Lai Lih-Shiuh, Wang Dai-Jung, Chang Chen-Tien, Wang Cheng-Hsin
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan 402, Republic of China.
J Agric Food Chem. 2006 Nov 1;54(22):8611-6. doi: 10.1021/jf060888w.
The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).
小麦草粗酶提取物在60℃加热30分钟，然后进行硫酸铵分级分离，并在聚缓冲剂交换剂（PBE 94）上进行等电聚焦色谱法纯化。然后对纯化的过氧化物酶的催化特性进行表征。结果发现，浓度为0.25 mM的AgNO₃以及浓度为5 mM的MnSO₄和EDTA显著抑制了小麦草过氧化物酶的活性。然而，浓度为5.0 mM的KCl、NaCl、CuCl₂、CaCl₂、ZnCl₂和MgCl₂以及浓度为0.25 mM的HgCl₂增强了酶活性。化学修饰显著影响了小麦草过氧化物酶的活性。特别是，N-溴代琥珀酰亚胺（5 mM）抑制了16%的酶活性，而N-乙酰咪唑（2.5 mM）、焦碳酸二乙酯（2.5 mM）和苯甲基磺酰氟（2.5 mM）使酶活性提高了18 - 29%。这些结果表明色氨酸、组氨酸、酪氨酸和丝氨酸残基与酶活性有关。小麦草过氧化物酶催化邻苯二胺（OPD）、儿茶酚、焦性没食子酸和愈创木酚氧化的最适pH分别为5.0、4.5、6.5和5.0。OPD、儿茶酚、焦性没食子酸和愈创木酚的表观Km值分别为2.9、18.2、2.5和3.8 mM。在最佳反应条件下，小麦草过氧化物酶催化OPD（一种芳香胺底物）氧化的速度比愈创木酚、儿茶酚和焦性没食子酸（苯环中含有一至三个羟基的酚类底物）快3 - 11倍。
