase
7ku
glutamate dehydrogenase
3ku
[0057] Reagent 2:
Dosage per liter
HEPES buffer, pH7.4, 20.0°C
50mM
Mannitol
60mM
ATP
10mM
[0058] Sodium azide
15.0mM
PEG2000
0.5g
Tween-20
3.0g
bovine serum albumin
1.0g
H-E-Aase
50ku
A-G-Mase
70ku
[0059] The co...
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