嗜热菌蛋白酶（Thermolysin）是由嗜热地芽孢杆菌（Geobacillus stearothermophilus，旧称：嗜热脂肪芽孢杆菌 Bacillus stearothermophilus）产生的热稳定金属蛋白酶。该酶可特异性水解亮氨酸 (Leu)、异亮氨酸 (Ile)、缬氨酸 (Val)、苯丙氨酸 (Phe) 等大体积疏水氨基酸残基氨基侧的肽键。嗜热菌蛋白酶结合 1 个锌离子维持酶活性，结合 4 个钙离子维持蛋白稳定性。该酶的氨基酸序列及三维空间结构已被解析。
产品特性
1. 性状：冻干无定形粉末（纯度：二次结晶）
2. 最适温度：65‑70 ℃
3. 最适 pH：8.0
4. 稳定 pH 范围：5.0‑8.5
5. 稳定温度：70 ℃以下稳定性良好
6. 抑制剂：EDTA、链霉菌金属蛋白酶抑制剂（SMPI）
7. 溶解度：在稀释缓冲液中约 0.02%；在 40‑50% 甘油中约 4%
8. 分子量：34600
9. E¹%₁cm（280 nm）：pH 7.0 条件下为 17.65
注：本产品酶学特性及酶活相关数据均为本实验室实验所得，不同实验条件下测定结果可能存在差异。
技术指标
蛋白水解活性（pH 7.2）：不低于 7000 PU/mg
检测方法
本检测方法基于 Hagihara 改良版 Anson 法。
酶活单位定义
一个蛋白酶单位（PU）定义为：在 35 ℃、pH 7.2 条件下，反应初期，每分钟从终浓度 0.5% 牛乳酪蛋白底物中释放出福林显色当量等同于 1 μg 酪氨酸的非蛋白消化产物所需的酶量。
试剂
1、底物：1% 牛乳酪蛋白（Hammarsten 级），含 1/15 M 磷酸氢二钠‑磷酸二氢钾缓冲液（pH 7.55），使用时体系 pH 应调至 7.2。
2、三氯乙酸混合液（TCA 混合液）：0.1 M 三氯乙酸（TCA），含 0.2 M 乙酸钠、0.3 M 乙酸。
3、0.4 M 碳酸钠溶液
4、稀释福林试剂：5 倍稀释
5、酶稀释液：0.01 M 硼酸盐缓冲液（pH 8.0），含 0.002 M 硫酸钙、0.005% Triton X‑100。
6、42% 甘油溶液（pH 8.0）：42% 甘油，使用 0.01 N 氢氧化钠调 pH 至 8.0，含 0.005% Triton X‑100。
供试品制备
精密称取约 100 mg 嗜热菌蛋白酶，用 42% 甘油溶液（pH 8.0）溶解，并用该甘油溶液定容至 100 mL。使用酶稀释液进行稀释，使最终稀释液每 1 mL 含 5‑10 PU 酶活力。
操作步骤
1、吸取 1.0 mL 供试品溶液置于试管中，35 ℃温度平衡。
2、加入 1.0 mL 预热至 35 ℃的底物溶液，混匀。
3、精确反应 10 min 后，加入 2.0 mL TCA 混合液终止反应，混匀后于 35 ℃继续孵育 30 min。
4、使用滤纸过滤混合液。
5、吸取 1.0 mL 滤液至试管，加入 5.0 mL 0.4 M 碳酸钠，再加入 1.0 mL 稀释福林试剂，混匀，35 ℃孵育 20 min。
6、在 660 nm 波长处测定吸光度（ODₜ）。
7、设置空白对照：将底物与 TCA 混合液的添加顺序颠倒，测定空白吸光度 (ODb) 。
计算公式
Activity(PU/ml)=(ODₜ−ODb)×4/A×10
4：体系总体积 (mL)
10：反应时间 (min)
A：1 μg/mL 酪氨酸溶液（溶于 0.2 N 盐酸）按照上述第 5、6 步操作测得的吸光度。
操作注意事项
1. 切勿吸入本品粉尘。
2. 若皮肤或眼睛直接接触，立即用大量清水冲洗。
3. 详细信息请查阅物质安全数据表（MSDS）。
一般性质
以下数据为该酶在部分缓冲液体系中测得，实际使用请根据实验需求确认合适反应条件。
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Thermolysin is a heat-stable metalloprotease produced by Geobacillus stearothermophilus (Former: Bacillus stearothermophilus). Thermolysin specifically hydrolyzes peptide bonds on the amino side of bulky hydrophobic residues such as Leu, Ile, Val, and Phe. Thermolysin has one zinc ion necessary for activity and four calcium ions for stability. The amino acid sequence and three-dimensional structure of this enzyme have been determined.
Features
- Amorphous powder, freeze-dried (Purity: Two times crystallized)
- Optimum temperature: 65 - 70°C
- Optimum pH: 8.0
- Stable pH range: 5.0 - 8.5
- Stable temperature: Considerably stable at below 70°C
- Inhibitors: EDTA, SMPI (Streptomyces metalloprotease inhibitor)
- Solubility: approx. 0.02% in dilute buffer solution; approx. 4% in 40-50% glycerin
- Molecular weight: 34,600
- E1% 1cm (280nm): 17.65 at pH 7.0
The data for genera enzyme features and enzyme activity are based only on our laboratory experiment results. Therefore, different experiment conditions may yield different results.
Specification
Protein digestive activity (pH 7.2): Not less than 7,000 PU/mg.
Assay method
This method is based on the Hagihara modification of the Anson method.
Unit definition
One protease unit (PU) is defined as the amount of the enzyme that will liberate non-proteinous digestion products from milk casein (final concentration 0.5%) to give a Folin's color equivalent to 1μg of tyrosine per minute at the early stage of the reaction at 35°C, pH
7.2.The assay method is available on request.
Reagents
- Substrate: 1% milk casein (according to Hammarsten) containing 1/15M Na₂HPO₄-KH₂PO₄ buffer (pH 7.55). The pH value of this solution should be 7.2.
- TCA mixture: 0.1M trichloroacetic acid (TCA) containing 0.2M sodium acetate and 0.3M acetic acid.
- 0.4M Na₂CO₃
- Diluted Folin’s reagent: 5-fold
- Enzyme diluents: 0.01M borate buffer, pH 8.0, containing 0.002M CaSO₄ and 0.005% Triton X-100.
- 42% Glycerin (pH 8.0): 42% glycerin adjusted to pH 8.0 with 0.01N NaOH, containing 0.005% Triton X-100.
Test preparation
Weigh accurately about 100mg of Thermolysin, dissolve it in 42% glycerin (pH 8.0), and bring to 100ml with 42% glycerin (pH 8.0). Dilute it with enzyme diluents so that 1ml of the final dilution will contain between 5 and 10 PU.
Procedure
- Pipette 1.0ml of the test preparation into a test tube, and equilibrate it to 35°C.
- Add 1.0ml of substrate pre-warmed to 35°C, and mix.
- After exactly 10 minutes, add 2.0ml of TCA mixture to stop the reaction and incubate the mixture for a further 30 minutes at 35°C.
- Filter the mixture with filter paper.
- Pipette 1.0ml of the filtrate into a test tube, add 5.0ml of 0.4M Na₂CO₃ and then 1.0ml of diluted Folin’s reagent, mix, and incubate the mixture for 20 minutes at 35°C.
- Read the optical density at 660nm (ODₜ)
- Run a blank in which the order of adding substrate and TCA mixture is reversed (OD<sub>b</sub>)
Calculation
Activity(PU/ml)=(ODₜ−OD<sub>b</sub>)×4/A×10
4: Total volume (ml)
10: Reaction time (min)
A: Optical density of 1μg/ml tyrosine solution (dissolved in 0.2N HCl) according to procedures 5 and 6
Handling caution
- Do not inhale.
- In case of direct contact with skin or eyes, immediately wash or rinse with plenty of water.
- Please refer to MSDS for details.
General properties
The following results were obtained for the enzyme solution in some buffers. Please check appropriate conditions for your applications.
ZJ26F1040005
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