Enhancing Production of Lignin Peroxidase from White Rot Fungi Employing Statistical Optimization and Evaluation of its Potential in Delignification of Crop Residues

Abstract

A high demand for fungal lignin peroxidases on account of their innumerable biotechnological applications necessitates enhanced production This paper describes the enhanced production of lignin peroxidase by three isolates (LPS1, LPS2 and LPS3) of white rot fungi for delignification of crop residues. Initial screening of medium components was performed using a Plackett-Burman design and the variables with statistically significant effects such as pH, temperature, carbon source, nitrogen source, inoculums size, incubation period, inhibitors on lignin peroxidase production were identified. The optimized temperature was 30 0 C with pH 3 for LiP production in all three isolates (LPS1, LPS2 and LPS3). Veratryl alcohol proved to be the best substrate for lignin peroxidase production. The best carbon source and nitrogen sources were glucose and sodium nitrite respectively. The lignin peroxidase activity was found to be maximum in LPS1 (280 µmoles/min), followed by LPS2 (233 µmoles/min) and LPS3 (220 µmoles/min). These variables were selected for further optimization studies of lignin peroxidase production by LPS1 using Response Surface Methodology. Optimized conditions for the production of LPS1 using design expert software were temperature of 30 0 C, pH of 4.2, glucose at 12 g concentration, Veratryl alcohol and H 2 O 2 of 100mM and 0.1mM concentrations respectively. The maximum LiP activity obtained by statistical optimization for LPS1 was 349.3 units which was 21% higher in comparison to LiP activity with unoptimized medium. Validation experiments proved that experimentally determined production values of 328.63 units obtained for LPS1 were in close agreement with statistically predicted ones, confirming the reliability of the model. Treatment of nine crop residues with lignin peroxidase produced under optimized conditions showed a reduction in lignin content ranging between 0.21 and 0.94 and increase in vitro dry matter digestibility ranging between 0.45 and 2.76, proving its delignification potential. K e y w o r d sLignin peroxidase, White rot fungi, Statistical, Optimization, Delignification

Lignin‐modifying enzymes: a green and environmental responsive technology for organic compound degradation

“…Also, it is characterized by its high redox potential (about 1.2 V at pH 3) in the presence of H 2 O 2 , which allows the oxidation of various aromatic and non‐phenolic compounds, with or without mediators 33,74 . In addition to lignin depolymerization, LiP is active in delignification due to its efficiency in removing lignin from lignocellulosic biomass 79,80 . In general, what differentiates LiP from classical peroxidases is the fact that LiP oxidizes aromatic rings that are activated by electron‐donating substrates, whereas classical peroxidases act only on already activated aromatic substrates 81 …”

A Secreted Lignin Peroxidase Required for Fungal Growth and Virulence and Related to Plant Immune Response

“…They belong to the auxiliary activities family 2 (AA2), which were first described in Phanerochaete chrysosporium [ 41 ]. However, most of them are focused on the biochemical function of enzyme activity, e.g., P. chrysosporium [ 42 , 43 , 44 ], Pseudomonas aeruginosa , and Serratia marcescens [ 45 ]. Among ligninolytic enzymes, only a few characterized as virulence factors, including laccase (Lac) and manganese peroxidase (MnP), e.g., Lac2 , a laccase gene that is involved in the appressorial melanization of Colletotrichum orbiculare [ 46 ], and MnP, have a function in fungal growth and development and stress response in Trametes trogii [ 47 ].…”