Catalysis Performance Of Fungal Laccase In Non-aqueous Media And Its Application In Coupling Polymerization

Laccase (benzenediol: oxygen oxidoreductase, EC1.10.3.2) is a family ofcopper-containing phenoloxidase widely distributed in nature. It directly catalyzes theoxidation of a variety of organic substrates including phenols, anilines and their derivatives.Fungi laccase has draw great attention due to its relatively high available amounts, high redoxpotential and simple procedures for purification.Along with the growing of biochemical engineering, more and more researchers devoteto develop enzyme-catalyzed synthesis. Owing to its advantages in mild working conditions,high selectivity and environmental friendly features, laccase catalysis is a promising greenprocess for producing polyphenols, polyamines and functional heterocylic compounds. In thiswork, a white laccase was produced and purified from a white-rot fungus Panus conchatusand the structures and properties were characterized. The scavenging of ABTS radicals byacetone was also discussed. The synthesis of2,3-diaminophenazine and lignin-basedhydrogels by laccase was also studied. This may help extend the applications of laccase andpromotes the value of lignin products.The structure and properties of P. conchatus laccase purified from solid-statefermentation culture were analyzed, including N-terminal amino acid sequence, metalcompositions in activity center, substrate specificity and enzyme activity. The results showthat the activity center of this laccase is Cu2FeZn, in which the two-copper cluster appearedbut the TI-Cu and TII-Cu are substituted by Zn and Fe respectively. This structure is differentfrom that of typical blue laccase. The molecular weight of P. conchatus laccase was56kDaand the isoelectric point was2.6. The N-terminal amino acid sequence in P. conchatus laccasewas similar with typical basidioycete fungi and tyrosine is the major amino acid. This laccaseshows high affinity to ABTS with Km of11.6μmol/L. It was stable in a varied range oftemperature and pH (in particular pH ranged from4-12).The oxidation of ABTS by laccase in aqueous acetone was studied using aspectrophotometer. The inhibitory effect of acetone to laccase-ABTS oxidation was obvious.The apparent reaction rate decreased dramatically with acetone content. Both Km and Vmaxvaried exponentially with acetone concentrations. Acetone scavenging ABTS radicals can be asupplemental mechanism that explains the above phenomena. In aqueous acetone (20:80,Vol%),95%of ABTS radicals were transformed to their reductive form in60min. Thescavenging rate decreased with time and the maximum was observed in the initial one minute. A recycling procedure was designed according to the results. ABTS can be recycled for6times at least. After each recycle,23-30%of active ABTS was lost. This recycle method mayhelp developing a more economic laccase/ABTS systemfor industrial use.2,3-diaminophenazine (DAP) was synthesized from o-phenylenediamine (OP) usingfungi laccase catalyzing in aqueous/organic solvent. Spectroscopic analysis including UV-Vis,FTIR,2D-NMR and MS were performed to characterize the structure of products. The resultsshow that laccase is an effective bio-catalyst that can oxidize OP to generate DAP. Theconversion rate of OP was relatively high (almost100%) under mild conditions with oxygen.The structure of products was confirmed according to the results of UV-Vis, FTIR,2D-NMRand MS. The yield of crude DAP was85%. After purification, the yield of DAP was63%,higher than the HRP-catalyzed product yield.Investigation of SPORL process parameters suggests that the heating determines therejects rate while the holding time determines the hydrolysis performance of pretreated fibers.The homogeneity of pretreated solids plays an important role in the enzymatic hydrolysis ofSPORL biomass. After cellulose conversion, enzyme residual lignin (ERL) was obtained,which can be catalyzed together with chitosan, gelatin, and catechol by laccase to formhydrogels complex. The lignin-based hydrogels show strong water absorption capacity, e.g.one gram of hydrogels that contains0.2%lignin absorbs47.2g water. Moreover, the presenceof lignin prevents the loss of water from hydrogels when heating. Lignin-based hydrogelshave anti-oxidant capacities, e.g. over92%of ABTS radicals were scavenged in30min. Thepresence of lignin improves the reduction of ABTS radicals. The presence of lignin alsoimproves the protein absorption capacities of hydrogels. A maximum amount reached63mg/gusing BSA as a standard protein. Even if lowering the content of catechol, the proteinabsorbance can also be improved by addition of more lignin. The lignin-based hydrogelssignificantly denature laccase, as a result of30min treatment,99.5%of laccase activity waslost.