Lignin peroxidase (LiP), which has been studied extensively in white-rot Basidiomycetes with regard to bio-pulping and bio-bleaching, plays a role in the biodegradation of plant cell wall lignin. In the current study, LiP obtained from a wild isolate of Phanerochaete chrysosporium immobilized on polyurethane foam cubes was purified 21-fold using ammonium sulphate precipitation and size exclusion chromatography. The enzyme with a molecular mass of 55 kDa exhibited a considerably higher pH tolerance and thermostability compared with the native enzyme. It showed a strong affinity for the substrate veratryl alcohol and had kinetic constant values of 142.86 µmol and 65 µM. Cysteine, sodium azide, mercaptoethanol, and silver nitrate inhibited the activity, while ethanol, EDTA, Cu2+, Mn+, Na+, and Fe2+ exhibited induction. Purified LiP completely decolorized (100%) bromo phenyl blue, bromothymol blue, and bromocresol green. The 96 and 72% degradation obtained with phenol and congo red was also higher compared to crude LiP. Treatment with LiP showed reduction in ADL as compared to untreated straws, with a maximum of 2.87 units obtained in JR followed by 2.66 units in PS. The digestibility of all straws increased, the response varying from a maximum of 21.27 units in PRM to a minimum of 12.32 units obtained in LM.

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

The scarcity of quality feed is a major constraint to livestock productivity, and it is attributed to recalcitrant lignin, which hinders the utilization of crop residues. White-rot lignicolous fungi have been used for enrichment of crop residues through fungal pre-treatment, but dry matter loss is inevitable. Versatile peroxidase oxidizes a diverse class of aromatics without mediators and plays a key role in ligninolysis. In this study, the efficacy of a novel versatile peroxidase isolated from a wild species was evaluated for its effect in the delignification of crop residues. The strain identified as Lentinus squarrosulus (TAMI004, BankIt2098576 MH172167) demonstrated predominant versatile peroxidase activity amongst screened isolates in solid-state and submerged fermentations; it displayed hybrid characteristic of manganese oxidation and manganese independent reactions on aromatic compounds. The manganese oxidizing peroxidase activity was 12 U/mL in submerged and 131 U/mL in solid-state fermentation. Treatment of crop residues with L. squarrosulus extract rich in versatile peroxidase showed a decrease in neutral detergent fiber, acid detergent fiber, and acid detergent lignin contents, prompting delignification. Thus, the use of versatile peroxidase in enhancing the digestibility of straws is substantiated through proximate and in vitro digestibility analysis. Thus, the potential use of versatile peroxidase in increasing the in vitro degradation of straws for enhancing feed utilization in ruminants was substantiated.

36Scarcity of quality feed is a major constraint concerning livestock productivity with recalcitrant 37 lignin hindering utilization of crop residues as quality animal feed. Degradation of lignin in