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.
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.
Media components for the production of lignolytic enzyme MnP in the submerged fermentation from Clitopilus scyphoides were screened by PB design of experiments using main effects of different components and their percent contribution towards the MnP production. Total 31 components were considered in this study at temperature 30°C and incubation period of 5 days, in which Galactose, Malt extract, KH2 PO4 , NH4 H2 PO4 , CaCO3 , Tween80, Tartaric acid and sodium acetate were found to be significant components for MnP production. Further, response surface methodology was employed to optimize the media containing screened components along with temperature, pH and incubation days. The optimized medium contained 1.5% Galactose, 0.06%Malt extract, 0.02%KH2 PO4, 0.15%NH4 H2 PO4 , 0.003%CaCO3, 0.8%Tween80,0.005% MnSO4,0.88%Tartaric acid and 3% sodium acetate at temperature 31°C and pH 5.2 incubated for 5 days increased MnP productivity by 43 folds (43.2±1.29 U mL-1)when compared to un-optimized media (1.1±0.087 U mL-1). MnP produced was tested for lignolytic effects on different crop residues. Digestibility studies of different crop residues treated with crude MnP rich extracts indicated that a significant decrease in the percentage value of lignin and increase in in-vitro digestibility matter (at confidence interval 95%) when compared with untreated straws.
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