香樟(Cinnamomum camphora)果实酪氨酸酶的分离纯化及特性分析

目的]检测并分离香樟果实中的酪氨酸酶。方法]从香樟成熟果实中分离和纯化出酪氨酸酶,研究该酶的酶活、蛋白质含量、最适pH值、最适温度、动力学特性和稳定性,并分析了不同金属离子、化合物和中药对其酶活的影响。结果]经分离和纯化,获得43 kD的酪氨酸酶。该酶的最适pH值和最适温度分别为7.5和50℃。以L-DOPA为底物,测得Km和Vmax分别为12.83 mmol/L和180.65U/mg。该酶在pH值4~7和较高温度时均较为稳定。Al3+和Cu2+对该酶活性具有抑制作用,而Zn2+、Mg2+等则有激活作用。250mmol/LEDTA对该酶有激活作用,抗坏血酸和1 mmol/Lβ-巯基乙醇有完全抑制作用。中药女贞对其有强激活作用,乌梅对其抑制作用最明显。结论]香樟果实中酪氨酸酶同工酶的存在为该酶的纯化带来一定难度。

Separation and Purification of Tyrosinase from the Friuits of Cinnamonum camphora its Characteristics Analysis

Objective] The research aimed to detect and separate tyrosinase in the fruits of Cinnamomum camphora.Method] Tyrosinase was separated and purified from mature fruits of C.camphora to study its activity,protein content,the optimum pH value,the optimum temperature,dynamic characteristics and stability.And the effects of different metal ions,compounds and traditional Chinese medicine on the enzyme activity were analyzed.Result] Through separation and purification,tyrosinase with the size of 43 kD was obtained.The optimum pH value and the optimum temperature of this enzyme were 7.5 and 50 ℃ resp.With L-DOPA as substrate,Km and Vmax were determined as 12.83 mmol/ L and 180.65 U/mg resp.The enzyme was more stable both at pH value of 4～7 and under higher temperature.Both Al3+ and Cu2+ had inhibition on the enzyme activity,while Zn2+,Mg2+ and so on had the activation effects on it.250 mmol/L EDTA had an activation effect on this enzyme activity,while ascorbic acid and 1 mmol/L β-mercaptoethanol had complete inhibition on it.Taditional Chinese medicine Ligusrtum lucidum Ait.had a stronger activation effect on this enzyme activity,while the inhibition of Fructus mume on it was most obvious.Conclusion] The existing of tyrosinase isozyme in the fruits of C.camphora brought certain difficulty for the purification of this enzyme.