Abstract
BackgroundCost-effective production of industrially important enzymes is a key for their successful exploitation on industrial scale. Keeping in view the extensive industrial applications of lignin peroxidase (LiP), this study was performed to purify and characterize the LiP from an indigenous strain of Trametes versicolor IBL-04. Xerogel matrix enzyme immobilization technique was applied to improve the kinetic and thermo-stability characteristics of LiP to fulfil the requirements of the modern enzyme consumer sector of biotechnology.ResultsA novel LiP was isolated from an indigenous T. versicolor IBL-04 strain. T. versicolor IBL-04 was cultured in solid state fermentation (SSF) medium of corn cobs and maximum LiP activity of 592 ± 6 U/mL was recorded after five days of incubation under optimum culture conditions. The crude LiP was 3.3-fold purified with specific activity of 553 U/mg after passing through the DEAE-cellulose and Sephadex-G-100 chromatography columns. The purified LiP exhibited a relatively low molecular weight (30 kDa) homogenous single band on native and SDS-PAGE. The LiP was immobilized by entrapping in xerogel matrix of trimethoxysilane (TMOS) and proplytetramethoxysilane (PTMS) and maximum immobilization efficiency of 88.6% was achieved. The free and immobilized LiPs were characterized and the results showed that the free and immobilized LiPs had optimum pH 6 and 5 while optimum temperatures were 60°C and 80°C, respectively. Immobilization was found to enhance the activity and thermo-stability potential of LiP significantly and immobilized LiP remained stable over broad pH and temperature range as compare to free enzyme. Kinetic constants Km and Vmax were 70 and 56 μM and 588 and 417 U/mg for the free and immobilized LiPs, respectively. Activity of this novel extra thermo-stable LiP was stimulated to variable extents by Cu2+, Mn2+ and Fe2+ whereas, Cystein, EDTA and Ag+ showed inhibitory effects.ConclusionsThe indigenously isolated white rot fungal strain T. versicolor IBL-04 showed tremendous potential for LiP synthesis in SSF of corncobs in high titters (592 U/mL) than other reported Trametes (Coriolus, Polyporus) species. The results obtained after dual phase characterization suggested xerogel matrix entrapment a promising tool for enzyme immobilization, hyper-activation and stabilization against high temperature and inactivating agents. The pH and temperature optima, extra thermo-stability features and kinetic characteristics of this novel LiP of T. versicolor IBL-04 make it a versatile enzyme for various industrial and biotechnological applications.
Highlights
Cost-effective production of industrially important enzymes is a key for their successful exploitation on industrial scale
In our previous studies [2,4,15,16], we have successfully investigated the extracellular ligninolytic enzymes (MnP, lignin peroxidase (LiP) and laccase) synthesis potential of different locally isolated indigenous White rot fungi (WRF) strains including Trametes versicolor IBL-04 under solid and liquid state fermentation based on varying lignocellulosic substrates and dye decolorization
Maximum LiP activity of 592 ± 6 U/mL was recorded when corncobs supplemented with glucose and yeast extract in 25:1 C/N ratio, 1 mL of 1% tween-80 as surface active agent, and ZnSO4 as metal ion source was inoculated with 5 mL freshly prepared spore suspension of T. versicolor IBL-04 and fermented for 5 days under still culture solid state fermentation (SSF) conditions at 30°C
Summary
Cost-effective production of industrially important enzymes is a key for their successful exploitation on industrial scale. Lignin modifying enzymes (LMEs) are rarely produced by bacteria, yeasts and most fungi but frequently occur in the fermented culture broth of white rot fungi [1,2]. White rot fungi (WRF) are so far exclusive in their potential to entirely degrade all the components of lignocellulosic materials and this capability is due to their extra cellular nonspecific LMEs which function together with H2O2 and secondary metabolites. Veratryl alcohol a secondary metabolite produced by ligninolytic WRF plays an important role in LiP catalysis. LiPs from various WRF including Trametes versicolor and Pleurotus ostreatu differ from the other oxidoreductases in that they have low pH optima varying between pH 2–5 and much higher redox potentials [7]
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