Zheng Suyue, Wang Hexiang, Zhang Guoqing
Hebei Engineering University, Handan, China.
Acta Biochim Pol. 2011;58(2):269-73. Epub 2011 Jun 16.
A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ∙ ml(-1) and 0.668 mg ∙ ml(-1) ∙ min(-1), respectively.
从野生可食用蘑菇鸡枞菌的新鲜子实体中分离出一种分子量为30 kDa、N端序列为GLQTNAPWGLARSS的蛋白酶。纯化方案包括在DEAE - 纤维素、Q - 琼脂糖、SP - 琼脂糖上进行离子交换色谱以及在Superdex 75上进行快速蛋白质液相色谱 - 凝胶过滤。该蛋白质不吸附于DEAE - 纤维素和Q - 琼脂糖，但吸附于SP - 琼脂糖。纯化酶的最适pH和温度分别为10.6和60℃。该酶在2%（v/v）吐温80和4 M尿素存在下稳定。在2%（v/v）Triton X 100中保留了超过80%的酶活性，在10 mM EDTA中保留了54%，在2%（w/v）SDS中保留了31%。该酶受到苯甲基磺酰氟（PMSF）的强烈抑制，但不受二硫苏糖醇（DTT）、胃蛋白酶抑制剂或利马豆胰蛋白酶抑制剂的抑制，表明它是一种丝氨酸蛋白酶而非类胰蛋白酶。该蛋白酶受到Hg(2+)、Cu(2+)和Fe(3+)离子的抑制。纯化酶对酪蛋白的K(m)和V(max)值分别为8.26 mg ∙ ml(-1)和0.668 mg ∙ ml(-1) ∙ min(-1)。
