Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase.
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.
Pérez-Gilabert
M
M
Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, E-30071 Murcia, Spain.
Morte
A
A
Honrubia
M
M
García-Carmona
F
F
D001203:Ascomycota / Q000201:enzymology*; D004156:Catechol Oxidase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D003902:Detergents / Q000737:chemistry*; D004789:Enzyme Activation; D006651:Histocytochemistry; D007700:Kinetics; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D003902:Detergents; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D004156:Catechol Oxidase