Characterisation of a Novel White Laccase from the Deuteromycete Fungus Myrothecium verrucaria NF-05 and Its Decolourisation of Dyes

A novel ‘white’ laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml−1 on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na+, Mn2+, Cu2+ and Zn2+ while inhibited by DTT, NaN3 and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications

Determination of Total Polyphenol Content in Food with the Flow-Injection

U ovom radu predložena je modificirana automatizirana metoda ubrizgavanja u protok za određivanje sadržaja ukupnih polifenola u namirnicama bazirana na Folin-Ciocalteauovoj reakciji u 0,5 mol L-1 NaOH. Metoda omogućuje automatiziranu analizu različitih uzoraka brzinom protoka 55 uzoraka na sat uz upotrebu galne kiseline kao standarda. Primjenom predložene metode na konkretne uzorke (bijelo i crno vino, zeleni, indijski te čaj od lipe, metvice i kamilice i bistri voćni sokovi od crnog ribiza i višnje), određen je njihov “indeks ukupnih polifenola” s većom repetibilnošću za razliku od ranije objavljenih metoda, manje ovisno o razrjeđenju uzorka. U odnosu na “batch” metodu, ova je metoda visoko tolerantna prema najčešćim interferentima (SO2, reducirajući šećeri i askorbinska kiselina). Rezultati dobiveni predloženom metodom pokazali su relativno slaganje s onima dobivenim referentnom Folin-Ciocalteauovom metodom.This paper describes an optimised flow-injection method for the determination of total polyphenol in food based on the Folin-Ciocalteau reaction in 0.5 mol L-1 NaOH. The method allows different types of samples to be analysed automatically at a rate of 55 samples per hour by using gallic acid as standard. By applying the proposed method to real samples (white and red wines, green, Indian, lime-tree, mentha and chamomile teas, and blackberry and cherry juices), their total polyphenol indices were determined with a higher reproducibility than obtained by earlier methods, whatever the dilution used. This method is highly tolerant towards the most common interferences (SO2, reducing sugars, and ascorbic acid) associated with the batch method. The results obtained by the proposed method relatively agree with those obtained using the referent Folin-Ciocalteau method