Kinetic Study of the Activation Process of a Latent Mushroom (Agaricus bisporus) Tyrosinase by Serine Proteases

Abstract

Latent mushroom tyrosinase can be considered as a zymogen when activated by proteases because the activation process fulfilled all of the kinetic dependencies predicted by a theoretical zymogen activation model previously reported. The activation was studied under two assay conditions: high and low ratio of latent tyrosinase/serine protease (trypsin and subtilisin Carlsberg) concentrations, in the presence and in the absence of a serine protease inhibitor (aprotinin). The size of the latent enzyme was 67 kDa, determined by denaturing SDS-PAGE electrophoresis and Western blot assays. After proteolytic activation, the size was 43 kDa, with an intermediate band of 58 kDa. The values of the catalytic () and Michaelis () constants for the active forms of tyrosinase resulting from the activation by subtilisin, trypsin, or sodium dodecyl sulfate on the substrate tert-butylcatechol were slightly different, which could support the idea of "one activator-one different active tyrosinase". Vacuum infiltration experiments tried to reproduce in vivo the role of mushroom serine proteases in the activation of latent tyrosinase. The use of serine protease inhibitors is proposed as a new alternative tool to prevent melanin formation.

The Tyrosinase-Encoding Gene ofLentinula edodes,Letyr, Is Abundantly Expressed in the Gills of the Fruit-Body during Post-Harvest Preservation

“…14) The deduced LeTyr had a molecular mass of 68 kDa, which corresponded to the size observed for the protyrosinases of these other fungi: the mature tyrosinases of A. bisporus 17,18) and P. sanguineus 19) were detected as proteins of about 45 kDa, suggesting a proteolytic mechanism of maturation. Halaouli et al 5) indicated that a tyrosine residue was highly conserved among some fungi in the consensus LYG sequence, which acts as a putative chymotrypsin cleavage site.…”

Consistency of Polyphenol Oxidase (PPO) Thermostability in Ripening Apricots (Prunus armeniaca L.): Evidence for the Presence of Thermostable PPO Forming and Destabilizing Mechanisms in Apricots

“…In the early literature it has been reported that the active forms of PPO in plants having molecular masses in the range of 40-50 kDa derive from the in situ proteolysis of 60-70 kDa precursors (36)(37)(38). Fraignier et al (31) reported that in apricots and in other Prunus fruits the main active PPO form is a 43 kDa enzyme.…”

Zymography of Monophenolase and o-Diphenolase Activities of Polyphenol Oxidase

“…Horseradish peroxidase (HRP) (EC 1.11.1.7) was obtained from Sigma (grade II, Lot 16H9522) and from Boehringer Mannheim (grade II, Lot 12486022-85). Crude extract of PPO from mushroom fruit bodies, prepared essentially as described by Espín et al (13), was a gift from Dr. Harry J. Wichers from Agrotechnological Research Institute, Wageningen, The Netherlands. Ungerminated grains of S. bicolor (L.) Moench var.…”