Dimethoxyphenol oxidase activity of different microbial blue multicopper proteins

Abstract

2,6-Dimethoxyphenol is a versatile substrate for Pyricularia oryzae laccase, PpoA from Marinomonas mediterranea, phenoxazinone synthase from Streptomyces antibioticus and mammalian ceruloplasmin. In addition, in cellular extracts of microorganisms expressing other blue multicopper proteins with no enzymatic activity previously described, such as Escherichia coli (copper resistance CueO), Pseudomonas syringae and Xanthomonas campestris (copper resistance CopA), Bacillus subtilis (sporulation protein CotA) and Saccharomyces cerevisiae (iron transporter Fet3p), laccase activity is detected under appropriate conditions. This oxidase activity can be spectrophotometrically followed by the oxidation of 2,6-dimethoxyphenol. Specific staining after SDS-PAGE is also possible for some of these proteins. This detection assay can facilitate the study of the multiple functions that such proteins seem to carry out in a variety of microorganisms.

Involvement of a novel copper chaperone in tyrosinase activity and melanin synthesis in Marinomonas mediterranea

“…In both cases, PCR amplification included the promoter region. pBppoB2 and pBppoB1 were cloned in E. coli S17-1 (lpir) and subsequently mobilized into the ppoB2 or ppoB1 mutant by conjugation (Solano et al, 2000). Conjugation plates were enriched with 0.5 mM IPTG, to promote transposition over recombination.…”

“…mediterranea expresses two copper PPO enzymes, PpoB1 and PpoA. PpoA shows laccase activity, which is measured using DMP as specific substrate for this enzyme (Solano et al, 2000). Previous results indicated that laccase activity in strain T105 is similar to that of the wild-type strain MMB1-R, suggesting that the absence of PpoB2 does not affect PpoA enzymic activity (Ló pez- Serrano et al, 2004).…”

Cloning of multicopper oxidase gene from <i>Ochrobactrum sp</i>. 531 and characterization of its alkaline laccase activity towards phenolic substrates

“…The Ni-affinity column was equilibrated with 2.5. Active Staining 10% native gel was used to separate the crude extract as described previously [25]. After electrophoresis, the minigel was first soaked in 30 ml of 50 mM TrisHCl (pH 7.5 or 8.0) for 15 minutes, and then stained in 50 mM TrisHCl buffer (pH 7.5 or 8.0) containing 0.02 mM DMP (or SGZ) and 0.2 mM CuSO 4 at room temperature for 15 minutes.…”

“…Like fungal and plant laccases, bacterial laccases also contain four copper atoms: type I or blue copper, type II or normal copper, and type III or coupled binuclear copper center [15]. Compared to fungal laccases, bacterial laccases show little overall amino acid sequence similarity, low oxidation rate, and supplementation of Cu 2+ [16]. Because of these differences, bacterial enzyme is often defined as "multicopper oxidase (MCO)", and its activity as "laccase-like enzyme".…”

“…The amount of fungal laccases does not meet current market requirements due to their expression difficulty in prokaryotic hosts in an active form, but bacterial laccases, unlike fungal ones, do not require any post-transcriptional modification, and are able to express in prokaryotic hosts as a soluble form. Bacterial laccases also act in a wide rang of pH, and have wide substrate specificities and good stability [16]. Its low activities can be improved, because the factors affecting enzyme activity can often be defined and optimized via enzymatic study and protein engineering [17][18][19][20].…”