荷斯坦阉牛瘤胃液中阿魏酸酯酶和乙酰酯酶的酶学特性研究

Enzymological characterization of feruloyl and acetyl esterases in the rumen fluids of Holstein steers

为研究瘤胃液中阿魏酸酯酶和乙酰酯酶的酶学特性,自安装永久瘤胃瘘管的成年荷斯坦阉牛瘤胃中采集瘤胃食糜液,4℃1 000g离心10min制备成粗酶液,测定了粗酶液中阿魏酸酯酶和乙酰酯酶活性及其酶促反应动力学参数。结果表明,瘤胃液中阿魏酸酯酶酶促反应最适pH为9.0,最适温度为40～50℃;用阿魏酸甲酯作为为酶促反应标准底物,测得米氏常数(Km)为0.76mmol/L,最大反应速度(Vmax)为5.43mU;乙酰酯酶酶促反应最适pH为8.0,最适反应温度为50℃。用对-硝基苯乙酰酯作为标准底物,测得Km为0.64mmol/L,Vmax为91.00mU。研究发现Fe2+可以促进阿魏酸酯酶酶促反应,而Cu2+等金属离子可以抑制阿魏酸酯酶酶活性;Mg2+、K+、Co2+、Ca2+和Mn2+则可促进乙酰酯酶酶促反应。尽管瘤胃内环境并不能为2种酯酶发挥最大酶效提供最适温度和pH条件,相比较而言,阿魏酸酯酶在瘤胃液中稳定性更优于乙酰酯酶。

To determine enzymological characteristics of feruloyl esterase（FAE） and acetyl esterase（AE）,rumen fluids collected from Holstein steers intalled flexible ruminal cannula were centrifuged at 1 000g at 4 ℃ for 10 min to prepare crude enzyme solutions.The optimal pH is 9.0 and optimal temperature for FAE is 40-50 ℃;and the Michaelis constants（Km） is 0.76 mmol/L and maximum velocities（Vmax） against methyl ferulate is 5.43 mU at pH 6.0 and 39 ℃.The optimal pH for AE is 8.0 and optimal temperature for AE is 50 ℃,and the Km and Vmax against p-nitrophenyl acetate at pH 7.0 and 39 ℃ were 0.64 mmol/L and 91.00 mU.Cations such as Mg2＋,Zn2＋,K＋,Cu2＋,Co2＋,Ca2＋ and Mn2＋ in this study inhibited the activities of esterases for FAE while Fe2＋ has opsite effects.The cations of Mg2＋,K＋,Co2＋,Ca2＋ and Mn2＋ had a stimulatory effect on AE.In general,FAE showed relatively more stable than AE in the rumen though both pH and temperature in rumen fluids may not be optimum for both esterases to exert their enzymological potentials in plant cell wall degradation.