【摘要】 重组的乳酸乳球菌X 脯氨酰 二肽酰基 氨基肽酶是一个工具酶 ,它对基因构建的神经肽或活性多肽的转活具有重要意义 .通过细菌细胞的破碎 ,洗涤 ,冷冻离心 ,透析 ,DEAE 纤维素 5 2柱层析等工艺过程达到电泳纯 .该酶比活为 11.92 6U mg ,纯化倍数为 14.37倍和总活性收率为5 5 .5 6% .通过SDS PAGE和凝胶柱层析法 ,测得该二肽酶有单肽链组成 ,分子量 89KD .在该酶的动力学研究中 ,针对特异性底物L 甘氨酰 L 脯氨酰 对 硝基苯胺 ,求得该酶的米氏方程式 1 V =0 0 4 8 [S]+ 0 2 5 66(r =0 994 ) .它的Km 值为 0 1871mmol L ,最大反应速度Vmax为 3 897μmol·L-1·min-1.该酶可被苯甲酰基磺酰氟 ,胰蛋白酶抑制剂和Mn2 +,Ba2 +,Cu2 +andZn2 +等金属离子抑制 ,但可被Mg2 +激活 .进一步试验显示 ,当Cu2 +和Zn2 +浓度增加到 3 72 6mmol L ,抑制作用明显增强 .低浓度的EDTA Na2 (≤ 0 62 12mmol L)不影响酶的活性 .因此 ,该X 脯氨酰 二肽酰基 氨基肽酶是一个金属离子非依赖性的丝氨酸蛋白酶更多还原

【Abstract】 The recombinant x-prolyl dipeptidyl aminopeptidase (PepX) from Lactococcus lactis,a tool enzyme responsible for the maturation of the neuropeptides or active polypeptides, was purified to homogeneity by means of cell disruption, washing, centrifugation, dialysis, single ion exchange column chromatography on DEAE-cellulose 52. The specific activity of the PepX was enriched about 14.37 folds and the yield of total activity was 55.56%. PepX was composed by single polypeptide chain with molecular weight 89 kD determined by SDS-PAGE and gel column chromatography. In the study of the kinetics and inhibition of PepX against the substrate Gly-Pro-p-NA, Michaelis-Menton equation on PepX against the substrate was 1/V=0.048/[S]+0.2566 (r=0.994). K m constant of 0.1871 mmol/L and V max of 3.897 μmol/L.min -1 were calculated. PepX activity was inhibited by PMSF, trypsin inhibitor and some metal cations such as Mn 2+, Ba 2+, Cu 2+ and Zn 2+, but stimulated by Mg 2+. Further experiment showed when Cu 2+ or Zn 2+ concentration was increased to 3.726 mmol/L, the PepX activity was significantly inhibited. Low final concentrations of EDTA (≤0.6212 mmol/L) had no influence on the enzyme activity. The inhibition of PMSF showed that the active site includes a serine residue. The results suggest that PepX is a metal-independent serine peptidase.更多还原