LIGNOCELLULOSE WASTE VALORIZATION BY AN ENZYMATIC COCKTAIL OF P. ERYNGII AND P. PULMONARIUS

The present study aimed to characterize Pleurotus eryngii and P. pulmonarius ligninolytic enzymes and to determine their potential for polymer degradation in common agroforestry residues. The peak of laccase activity (36052.33 U L-1) was observed after P. pulmonarius cultivation on oak sawdust. The maximal Mn-dependent peroxidase activity was reached by P. eryngii (2511.36 U L-1), while the highest level of versatile peroxidase activity was noted in P. pulmonarius (3053.03 U L-1), after fermentation of corn stalks. The highest level of lignin loss (46.28%) was achieved after cultivation of P. pulmonarius on corn stalks, but the most selective degradation of lignocellulose polymers was observed after P. eryngii cultivation on wheat straw. The obtained results lead to the conclusion that the studied P. eryngii and P. pulmonarius strains are good producers of ligninolytic enzymes and effective and selective depolymerizers of agroforestry residues, and therefore their use would be beneficial in numerous environmentally friendly technologies.

Functional Characterization of Melanin Decolorizing Extracellular Peroxidase of Bjerkandera adusta

Melanin pigmentation in the human skin results from complicated cellular mechanisms that remain to be entirely understood. Uneven melanin pigmentation has been counteracted by inhibiting synthesis or transfer of melanin in the skin. Recently, an enzymatic approach has been proposed, wherein the melanin in the skin is decolorized using lignin peroxidase. However, not many enzymes are available for decolorizing melanin; the most studied one is lignin peroxidase derived from a lignin degrading fungus, Phanerochaete chrysosporium. Our current study reveals that versatile peroxidase from Bjerkandera adusta can decolorize synthetic melanin. Melanin decolorization was found to be dependent on veratryl alcohol and hydrogen peroxide, but not on Mn2+. The degree of decolorization reached over 40% in 10 min at 37 °C and a pH of 4.5. Optimized storage conditions were slightly different from those for the reaction; crude enzyme preparation was the most stable at 25 °C at pH 5.5. Since the enzyme rapidly lost its activity at 50 °C, stabilizers were screened. As a result, glycerol, a major component in several cosmetic formulations, was found to be a promising excipient. Our results suggest that B. adusta versatile peroxidase can be considered for future cosmetic applications aimed at melanin decolorization.

A Versatile Peroxidase from the Fungus Bjerkandera adusta Confers Abiotic Stress Tolerance in Transgenic Tobacco Plants

White-rot fungi are efficient lignin degraders due to the secretion of lignin peroxidase, manganese peroxidase, laccase, and versatile peroxidase (VP) on decayed wood. The VP is a high-redox-potential enzyme and could be used to detoxify reactive oxygen species (ROS), which accumulate in plants during biotic and abiotic stresses. We cloned the VP gene and expressed it via the Agrobacterium transformation procedure in transgenic tobacco plants to assay their tolerance to different abiotic stress conditions. Thirty independent T2 transgenic VP lines overexpressing the fungal Bjerkandera adustaVP gene were selected on kanamycin. The VP22, VP24, and VP27 lines showed significant manganese peroxidase (MnP) activity. The highest was VP22, which showed 10.87-fold more manganese peroxidase activity than the wild-type plants and led to a 34% increase in plant height and 28% more biomass. The VP22, VP24, and VP27 lines showed enhanced tolerance to drought, 200 mM NaCl, and 400 mM sorbitol. Also, these transgenics displayed significant tolerance to methyl viologen, an active oxygen-generating compound. The present data indicate that overproducing the VP gene in plants increases significantly their biomass and the abiotic stress tolerance. The VP enzyme is an effective biotechnological tool to protect organisms against ROS. In transgenic tobacco plants, it improves drought, salt, and oxidative stress tolerance. Thus, the VP gene represents a great potential for obtaining stress-tolerant crops.

Augmenting versatile peroxidase production from Lentinus squarrosulus and its role in enhancing ruminant feed

Lentinus squarrosulus, a white-rot Basidiomycete, produces versatile peroxidase under nutrient-limiting conditions. This study was undertaken to enhance the yield of versatile peroxidase from the fungus by optimizing the physiological factors of nutrients and their concentration, inducers, temperature, and pH. Optimal medium was devised using a central composite design – response surface methodology and experimental validation. The fungus produced high levels of versatile peroxidase at a C:N ratio of approximately 100, temperature of 35 °C, and initial pH of 7. Immobilization of Lentinus squarrosulus on inert polyurethane foam (PUF) with optimized medium for production enhanced the versatile peroxidase yield multifold. Maximal yield of versatile peroxidase achieved through optimization and immobilization strategies was 116 U/mL. Efficacy of crude versatile peroxidase on aromatics degradation was illustrated through Fourier transform infrared analysis. The results demonstrated the improvised yield of versatile peroxidase from Lentinus squarrosulus and its efficiency in delignification of crop residues, substantiating the role of this enzyme in biotechnological applications especially for enriching ruminant feed.

Purification and characterization of versatile peroxidase from Lentinus squarrosulus and its application in biodegradation of lignocellulosics

Versatile Peroxidases are high redox potential peroxidases capable of degrading lignin of lignocellulosic crop residues. Hence Versatile Peroxidases are prominent biocatalysts in upgrading lignocellulosic biomass for biotechnological applications. In the interest of exploiting the potential of Versatile Peroxidase in improving the digestibility of crop residues through delignification, a novel Versatile Peroxidase was purified and characterized from the immobilized cultures of native isolate Lentinus squarrosulus. The enzyme was purified with a specific activity of 62 U/mg through ion exchange and gel filtration chromatographic procedures. The enzyme possessed high affinity towards RB5 and manganese with a Km value of 6.84 µM for RB5 and 0.15 mM for manganese. The optimum temperature for oxidation was identified to be 30°C and optimum pH for manganese and RB5 oxidation was 5 and 3 respectively. Reactivity of the enzyme towards diverse substrates was investigated besides studying the effect of metal ions and inhibitors on RB5 oxidation. The enhanced potential of this purified Versatile Peroxidase in biodegradation of crop residues was demonstrated through augmentation of digestibility of finger millet and paddy straws by 20%.The results demonstrated that Versatile Peroxidase from Lentinus squarrosulus is capable of enhancing the nutritive value of crop residues through delignification