Pérez-Gilabert M, Morte A, Honrubia M, García-Carmona F
Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, E-30071 Murcia, Spain.
J Agric Food Chem. 2001 Apr;49(4):1922-7. doi: 10.1021/jf001009n.
In the present paper, a fully latent polyphenol oxidase (PPO) from desert truffle (Terfezia claveryi Chatin) ascocarps is described for the first time. The enzyme was partially purified by using phase partitioning in Triton X-114 (TX-114). The achieved purification was 2-fold from a crude extract, with a 66% recovery of activity. The interfering lipids were reduced to 13% of the original content. In addition, the purification gave rise to a reduction of phenolic compounds to only 37.5%, thus avoiding the postpurification tanning of the enzyme. Latent PPO was activated by the anionic surfactant sodium dodecyl sulfate (SDS) or by incubation with trypsin. The amount of SDS necessary to obtain a maximum activation was dependent on the nature of the substrate. The use of SDS also permitted the histochemical localization of the latent enzyme within the ascocarp. Terfezia polyphenol oxidase was kinetically characterized using two phenolic substrates (L-DOPA and tert-butylcatechol). The latter substrate presented inhibition at high substrate concentration with a K(si) of 6.3 mM. Different inhibiting agents (kojic and cinnamic acid, mimosine and tropolone) were also studied, tropolone being the most effective.
在本论文中，首次描述了来自沙漠块菌（Terfezia claveryi Chatin）子囊果的一种完全潜伏态的多酚氧化酶（PPO）。该酶通过在Triton X-114（TX-114）中进行相分配进行部分纯化。从粗提物中实现了2倍的纯化，活性回收率为66%。干扰脂质减少至原始含量的13%。此外，纯化使酚类化合物仅减少至37.5%，从而避免了酶纯化后的鞣化。潜伏态PPO可通过阴离子表面活性剂十二烷基硫酸钠（SDS）或与胰蛋白酶孵育来激活。获得最大激活所需的SDS量取决于底物的性质。SDS的使用还允许在子囊果内对潜伏酶进行组织化学定位。使用两种酚类底物（L-多巴和叔丁基儿茶酚）对Terfezia多酚氧化酶进行了动力学表征。后一种底物在高底物浓度下表现出抑制作用，K(si)为6.3 mM。还研究了不同的抑制剂（曲酸和肉桂酸、含羞草碱和托酚酮），托酚酮是最有效的。
