Li Jiang, Shi Peng-Jun, Han Xiao-Yu, Meng Kun, Yang Pei-Long, Wang Ya-Ru, Luo Hui-Ying, Wu Ning-Feng, Yao Bin, Fan Yun-Liu
Microbial Engineering Department, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Protein Expr Purif. 2007 Jul;54(1):79-86. doi: 10.1016/j.pep.2007.02.012. Epub 2007 Feb 28.
We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.
我们报道了sfp2基因的初步表征和表达，该基因编码来自弗氏链霉菌变种k11的一种角蛋白分解丝氨酸蛋白酶。重组SFP2在毕赤酵母中表达并分泌，诱导25小时后最终产量为78 mg/L（酪蛋白分解活性为136.2 U/mL）。重组酶通过硫酸铵沉淀和凝胶过滤色谱法纯化至电泳纯，其糖基化程度合适，分子量为26.0 kDa。对纯化的重组SFP2进行了表征。SFP2分解酪蛋白和角蛋白天青的最佳pH值和温度分别为pH 10.0、60℃和pH 9.0、55℃。SFP2的活性在pH 3.0至pH 11.0范围内稳定。该酶的活性受到Co(2+)和Cr(3+)的抑制，而Ni(2+)和Cu(2+)则增强了其活性。以N-琥珀酰-Ala-Ala-Pro-Phe-pNA为底物计算得出K(m)为0.45 mmol/L，V(max)为19.84 mmol/min mg。我们测试了SFP2对可溶性和不溶性底物的活性；与蛋白酶K和其他商业蛋白酶相比，SFP2对角质底物更具特异性。
