A laccase-producing hyper performer, Schizophyllum commune, a white-rot fungus, was evaluated for its ability to selectively degrade lignin of diverse crop residues in vitro. Relative analysis of crop residue treatment using laccase obtained from immobilized cells demonstrated degradation of 30–40% in finger millet straw and sorghum stover, 27–32% in paddy straw, 21% in wheat straw, and 26% in maize straw, while 20% lignin degradation was observed when purified and recombinant laccase was used. Further investigations into in vitro dry matter digestibility studies gave promising results recording digestibility of 54–59% in finger millet straw 33–36% in paddy straw and wheat straw, 16% in maize straw for laccase obtained from cell immobilization method, whereas 14% digestibility was observed when purified and recombinant laccase was used. Sorghum stover recorded digestibility of 13–15% across all straws treated with laccase. The results obtained elucidated the positive influence of laccase treatment on lignin degradation and in vitro dry matter digestibility. The present research gave encouraging figures confirming the production of laccase using the cell immobilization method to be an efficient production method commensurate with purified and recombinant laccase under conditions of submerged cultivation, proclaiming a cost-effective, environmentally safe green technology for effectual lignin depolymerization.

White rot fungi (WRF) produce lignolytic enzymes comprised by laccases and peroxidases responsible for mineralization of recalcitrant lignin. Because of the so-called lignin modifying enzymes(LME’s), these fungi have potential applications in biodegradation and bioremediation processes. Increased demand for lignolytic enzymes to exploit their various applications has sparked interest in identifying and characterizing new novel strains of WRF. Despite this undisputed biotechnological significance, molecular identification of WRF, remains a daunting task for researchers as genomic DNA isolation is a tedious process, unsuccessful many a times because of their rigid and resistant cell walls. A rapid, effective and efficient method to identify the innumerable fungal strains within no time is the need of the hour. The fungal mycelia of various unknown as well as know isolates of WRF, after alternative washing with TE buffer and sterile water were suspended in TE buffer. Fungi in solution were then exposed to microwave. The crude extract contained genomic DNA which was extracted and amplified using ITS primers for further identification. Based on sequencing results the identity of known cultures was confirmed, while the unknown cultures were identified as Clitopilus scyphoides (AGUM004, BankIt2098576 MH172163); Ganoderma rasinaceum (AGUM007, BankIt2098576 MH172163); Schizophyllum sp (KONA001 BankIt2098576 MH172164; AGUM011 BankIt2098576 MH172165and AGUM021 BankIt2098576 MH172166respectively), Coprinellus disseminatus (BANG001, BankIt2098576 MH172167) and Lentinus squarrosulus (TAMI004, BankIt2098576 MH172167). The microwave method described for isolating quality DNA of WRF without further purification steps proved a novel method requiring less than ten minutes and minimized the chances of the presence of PCR inhibitors.IMPORTANCEWhite rot fungi which decay wood, possess selective lignin degrading enzymes responsible for degrading a wide variety of environmental pollutants, xenobiotic compounds in addition to mineralizing chemicals that are insoluble and recalcitrant. Lignolytic enzymes hold potential towards replacing conventional chemical processes and their increased demand in the market has ignited interest in identifying and characterizing new strains of WRF. A rapid, efficient method capable of quickly identifying fungal isolates is a constraint. The microwave method is a novel quick method for isolating superior quality DNA. Its adoption circumvents the initial purification steps and /or interference of PCR inhibitors, which are encompassed in the use of conventional methods. The microwave method thus permits the thorough amplification of the ITS region thereby aiding in the easy identification of unknown species. Use of the microwave method will permit researchers to obtain DNA from fungi very quickly for further application in molecular studies.

Manganese peroxidase (MnP), a crucial enzyme in biodegradation of lignin, is synthesized by most white rot fungi. To obtain novel enzymes with superior biodegradation potential, MnP-producing wild isolates were evaluated for their ability to degrade recalcitrant azo dyes, sulfonephthalein dyes, and kraft lignin. Of 30 wild isolates screened, 18 tested positive for lignin modifying enzymes (LMEs). Thirteen of these isolates were positive for both laccase and MnP, whereas four produced only laccase, and one produced lignin peroxidase alone. The isolates were identified as Clitopilus scyphoides MH172162 (AGUM004), Ganoderma rasinaceum MH172163 (AGUM007), and three Schizophyllum species: MH172164, MH172165, and MH172166 (KONA001, AGUM0011, and AGUM021). The Fourier-transform infrared spectroscopy (FTIR) analysis of dye degradation and kraft lignin samples with AGUM004 and AGUM007 revealed biotransformation. The former could not completely degrade Reactive Black 5 and bromocresol green, but it could completely (100%) decolorize bromophenyl blue, bromothymol blue, and Remazol brilliant blue R. The latter efficiently degraded almost all tested dyes. Both degraded kraft lignin. The screened hyper MnP-producing wild AGUM004 and AGUM007 were shown to be potential dye degraders in addition to having lignin degrading abilities.

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

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