State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China.
J Biosci Bioeng. 2010 Jan;109(1):20-4. doi: 10.1016/j.jbiosc.2009.06.022. Epub 2009 Jul 21.
A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. It was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 degrees C, with about 40% activity remaining at 87 degrees C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14600 U/mg toward casein. The K(m) of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 degrees C. The V(max) was 5.35x10(-2) mg ml(-1) min(-1). It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg(2+), Fe(2+) and Mn(2+), but was curtailed by Cu(2+), Hg(2+) and Fe(3+). It was devoid of antifungal and ribonuclease activities.
一种 33.5kDa 的丝氨酸蛋白酶，被命名为 helvellisin，从野生子囊菌蘑菇 Helvella lacunosa 的干燥子实体中分离出来。它通过使用包括 DEAE-纤维素、CM-Sepharose、Q-Sepharose 离子交换层析和 FPLC-凝胶过滤 Superdex 75 的程序进行纯化。该蛋白酶具有独特的 N-末端氨基酸序列、热稳定性和 pH 稳定性。该蛋白酶在 pH 11.0 和 65°C 时表现出最佳活性，在 87°C 和 pH 5 和 13 时仍保留约 40%的活性。Helvellisin 对酪蛋白的蛋白酶活性为 14600 U/mg。在 pH 11.0 和 37°C 时，纯化蛋白酶对酪蛋白的 K(m)为 3.81mg/ml。V(max)为 5.35x10(-2)mg ml(-1) min(-1)。它受到苯甲基磺酰氟的抑制，表明它是一种丝氨酸蛋白酶。该蛋白酶的活性受 Mg(2+)、Fe(2+)和 Mn(2+)的增强，但受 Cu(2+)、Hg(2+)和 Fe(3+)的抑制。它没有抗真菌和核糖核酸酶活性。
