酵母裂解酶（Zymolyase）由藤黄节杆菌（Arthrobacter luteus）深层发酵培养制得，对活酵母细胞壁具有强裂解活性，可制备多种酵母菌株的原生质体或球状体。酵母裂解酶发挥裂解活性的核心酶组分为 β-1,3 - 葡聚糖昆布五糖水解酶。该酶可水解具有 β-1,3 糖苷键的线性葡萄糖多聚体，特异性释放昆布五糖作为主要且最小的水解产物单元。
产品信息
| 性状 | 冻干粉 | | 纯化方式 | 硫酸铵沉淀法 | | 活性 | 20000 U/g | | 核心酶组分 | β‑1,3‑葡聚糖昆布五糖水解酶 | | 含有的其他酶活性（※1） | β‑1,3‑葡聚糖酶：约 1.5 × 10⁶ U/g
蛋白酶：约 1.0 × 10⁴ U/g
甘露聚糖酶：约 1.0 × 10⁶ U/g | | 杂质酶（淀粉酶、木聚糖酶、磷酸酶） | 痕量 | | 最适 pH 与温度 | pH 7.5，35℃（裂解活酵母细胞）
pH 6.5，45℃（水解酵母葡聚糖） | | 稳定性（2℃） | 保存 1 年活性无损失 | | 热稳定性 | 30℃：存放 3 个月，裂解活性损失 70%
60℃：孵育 5 分钟，裂解活性完全丧失 | | 底物特异性（裂解谱） | 阿舒囊霉属、假丝酵母属、德巴利酵母属、伊蒙酵母属、内孢霉属、汉逊酵母属、汉森有孢酵母属、克勒克酵母属、克鲁维酵母属、脂酵母属、梅奇酵母属、毕赤酵母属、普鲁兰酵母属、球拟酵母属、酵母菌属、复膜孢酵母属、酵母样菌属、施万酵母属等 |
（※1）各酶活单位定义参见文献：Kitamura, K., Kaneko, T., Yamamoto, Y., J. Gen. Appl. Microbiol., 18, 57 (1972) |
单位定义
裂解活性的 1 个单位定义为：在下述反应条件下，可使反应混合液在 800 nm 处吸光度（A₈₀₀）下降 30% 所需的酶量。
反应混合液
| 酶溶液 | 1 mL（酵母裂解酶浓度 0.05–0.1 mg/mL） |
| 啤酒酵母细胞悬液 | 3 mL（2 mg/mL） |
| 1/15M 磷酸盐缓冲液 | 5 mL（pH7.5） |
| 蒸馏水 | 1 mL |
在 25℃条件下温和振荡孵育 2 小时后，测定混合液在 800 nm 处的吸光度 A₈₀₀。若反应体系中 A₈₀₀下降 60%（相当于 2 个酶活单位），则啤酒酵母细胞完全裂解；即 1 单位酵母裂解酶可裂解 3 mg 干重啤酒酵母。
1/15 M 磷酸盐缓冲液配制（配制 1 L pH 7.5 缓冲液）
1. 称取 19.1 g 十二水合磷酸氢二钠，溶于蒸馏水，定容至 800 mL。
2. 称取 1.814 g 磷酸二氢钾，溶于蒸馏水，定容至 200 mL。
3. 将两份溶液混合。
下述 pH 7.5 磷酸盐缓冲液，经测试均可兼容本实验。
| 缓冲液 | 浓度 | 酵母裂解活性（U/g） |
|---|
| 钾 / 钠磷酸盐缓冲液 | 1/15 M | 21000 |
| 磷酸钾缓冲液 | 5 mM
10 mM
50 mM | 20300
21400
21000 |
| 磷酸钠缓冲液 | 5 mM
10 mM
50 mM | 20500
21200
21200 |
Zymolyase, produced by a submerged culture of Arthrobacter luteus, has strong lytic activity against living yeast cell walls, to produce protoplast or spheroplast of various strains of yeast cells. An essential enzyme for the lytic activity of Zymolyase is β-1,3-glucan laminaripentaohydrolase. It hydrolyzes linear glucose polymers with β-1,3-linkages and releases specifically laminaripentaose as the main and minimum product unit.
Product Information
| Form | Lyophilized Powder | | Purification | Ammonium Sulfate Precipitation | | Activity | 20,000 units/g | | Essential enzyme | β-1,3-glucan laminaripentaohydrolase | Other activities
contained (※1)
| β-1,3-glucanase: approx. 1.5 × 10⁶ units/g
Protease: approx. 1.0 × 10⁴ units/g
Mannanase: approx. 1.0 × 10⁶ units/g | | Contaminants (Amylase, Xylanase, Phosphatase) | Trace amount | | Optimum pH and Temp. | pH 7.5, 35°C (for lysis of viable yeast cells)
pH 6.5, 45°C (for hydrolysis of yeast glucan) | | Stability (2°C) | No loss of activity was found after storage for 1 year | | Heat stability | 30°C:
70% of the lytic activity is lost after storage for 3 months
90% of the lytic activity is lost after storage for 3 months
60°C:
Lytic activity is lost on incubation for 5 minutes | | Specificity (Lytic Spectrum) | Ashbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloeckera, Kluyveromyces, Lipomyces, Metschnikowia, Pichia, Pullularia, Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes, Schwanniomyces, etc. |
(※1) See reference, Kitamura, K., Kaneko, T., Yamamoto, Y., J. Gen. Appl. Microbiol., 18, 57 (1972) as to the definition of each enzyme units. |
Unit Definition
One unit of lytic activity is defined as that amount which indicates 30% of decrease in absorbance at 800 nm(A800) of the reaction mixture under the following condition.
Reaction Mixture
| Enzyme solution | 1 ml (0.05–0.1 mg/ml for Zymolyase) | | Brewer's yeast cell suspension | 3 ml (2 mg/ml) | | 1/15M Phosphate buffer | 5 ml (pH7.5) | | Distilled water | 1 ml |
|
After incubation for 2 hours at 25°C with gentle shaking, A800 of the mixture is determined. When 60% of A800 decrease, equivalent to 2 units, is observed in the reaction system, the brewer’s yeast cells are completely lysed, namely 1 unit of Zymolyase lyses 3 mg dry weight of brewer’s yeast.
Preparation of 1/15 M Phosphate Buffer (1L of pH 7.5 buffer)
- Dissolve 19.1 g of di-sodium hydrogenphosphate 12-hydrate in distilled water to make an 800 mL solution.
- Dissolve 1.814 g of potassium dihydrogenphosphate in distilled water to make a 200 mL solution.
- Mix the two solutions.
The below phosphate buffers (pH 7.5) have been tested for compatibility.
ZJ26F0535439
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