Proteinase K is a broad-spectrum serine protease and our product is extracted from Pichia pastoris cells with cloned gene encoding Engyodontium album (Tritirachium album) endolytic protease. It is a highly reactive protease frequently used for digesting various proteins and enzymes (including endonuclease, exonuclease, DNase or RNase) .Therefore, it is usually used in DNA preparations without impairing the integrity of the isolated DNA. It has a superior performance under a broad range of conditions: pH, buffer, detergents (such as SDS), chelator (such as EDTA), and temperature. Proteinase K hydrolyzes peptide bonds preferentially adjacent to carboxyl group of hydrophobic amino acids (aliphatic, aromatic, and others).
Except the isolation of genome, it can also take a job in detection of enzyme localization or removal of enzymes from DNA to improve cloning efficiency.
Appropriate working concentration of proteinase K is always among the range of 0.05 to 1 mg/mL. The activity of the enzyme can be stimulated by 0.2 to 1% SDS or by 1 to 4 mol urea. It is activated by calcium (1-5mM), although calcium ions do not affect the enzyme activity, but it contributes to the thermal stability and protects the proteinase from autolysis. Proteinase K has two binding sites for Ca2+, which are located close to the active center, but are not directly involved in the catalytic mechanism. So calcium ion has a regulatory function for the substrate binding site of proteinase K. The enzyme is inactivated by DIFP or PMSF. However, it is not inhibited by EDTA, iodoacetic acid, trypsin-specific inhibitor TLCK, chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.
We recommend an optimum pH of 7.5 to 8.0 and optimum temperature at 50 to 55°C. Rapid denaturation will occur at temperatures above 65°C. You can hold it under 95°C for 10 min as a heat inactivation.
References:
[1]. Kraus, E; et.al. Proteinase K from the Mold Tritirachium album limber, Specificity and Mode of Action. Z. Physiol. Chem., 357:939; 1976.
[2]. Jany,KD, et al. Amino Acid Sequence of Proteinase K from the Mold, Tritirachium albumlimber. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letter, 199,139.1986.
蛋白酶K是一种广谱的丝氨酸蛋白酶，我们的产品是从克隆Engyodontium album（Tritirachium album）内切蛋白酶基因的毕赤酵母细胞中提取的。它是一种高度反应性的蛋白酶，常用于消化各种蛋白质和酶（包括内切酶、外切酶、DNase或RNase等）。因此，在DNA制备中通常使用它而不会损害所分离的DNA的完整性。它在广泛的条件下具有优越的性能：pH，缓冲液，清洁剂（如SDS），螯合剂（如EDTA）和温度。蛋白酶K首选水疏性氨基酸（脂肪族、芳香族等）的羧基侧链相邻的肽键进行水解。
除了分离基因组外，它还可以在酶定位检测或去除酶从而提高克隆效率方面发挥作用。
适当的蛋白酶K工作浓度始终在0.05至1 mg/mL范围内。酶活性可以通过0.2至1％ SDS或1至4 mol尿素来刺激。它被钙（1-5mM）激活，尽管钙离子不影响酶活性，但它有助于热稳定性并保护蛋白酶不受自溶的影响。蛋白酶K有两个钙离子结合位点，位于活性中心附近，但不直接参与催化机制。因此，钙离子对蛋白酶K的底物结合位点具有调节作用。该酶被DIFP或PMSF失活。但是，它不受EDTA，碘乙酸，胰蛋白酶特异性抑制剂TLCK，胰蛋白酶特异性抑制剂TPCK和对氯汞苯甲酸盐的抑制。
我们推荐最适宜的pH为7.5至8.0，最适宜的温度为50至55°C。在65°C以上的温度下会发生快速变性。您可以将其保持在95°C下进行10分钟的热失活。