CAJ | 학술논문

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-52柱层析等工艺过程达到电泳纯. 该酶比活为11.926 U/mg, 纯化倍数为14.37倍和总活性收率为55.56%.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89 KD. 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=0.048/[S]+0.2566(r=0.994). 它的Km值为0.1871 mmol/L, 最大反应速度Vmax为3.897 μmol*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+ and Zn2+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3.726 mmol/L, 抑制作用明显增强. 低浓度的EDTA-Na2 (≤0.6212 mmol/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.
중조적유산유구균X-포안선-이태선기-안기태매시일개공구매, 타대기인구건적신경태혹활성다태적전활구유중요의의. 통과세균세포적파쇄, 세조, 냉동리심, 투석, DEAE-섬유소-52주층석등공예과정체도전영순. 해매비활위11.926 U/mg, 순화배수위14.37배화총활성수솔위55.56%.통과SDS-PAGE화응효주층석법, 측득해이태매유단태련조성, 분자량89 KD. 재해매적동역학연구중, 침대특이성저물L-감안선-L-포안선-대-초기분알, 구득해매적미씨방정식1/V=0.048/[S]+0.2566(r=0.994). 타적Km치위0.1871 mmol/L, 최대반응속도Vmax위3.897 μmol*L-1*min-1. 해매가피분갑선기광선불, 이단백매억제제화Mn2+, Ba2+, Cu2+ and Zn2+등금속리자억제, 단가피Mg2+격활. 진일보시험현시, 당Cu2+화Zn2+농도증가도3.726 mmol/L, 억제작용명현증강. 저농도적EDTA-Na2 (≤0.6212 mmol/L)불영향매적활성. 인차, 해X-포안선-이태선기-안기태매시일개금속리자비의뢰성적사안산단백매.
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). Km constant of 0.1871 mmol/L and Vmax of 3.897 μmol/L.min-1 were calculated. PepX activity was inhibited by PMSF, trypsin inhibitor and some metal cations such as Mn2+, Ba2+, Cu2+ and Zn2+, but stimulated by Mg2+. Further experiment showed when Cu2+ or Zn2+ 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.