Screening of fungal enzymes for fine chemical transformations: laccases and enoate reductases.

Due to the increasing industrialization and the growing sensibility about pollution problems, the identification of energetically and environmentally sustainable processes are currently needed. Nowadays, several industrial sectors are developing bio-based technologies to reduce the high costs and environmental impact of traditional chemical processes. Fungi and their enzymatic derivatives become then interesting for the production of fine chemicals, pharmaceuticals and agrochemical intermediates. Laccases are among the most studied oxidoreductases and fungi have been fully accepted as the most interesting microbial source. Up to date, most of the researches has dealt with Basidiomycetes but little is known about Ascomycetes. The aim of the present study was to investigate the enzymatic productivity of Ascomycetes, isolated in sea water, industrial wastewaters, Alpine rocks, compost or contaminated soil, and including also lichen and marine algae symbionts. Solid and liquid screenings were performed and some cultural media were used. Among the almost tested 100 strains, those isolated from marine environments and compost were the most active ones, mediating an aspecific, wide and rapid oxidation of RBBR, ABTS, naphthol and guaiacol. Black microcolonial fungi and lichen symbionts showed unexpected oxidative activity: even in presence of a deeply scarce growth, ABTS plates completely tacked to green in a restrained period of time (2-4 days). When laccase production was stimulated in liquid by the addition of copper, Acremonium tubakii (marine), Cladosporium herbarum (compost) and Coniosporium uncinatum (rocks) produced the largest amount of enzymes. Laccases of A. tubakii were used for the biotransformation of caffeic acid, whose oxidation leads to the formation of dimers with high value in pharmaceutical field. Dimer formation was obtained already after 1 h and undesired byproducts did not form. Ene-reductases are able to reduce C=C double bonds of carboxylic acids and esters, conjugated with different electron-withdrawing groups (EWG) such as carbonyl, nitro and ester and carboxylic acid. This reaction is a crucial step in organic chemistry, currently performed by highly polluting and expensive metal catalysts. To date, most of the information about this enzymatic class comes from bacteria and yeasts, meanwhile filamentous fungi have been poorly investigated. Having such scarce data about their possible occurrence within the Fungi Kingdom, several species of filamentous fungi belonging to Ascomycota, Basidiomycota and Zygomycota were here taken into consideration for the conversion of cyclohexenone, α-methylnitrostyrene, α-methylcinnamaldehyde and methyl cinnamate. Almost all the 28 strains showed ene-reductase activity and transformed the substrates producing molecules which can be used for instance in the flavors production (i.e. methyldihydrocinnamyl alcohol from α-methylcinnamaldehyde). In particular Mucor circinelloides, M. plumbeus and Syncephalastrum racemosum resulted versatile and effective, reducing all the substrates within the first 1-3 days. Interestingly, in addition to the ene-reductase activity, they showed also carboxylic acid reductase activity which appears to be well sought in several industrial sectors whether its occurrence is almost unknown in filamentous fungi. Further studies will be design to get additional information on the intraspecific variability, the role of the each EWG on the reaction efficiency and the enantioselectivity of the reaction. Moreover molecular analyses are also in progress to define the genes coding for ene-reductases in the genome of the fungus M. circinelloides.