Screening, Production and Partial Characterization of a Thermostable Laccase from Trametes sp. Isolate B7 with Biotechnological Potentials

Abstract

The search for efficient and green oxidation technologies has increased interest in utilization of laccases in non conventional methods. Laccases catalyze a wide range of substrates due to low substrate specificity and strong oxidative potentials. Challenges to large-scale enzyme utilization include, low enzyme activity and instability which restrict use in many areas of biotechnology. In the study, 59 fungi comprising Aspergillus niger (40%), Trichoderma harzianum (31%), Aspergillus flavus (9.0%), Trichoderma viride (5.0%), Fusarium oxysporum (5.0%), Rhizopus stolonifer (5.0%), Trametes sp. (3.0%) and Aspergillus nidulans (2.0%) were isolated and screened for laccase production. Plate screening test showed 57.5%, 34.0% and 8.5% of fungi were laccase-positive on ABTS, Guaiacol, and α-naphthol agar respectively. Isolates were further screened in liquid cultures, and the highest laccase producer identified molecularly. Trametes sp isolate B7 was selected for solid state fermentation (SSF). Laccase production in SSF was highest at pH 5.0 (2356 U/mL). The purified laccase showed high activity (pH 3.0 - 6.0) and stability (pH 3.0 - 8.5) using ABTS. It was active (20 - 80°C) and thermostable (30 - 80°C) with optimum stability at 70°C (100% for 1 hour). The percentage decolourization of Phenol red were 28% and 36% using 1000 U/mL and 2000 U/mL crude laccases respectively. Similarly, RBBR (100%), Congo red (75%) and Malachite green (62%), and 77.4%, 64% and 28% were decolourized using 1000 U/mL and 2000 U/mL crude laccases respectively. ABTS agar was very reliable in large-scale screening for laccase which possessed thermostable property and degraded synthetic dyes without use of enzyme mediators. These attribute made the enzyme suitable for application in industry and biotechnology.

Production of Fungal Laccase under Solid State Bioprocessing of Agroindustrial Waste and Its Application in Decolourization of Synthetic Dyes

“…In many studies, different chromogenic indicators such as Guaiacol, Tannic acid, α-Naphthol and ABTS have been used to detect production of laccases in cultures. The use of ABTS and Guaiacol during primary screening for laccase production provides rapid detection and confirmation of the enzyme [10]. It has been established that most laccases of fungal origin show higher sensitivity using ABTS as substrate compared to other chromogenic indicators such as Guaiacol and α-Naphthol [10,41].…”

“…Trametes versicolar, Trametes sp. isolate B7, Pleurotus ostreatus, Pleurotus eryngii, Agaricus blazei, and Clitocybe maxima secret high titres of laccases while Ganoderma lucidum efficiently produces laccases, manganese and lignin peroxidases [9,10].…”