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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
阿魏酸酯酶 (FAE，EC3.1.1.73) 又称肉桂酸酯酶或肉桂酸水解酶，是一种胞外羧酸酯酶，阿魏酸酯酶主要降解细胞壁多糖或果胶中阿拉伯糖或半乳糖或羟基肉桂酸之间的酯键。
阿魏酸酯酶 (FAE) 催化底物阿魏酸甲酯分解，通过检测阿魏酸甲酯于340nm的下降速率即可得出阿魏酸酯酶 (FAE) 酶活力大小。
反应方程式：Feruloyl-Polysaccharide+H₂O=Ferulate+Polysaccharide
注：本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
自备仪器和试剂
操作步骤（仅供参考）：
建议先选取1-3个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实 验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1. 样本提取
① 组织样本：
取约0.1g组织 (水分充足的样本可取0.5g)，加入1mL 提取液，4°C×12000rpm离心15min，取上清液待测。
[注]: 若增加样本量，可按照组织质量(g): 提取液体积(mL)为1:5~10的比例提取。
② 液体样本：可直接测定，或者适当稀释后测定。若浑浊，离心后取上清检测。
③细菌/细胞样本：先收集细菌或细胞到离心管内，离心后弃上清；取约500万细菌或细胞加入1mL提取液，超声波破碎细菌或细胞 (冰浴，功率200W，超声3s，间隔10s，重复30次)；12000rpm 4°C离心10min，取上清，置冰上待测。
[注]: 若增加样本量，可按照细菌/细胞数量(10⁴)：提取液(mL)为500~1000:1的比例进行提取。
2. 检测步骤：
① 酶标仪预热 30 min (等仪器过自检程序亦可)，调节波长为340nm。
② 所有试剂于25℃水浴中预热 10min。
③ 在96孔板中依次加入下列试剂：
| 试剂组分 (μL) | 测定管 |
| 样本 | 50 |
| 试剂一 | 130 |
| 试剂二 | 20 |
混匀，立即于340nm处读取吸光值A1，40°C孵育30min后读取A2。ΔA=A1-A2。
[注]: ①若ΔA的值非常低在零附近，可增加样本量V₁ (如增至100μL，则试剂一相应减少) 或延长反应时间T (如增至60min 或更长)，则重新调整的V₁和T代入公式重新计算。 ② 若A1的值超过1.5，可减少样本量V₁(如减至30μL，则试剂一相应增加)，则调整后的V₁代入公式重新计算。
结果计算：
1. 标准曲线：y =0.033x-0.0034，x是标准品摩尔质量 (nmol)，y是ΔA。
2. 按照样本质量计算：
酶活定义：每克组织每分钟水解1nmol底物阿魏酸甲酯定义为一个酶活单位。
FAE (nmol/min/g 鲜重)=[(ΔA+0.0034)÷0.033]÷(W×V₁÷V)÷T=20.2×(ΔA+0.0034)÷W
3. 按照样本蛋白浓度计算：
酶活定义：每毫克蛋白每分钟水解1nmol底物阿魏酸甲酯定义为一个酶活单位。
FAE (nmol/min/mg prot)=[(ΔA+0.0034)÷0.033]÷(Cpr×V₁)÷T=20.2×(ΔA+0.0034)÷Cpr
4. 按按液体体积计算：
酶活定义：每毫升液体每分钟水解1nmol底物阿魏酸甲酯定义为一个酶活单位。
FAE (nmol/min/mL)=[(ΔA+0.0034)÷0.033]÷V₁÷T=20.2×(ΔA+0.0034)
W：样品质量，g V：提取液体积，1 mL
V₁：上清液体积 (mL)，0.05mL
T：反应时间，30 min 500：细胞数量，万
Cpr：上清液蛋白质浓度，mg/mL；建议使用本公司的BCA蛋白质含量测定试剂盒（B665595）。
标准曲线制作过程：
1. 标曲为非必做实验，用户可根据实验需求制作标曲，亦可直接采用说明书计算公式进行结果计算。
2. 制备标准品母液（10μmol/mL）：向标准品EP管里面加入1mL乙醇溶解。
3.将母液用乙醇稀释成六个浓度梯度的标准品，例：0, 0.1, 0.2, 0.3, 0.4, 0.5μmol/mL。也可根据实际样本调整标准品浓度。
4. 标品稀释参照表如下：
吸取标准品母液100μL，加入1.9mL乙醇，混匀得到0.5μmol/mL的标品稀释液待用。
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5. 依据测定管的加样表操作，根据结果，以各浓度吸光值减去0浓度吸光值，过0点制作标准曲线。
| 试剂组分（μL） | 标准管 | 0浓度管（仅做一次） |
| 标品 | 50 | / |
| 乙醇 | / | 50 |
| 试剂一 | 150 | 150 |
混匀，立即于340nm处读取吸光值A1，40°C孵育30min后读取A2。ΔA=标准管(A1-A2)-0浓度管(A1-A2)。
Feruloyl esterase (FAE, EC 3.1.1.73), also known as cinnamoyl esterase or cinnamic acid hydrolase, is an extracellular carboxylesterase. It primarily hydrolyzes the ester bonds between arabinose, galactose and hydroxycinnamic acids in cell wall polysaccharides and pectin. FAE catalyzes the degradation of methyl ferulate substrate. The activity of feruloyl esterase can be determined by detecting the decreasing rate of absorbance of methyl ferulate at 340 nm.
Reaction equation: Feruloyl-Polysaccharide+H2O=Ferulate+Polysaccharide
Note: For research use only. Not for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
⁕Full-wavelength microplate reader (capable of detecting absorbance at 340 nm)
⁕Mortar (homogenizer), ice box (ice maker), tabletop centrifuge, adjustable pipette, water bath (drying oven, incubator, metal bath), 96-well plate, centrifuge tube
⁕1 mL cuvette, centrifuge tube
⁕Distilled water (deionized water and ultrapure water are acceptable).
Protocol (for reference only):
It is recommended to select 1–3 representative samples with significant differences (e.g., different types or groups) for a pre-experiment to familiarize with the operation process. Adjust the sample concentration according to the pre-experiment results to avoid unnecessary waste of samples and reagents.
1. Sample Extraction
① Tissue samples:
Weigh approximately 0.1 g of tissue (0.5 g for high-moisture samples), add 1 mL of extraction solution, and centrifuge at 12,000 rpm at 4 °C for 15 min. Collect the supernatant for testing.
Note: For increased sample dosage, perform extraction at a tissue mass (g) to extraction solution volume (mL) ratio of 1:5~10.
② Liquid samples:
Detect directly, or determine after appropriate dilution. If the solution is turbid, collect the supernatant by centrifugation for detection.
③ Bacterial / cell samples:
Collect bacteria or cells into a centrifuge tube and discard the supernatant after centrifugation. Add 1 mL of extraction solution to approximately 5 million bacteria or cells, and perform ultrasonic disruption under ice bath conditions (power: 200 W, sonication for 3 s, interval 10 s, repeat 30 times). Centrifuge at 12,000 rpm and 4 °C for 10 min, then collect the supernatant and place it on ice for testing.
Note: For increased sample dosage, extract at a ratio of bacteria/cell count (10⁴) to extraction solution (mL) of 500~1000:1.
2. Detection Procedures:
① Preheat the microplate reader for 30 min (instrument self-check is also acceptable), and set the wavelength to 340 nm.
② Preheat all reagents in a 25 °C water bath for 10 min.
③ Add the following reagents sequentially into a 96-well plate.
| Reagent (μL) | Assay Tube |
| Sample | 50 |
| Reagent 1 | 130 |
| Reagent 2 | 20 |
Mix well and immediately record the absorbance A1 at 340 nm. After incubation at 40 °C for 30 min, measure the absorbance A2. ΔA=A1-A2。
Note: ① If the ΔA value is extremely close to zero, appropriately increase the sample volume V₁ (e.g., increase to 100 μL with the corresponding reduction of Reagent 1), or extend the reaction time T (e.g., extend to 60 min or longer). Substitute the adjusted V₁ and T into the formula for recalculation.
② If the value of A1 exceeds 1.5, reduce the sample volume V₁ (e.g., decrease to 30 μL with the corresponding increase of Reagent 1), and substitute the adjusted V₁ into the formula for recalculation.
Result Calculation:
1 Standard curve equation: y =0.033x-0.0034,
1. 标准曲线：y =0.033x-0.0034, Where x = refers to the molar mass of the standard (nmol), y=ΔA.
2. Calculation based on sample weight:
Enzyme activity definition: One unit of FAE activity is defined as the amount of enzyme that hydrolyzes 1 nmol of methyl ferulate substrate per minute per gram of tissue.
FAE (nmol/min/g Fresh weight)=[(ΔA+0.0034)÷0.033]÷(W×V₁÷V)÷T=20.2×(ΔA+0.0034)÷W
3. Calculation based on sample protein concentration:
Standard Curve Preparation:
1. The standard curve preparation is optional. Users may prepare the standard curve according to experimental requirements, or directly calculate results using the formula provided in the instruction manual.
2. Preparation of standard stock solution (10 μmol/mL): Add 1 mL of ethanol to the EP tube containing the standard for complete dissolution.
3. Dilute the stock solution with ethanol to prepare six standard concentration gradients: 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μmol/mL. The standard concentration can be adjusted according to actual sample conditions.
4. Refer to the table below for standard dilution instructions:
Pipette 100 μL of standard stock solution, add 1.9 mL of ethanol, and mix well to prepare the 0.5 μmol/mL standard diluent for later use.
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5. Operate in accordance with the pipetting table for the determination tubes. Subtract the absorbance of the 0 concentration group from the absorbance of each standard concentration, and plot a zero‑based standard curve.
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| Item No. | Appearance | Components | 96T | Storage | Precautions |
| F1522256A | Liquid | 提取液 | 120 mL | 2-8℃. | / |
| F1522256B | Liquid | 试剂一 | 15mL | 2-8℃. | / |
| F1522256C | Solid | 试剂二 | 1 EA | 2-8℃. Store in the dark. | 1. 临用前加4mL无水乙醇混匀溶解； 2. 4℃保存。 |
| F1522256D | Solid | 标准品 | 1 EA | 2-8℃. Store in the dark. | 1. 若重新做标曲，则用到该试剂； 2. 按照说明书中标曲制作步骤进行配制； 3. 溶解后的标品一周内用完。 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838446 |
分析证书 | F1522256 |
| ZJ26F0636156 |
分析证书 | F1522256 |
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