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A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXAlb) whose sequences are known.

Extracellular enzymes in soil often occur in immobilised forms, a state that may alter their interactions with substrates in comparison with enzymes in the solution phase. Sonication was evaluated for its usefulness in studying immobilised acid phosphatase by dispersing soil aggregates. Factors affecting soil dispersion during ultrasound application were soil extraction ratio, total applied energy and power output ml−1 of sonicated soil slurry. For the clay loam soil used, optimal values for these variables were, respectively, 1:6 (w/v) and, at least, 1800 J ml−1 and 15 W ml−1. At the optimal sonication conditions for soil dispersion a substantial increase in phosphatase activity (up to 156% greater than the non-sonicated control) was induced by sonication. This increase in activity with sonication, which coincided with a release of soil chromophores, might be related to the exposure or release from aggregates of the extracellular enzyme fraction immobilised on humic colloids. Analysis of multiple regression between the phosphatase activity (dependent variable) and chromophore solubilisation and ATP release (independent variables) suggested the increased activity was from complexed enzymes that were released and not due to cell lysis. Soil treatment with sonication appeared to have liberated a large dormant portion of acid phosphatase activity. Coefficients of variation of the activity decreased greatly (from 20% in control soil to 4% as an average after sonication).

A stable acid phosphatase-polyresorcinol complex, retaining about 90% of the original enzymatic activity, was synthesized in the presence of peroxidase. The complex was disturbed by chaotropic agents, as 4 M urea, indicating that the enzyme linked the phenolic moiety by non-covalent bonds. The immobilized phosphatase was more stable than the free enzyme towards pH, temperature and proteolysis: however, when added to soil, free and immobilized phosphatase showed comparable stability. It appears that the polymerization conditions used, favouring the interaction between enzyme and polyphenol through secondary linkages, give rise to a fully active complex resembling those which naturally generate in soil.

Pleurotus ostreatus mycelium produced extracellular cellulases when grown on ground wheat straw. No cellulase activity was detectable when glucose or aqueous extracts of vegetable material were used as substitutes for straw in culture media. The specific activity of excreted cellulases did not vary significantly by increasing the straw concentration from 1 to 6%. Chromatographic fractionation of extracellular proteins gave rise to five fractions with cellulolytic activity. The hydrolytic properties of these partially purified fractions were analysed by using several substrates (carboxymethylcellulose, cellobiose,p-nitrophenyl-β-d-cellobioside,p-nitrophenyl-β-d-lactoside). The results indicate that the cellulase system ofP. ostreatus includes at least a β-glucosidase, two endocellulases, an exoglucohydrolase and an exocellobiohydrolase.

The best growth conditions for increasing ligninolytic enzyme production and biomass yield of Pleurotus ostreatus strain florida have been studied. The cultural conditions for scaling up mycelial growth from flask cultures to 2 and 15 litre fermenters, where pH could be controlled, were optimized. The effect in bioreactors of aeration and stirring on growth and enzymatic expression was also examined.

Pleurotus ostreatus mycelium was grown in the presence of tobacco and wheat straw juices, ground wheat straw and acid-precipitable polymeric lignin with the aim of increasing the production of extracellular enzymes potentially implicated in lignin degradation. The highest production of diphenol oxidases was obtained when culture broths were supplemented with solid straw or tobacco juice; cellulase and veratryl alcohol oxidase were produced in detectable amounts in the presence of solid straw and in the absence of any inducer, respectively. No lignin peroxidase was found under any growth condition. Chromatographic fractionation of extracellular proteins and kinetic analysis of partially purified fractions showed that, in the absence of inducers, o -, p - and m -diphenol oxidases could be detected in column eluates, whereas all the inducers tested increased selectively the production of o -diphenol oxidases.

A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201. This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide. Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and Trametes C30 (98% similarity). The expression of lcc1 was analysed under different growth conditions; transcription of this gene was enhanced by the addition of organic nitrogen to the medium. The level of lcc1 transcription was higher when T. trogii was grown on synthetic medium supplemented with yeast extract rather than mycological peptone or tryptone. The transcription data were in agreement with total laccase activity measured in the supernatant and suggested that laccase production and lcc1 transcription are coordinately regulated in this organism. The lcc1 cDNA was expressed in the methylotrophic yeast Pichia pastoris and the detection of laccase activity indicated that this cDNA encodes a laccase.

The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70 kDa, acidic isoelectric point and N-terminal sequence homologous to that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant molecules may be converted into substrates by introducing, in the correct position, o- or p-orienting ring-activating groups.

We have designed and implemented on a personal computer a program for identifying and quantifying the fragments present in a peptide mixture obtained by hydrolysing a polypeptide of known sequence using digesting agents. The qualitative data utilized by the main algorithm consist of the target sequence of the intact molecule and the amino acid residues identified at each step of the automatic sequence analysis of the unfractionated digestion mixture. In this way, the sequence of each fragment present in the mixture is quickly reconstructed. Furthermore, if the quantitative data of the amino acid residues identified at each step of the sequence analysis are utilized, the program will correlate the sequence of each fragment to its amount. We furnish an example of the application intended for the rapid identification and characterization of the extracellular proteinases produced by a basidiomycete fungus, utilizing the bovine insulin beta-chain as target substrate. A variety of uses for the method are discussed.

Background: Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts. Results: In order to obtain a simple and efficient source of laccase, the lcc1 cDNA isolated from the white-rot fungus Trametes trogii has been successfully expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol induced alcohol oxidase promoter P AOX1 . The recombinant Lcc1 was produced as a secreted protein with the native N-terminal prepropeptide for signal trafficking, and thus easily recovered from the culture medium. At the 1-liter scale, as calculated on the basis of the specific activity, the recombinant protein was produced at a yield of 17 mg/l. The highest production level obtained in fed-batch culture was 2520 U/l, corresponding to a specific productivity of 31.5 U/g biomass. The purified recombinant laccase exhibited a behaviour similar to the main laccase produced by T. trogii. Lcc1 showed high activity in the presence of organic solvents and a high decolourization capacity towards azo, triarylmethane, indigo carmine and anthraquinonic dyes, that could be significantly enhanced in the presence of the redox mediators 1-hydroxybenzotriazole and violuric acid. Conclusion: Heterologous expression of T. trogii laccase lcc1 in the methylotrophic yeast P. pastoris was successfully achieved. The biochemical and kinetic characterization of the recombinant protein suggests potential technological applications for this enzyme.

A protein inhibitor which is active against both exogenous and endogenous trypsin has been purified from wheat endosperm. The inhibitor is a single-chain protein of M r 8500 and isoelectric point 8.7. It contains 10 half-cystine residues per mol, all of them linked in disulphide bridges. The N -terminal sequence, 25 residues long, has been determined. The inhibitor is very active against bovine, porcine, cod and wheat trypsin, and inactive against animal chymotrypsin and bacterial subtilisin. The inhibitor binds bovine trypsin with a 1:1 molar stoichiometry.

Cytosolic aspartate aminotransferase (EC 2.6.1.1) from ox heart has been fully characterized; the most significant results have been reviewed. An investigation is now in progress to establish structural and evolutionary relationships between this enzyme and the homologous one from pig heart, whose complete primary structure has been reported. A more complete picture from an evolutionary point of view should also arise from a comparison of the mitochondrial enzymes. In fact the primary structure of the mitochondrial porcine enzyme has been reported. Structural investigations on cytosolic and mitochondrial chicken aspartate aminotransferases are being pursued. Therefore we have undertaken the purification and characterization of mitochondrial bovine aspartate aminotransferase. Preliminary sequence and crystallographic data are reported here.

Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs2 while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.