Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus.

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.

The crystal structure of an extracellular catechol oxidase from the ascomycete fungus Aspergillus oryzae.

Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled type 3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. The crystal structures of a limited number of bacterial and fungal tyrosinases and plant catechol oxidases have been solved. In this study, we present the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4) at 2.5-Å resolution. AoCO4 belongs to the newly discovered family of short-tyrosinases, which are distinct from other tyrosinases and catechol oxidases because of their lack of the conserved C-terminal domain and differences in the histidine pattern for CuA. The sequence identity of AoCO4 with other structurally known enzymes is low (less than 30 %), and the crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region. A diatomic oxygen moiety was identified as a bridging molecule between the two copper ions CuA and CuB separated by a distance of 4.2-4.3 Å. The UV/vis absorption spectrum of AoCO4 exhibits a distinct maximum of absorbance at 350 nm, which has been reported to be typical of the oxy form of type 3 copper enzymes.

Crystallization and preliminary X-ray analysis of Aspergillus oryzae catechol oxidase.

Catechol oxidase is an enzyme that catalyzes the oxidation of o-diphenols to the corresponding o-quinones. It is a copper-containing enzyme with a binuclear copper active site. Here, the crystallization and multiple-wavelength anomalous dispersion data collection of catechol oxidase from the mould fungus Aspergillus oryzae are described. During the purification, three forms of the enzyme (39.3, 40.5 and 44.3 kDa) were obtained. A mixture of these three forms was initially crystallized and gave crystals that diffracted to 2.5 Šresolution and belonged to space group P3(2)21, with unit-cell parameters a = b = 118.9, c = 84.5 Å, α = ß = 90, γ = 120°. A preparation containing only the shorter form (39.3 kDa) produced crystals that diffracted to 2.9 Šresolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 51.8, b = 95.3, c = 139.5 Å, α = ß = γ = 90°.

Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase.

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350-400 and 400-600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L(-1) production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on L-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60 degrees C. The UV-visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.

Biotransformation of acyclic monoterpenoids by Debaryomyces sp., Kluyveromyces sp., and Pichia sp. strains of environmental origin.

Sixty yeast strains, which belong to 32 species of the genera Debaryomyces, Kluyveromyces, and Pichia, and which were isolated from plant-, soil- or insect-associated habitats, were screened for their ability to biotransform the acyclic monoterpenes geraniol and nerol. The aptitude to convert both compounds (from 2.6 to 30.6, and from 2.7 to 29.1%/g cell DW (=dry weight), resp.) was apparently a broad distributed character in such yeasts. Depending upon the substrate used, the production of linalool, alpha-terpineol, beta-myrcene, D-limonene, (E)-beta-ocimene, (Z)-beta-ocimene, or carene was observed. Linalool was the main product obtained from geraniol, whereas linalool and alpha-terpineol were the main products obtained through the conversion of nerol. Yet, differently from nerol, the aptitude to exhibit high bioconversion yields of geraniol to linalool was an apparently genus-related character, whereas the ability to produce other monoterpenes was a both genus- and habitat-related character. The possible pathways of bioconversion of geraniol or nerol to their derivatives were proposed/discussed.

Crystal structure of an ascomycete fungal laccase from Thielavia arenaria--common structural features of asco-laccases.

Laccases are copper-containing enzymes used in various applications, such as textile bleaching. Several crystal structures of laccases from fungi and bacteria are available, but ascomycete types of fungal laccases (asco-laccases) have been rather unexplored, and to date only the crystal structure of Melanocarpus albomyces laccase (MaL) has been published. We have now solved the crystal structure of another asco-laccase, from Thielavia arenaria (TaLcc1), at 2.5 Å resolution. The loops near the T1 copper, forming the substrate-binding pockets of the two asco-laccases, differ to some extent, and include the amino acid thought to be responsible for catalytic proton transfer, which is Asp in TaLcc1, and Glu in MaL. In addition, the crystal structure of TaLcc1 does not have a chloride attached to the T2 copper, as observed in the crystal structure of MaL. The unique feature of TaLcc1 and MaL as compared with other laccases structures is that, in both structures, the processed C-terminus blocks the T3 solvent channel leading towards the trinuclear centre, suggesting a common functional role for this conserved 'C-terminal plug'. We propose that the asco-laccases utilize the C-terminal carboxylic group in proton transfer processes, as has been suggested for Glu498 in the CotA laccase from Bacillus subtilis. The crystal structure of TaLcc1 also shows the formation of a similar weak homodimer, as observed for MaL, that may determine the properties of these asco-laccases at high protein concentrations.

Crosslinking food proteins for improved functionality.

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (Tuber melanosporum Vitt.) and white (Tuber magnatum Pico) truffles.

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated L: -methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.