葡萄糖脱氢酶 (GDH-FAD) ：一种酶，可用作再生辅因子，将葡萄糖与 NAD (P) 转化为 NAD (P) H 和葡萄糖酸。
来源：米曲霉
催化作用：
规格
活力：≥100 单位每毫克粉末
外观：黄色粉末，冻干品
稳定性：在 -20℃下稳定保存 36 个月
添加剂：未添加
特性
| 分子量 | 180,000（凝胶过滤法） |
| | 等电点 | 6.5 |
| | 米氏常数（葡萄糖） | 15.0×10⁻³ M |
| | 抑制剂 | Ag⁺、Hg²⁺ |
| | 激活剂 | Triton X - 100 |
| | 最适 pH | 7.0 | Fig.1
| | 最适温度 | 55℃ | Fig.2
| | 稳定 pH 范围 | 4.0 - 7.0 | Fig.3
| | 稳定温度范围 | 40℃以下 | Fig.4
| | 底物特异性 |
| Table 1
| | 介体偏好性 |
| Fig.5
|
|
应用
这种酶用于通过葡萄糖传感器等对血液或尿液中的葡萄糖进行酶促测定。
葡萄糖脱氢酶(GDH-FAD)的测定方法
原理
1*非那定甲硫酸盐，2*硝基四氮唑蓝
通过分光光度法在 570nm 波长下测定甲臜（diformazan）的生成量。
单位定义：
1 个单位定义为：在以下所述条件下，每分钟生成 0.5 微摩尔（μmol）甲臜（diformazan）所需的酶量。
试剂
A. 曲拉通 X-100 溶液（5% 曲拉通 X-100 溶液）
称取 5.0g 曲拉通 X-100，加热溶解于 30mL去离子水中。冷却后，用去离子水定容至 100 毫升。（室温下储存，有效期 1 个月）
B. 工作液（含 0.5% 曲拉通 X-100 的 50 mmol/L PIPES - NaOH 缓冲液，pH 6.5）
称取 1.51 克 PIPES，溶解于约 70 毫升去离子水中。加入 10 毫升曲拉通 X-100 溶液（A 液），随后用 4 当量（N）的氢氧化钠溶液调节 pH 至 6.5，再用去离子水定容至 100 毫升。（2~8℃下储存，有效期 1 个月）
C. NTB 溶液（6.6 mmol/L）
称取 54 mg Nitorotetrazorium blue，溶解于 10 毫升去离子水中。该溶液需装入避光管中，避免光照。（2~8℃下储存，有效期 14 天）
D. PMS 溶液（3.0 mmol/L）
称取 9 mg phenazine methosulfate，溶解于 10 毫升去离子水中。该溶液需装入避光管中，避免光照。（2~8℃下储存，有效期 14 天）
E. 底物溶液（1.0 mol/L葡萄糖溶液）
称取 3.6 克 D - 葡萄糖，溶解于去离子水中，并用去离子水定容至 20 毫升。（室温下储存，有效期 14 天）
F. 酶稀释液
称取 3.02 克 PIPES、0.2 克牛血清白蛋白（低盐型）以及 29.4 毫克 CaCl₂・2H₂O，加入至约 160 毫升去离子水中溶解。加入 4 毫升曲拉通 X-100 溶液（A 液），随后用 4 当量氢氧化钠溶液调节 pH 至 6.5，再用去离子水定容至 200 毫升。（2~8℃下储存，有效期 1 个月）
G. 酶溶液
称取 25 毫克 葡萄糖脱氢酶 (GDH-FAD)，溶解于预冷的酶稀释液（F 液）中。酶溶液的配制需满足：2 分钟内的吸光度变化值（ΔOD/2minutes）处于 0.058±0.026 范围内。
H. 混合液
将 26 毫升工作液（B 液）、1 毫升 NTB 溶液（C 液）、2 毫升 PMS 溶液（D 液）与 1 毫升底物溶液（E 液）混合均匀。该溶液需装入避光管中，避免光照。（2~8℃下储存，有效期 6 小时）
实验步骤
1. 移取 3 毫升混合液（H 液）至一次性塑料比色皿（光程 d=10 毫米）中，在 37±0.5℃条件下保温 10 分钟。
2. 随后，移取 0.1 毫升酶溶液（E 液）加入比色皿中，立即充分混匀，继续在 37±0.5℃条件下保温。
3. 在加入酶溶液后，分别于精确的第 3 分钟和第 5 分钟，测定反应混合液在 570 纳米（nm）波长下的吸光度，记为 A3（第 3 分钟吸光度）和 A5（第 5 分钟吸光度）。
4. 空白对照操作：另取一只一次性塑料比色皿（光程 d=10 毫米），加入酶稀释液（F 液）替代酶溶液（G 液），其余操作与上述步骤一致，测定得到空白组第 3 分钟吸光度（Ab3）和第 分钟吸光度（Ab5）。
计算
2: 反应时间
20.1: 570nm波长下甲臜（diformazan）的半毫摩尔消光系数
3.1: 反应混合物的最终体积
0.1: 酶溶液的体积
Dm: 酶溶液的稀释倍数
FAD-dependent glucose dehydrogenase is an enzyme used as a regeneration cofactor to convert glucose and NAD(P) into NAD(P)H and gluconic acid.
ORIGIN: Aspergillus oryzae
CATALYSIS:
SPECIFICATIONS
| Activity | ≥100 U/mg powder
|
|---|
| Appearance | Yellow powder, lyophilized | | Stability | Stable for 36 months at -20 ℃ | | Additives | Not Added |
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CHARACTERISTICS
| Molecular weight | 180,000 (Gel filtration) | |
|---|
| Isoelectric point | 6.5 | | | Km (Glucose) | 15.0 × 10⁻³ M | | | Inhibitors | Ag⁺, Hg²⁺ | | | Activator | Triton X-100 | | | Opt. pH | 7.0 | Fig.1 | | Opt. temperature | 55 ℃ | Fig.2 | | Stable pH range | 4.0-7.0 | Fig.3 | | Stable temp. range | below 40 ℃ | Fig.4 | | Substrate specificity | | Table 1 | | Mediator Preference | | Fig.5 |
|
APPLICATIONS
This enzyme is used for enzymatic determination of glucose in blood or urine by glucose sensor etc.
Assay Method of Glucose Dehydrogenase (GDH-FAD)
Principle
1* phenadine methosulfate, 2* Nitorotetrazorium blue
The appearance of diformazan is measured at 570nm by spectrophotometry.
Unit Definition
One unit is defined as the enzyme quantity which produces 0.5 μmol of diformazan per minute under the conditions described below.
Reagents
A. Triton X-100 Solution (5% Triton X-100 solution)
Weigh 5.0 g of Triton X-100 and dissolve in 30 mL of deionized water with heating. After cooling, fill up to 100 mL with deionized water. (Expires after 1 month at room temperature)
B. Working Solution (50 mmol/L PIPES-NaOH (pH 6.5) containing 0.5 % Triton X-100)
Weigh 1.51 g of PIPES and dissolve in approx. 70mL of deionized water. Add 10 mL of Triton X-100 solution (A), then adjust the pH to 6.5 with 4N NaOH. Fill up to 100 mL with deionized water. (Expires after 1 month at 2~8℃)
C. NTB Solution (6.6 mmol/L)
Weigh 54 mg of Nitorotetrazorium blue and dissolve in 10 mL of deionized water. This solution should be kept in a light-proof tube to avoid exposure to light. (Expires after 14 days at 2~8℃)
D. PMS Solution (3.0 mmol/L)
Weigh 9 mg of phenazine methosulfate and dissolve in 10 mL of deionized water. This solution should be kept in a light-proof tube to avoid exposure to light. (Expires after 14 days at 2~8℃)
E. Substrate Solution (1.0 mol/L Glucose)
Weigh 3.6 g of D-glucose and dissolve in deionized water. Fill up to 20 mL with deionized water. (Expires after 14 days at room temperature)
F. Enzyme Diluent
Weigh 3.02 g of PIPES and 0.2 g of bovine serum albumin and 29.4 mg of CaCl₂·2H₂O in approx. 160 mL of deionized water. Add 4 mL of Triton X-100 solution (A), then adjust the pH to 6.5 with 4N NaOH. Fill up to 200 mL with deionized water. (Expires after 1 month at 2~8℃)
G. Enzyme Solution
Weigh 25 mg of Glucose Dehydrogenase (GDH-FAD) and dissolve in chilled Enzyme Diluent (F). Enzyme Solution should be prepared so that the value of ΔOD/2minutes becomes in the range of 0.058±0.026.
H. Mix Solution
Mix 26 mL of Working Solution (B), 1 mL of NTB Solution (C), 2 mL of PMS Solution (D) and 1 mL of Substrate Solution (E). This solution should be kept in a light-proof tube to avoid exposure to light. (Expires 6 hours at 2~8℃)
Procedure
Pipette 3 mL of Mix Solution (H) into a disposable plastics cuvette (d=10 ~mm) and keep at 37±0.5 ℃ for 10 minutes. Then, pipette 0.1 mL of Enzyme Solution (E) into the cuvette and mix well immediately. Keep the reaction mixture at 37±0.5 ℃. Exactly at 3 minutes and 5 minutes after the addition of Enzyme Solution (E), measure the absorbances of the reaction mixture at 570 nm. (A3 and A5) As a blank, pipette Enzyme Diluent (F) into another disposable plastics cuvette (d=10 ~mm) instead of Enzyme Solution (E), and take the same procedure described above. (Ab3 and Ab5)
Calculation
2: Reaction time
20.1: Half of millimolar extinction coefficient of diformazan at 570 nm
3.1: Final volume of the reaction mixture
0.1: Volume of Enzyme Solution
Dm: Dilution multiple of Enzyme Solution
ZJ25F1028501
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