Screening of Schizophyllum commune strains for laccase and peroxidase production

Abstract

The study entitled “Screening of Schizophyllum commune strains for laccase and peroxidase production” was conducted at College of Agriculture, Vellayani during 2023-2024. The objectives of the study were to evaluate three strains of S. commune for their laccase and peroxidase activity during submerged and solid-state cultivation; and expression profiling of the best strain for its lignolytic potential at different developmental stages using quantitative Real-Time PCR. The three strains of S. commune viz., DMRX-2156, DMRX-2157 and DMRX-2160 were procured from All India Coordinated Research Project (AICRP) on Mushrooms, Department of Plant Pathology, College of Agriculture, Vellayani and maintained on potato dextrose agar medium. Among the S. commune strains, DMRX-2160 took the minimum period of 9.23 days for complete coverage on petridish with maximum radial mycelial growth (0.97 cm/day). The mycelial growth was white, thick and fluffy with smooth margins. Hyphae of Schizophyllum strains were septate, branched, hyaline, of average size 2.1μm with distinct clamp connection and spicules. All the three tested strains reacted positively to the qualitative plate assay for laccase and peroxidase activity. DMRX-2160 showed the highest efficiency of laccase production in growth media supplemented with 0.04% guaiacol and gave the maximum ratio of reddish zone diameter (Z) to colony diameter (C) value as 0.86. Addition of 1% pyrogallol and 0.4% hydrogen peroxide mixture to fully grown cultures of Schizophyllum strains resulted in development of yellowish brown colour, thus confirming its peroxidase activity. Quantitative estimation of laccase and peroxidase enzymes from mycelia and broth of S. commune submerged cultures in three different media viz., Stock Basal Media (SBM), Potato Dextrose Broth (PDB) and Potato Malt Peptone Broth (PMPB) was done using substrates viz., guaiacol and pyrogallol for laccase and peroxidase, respectively. DMRX-2156 recorded the highest intracellular laccase activity (30.55±3.66 U/g) in PMPB and peroxidase activity (242.34±36.66 U/g) in PDB at 5 days after inoculation (DAI) whereas, DMRX-2160 recorded the highest extracellular laccase activity (15.58±0.74 U/ml) in SBM and peroxidase activity (52.10±1.34 U/ml) in PMPB at 15 DAI. DMRX-2160 took the minimum time for mycelial run in paddy grains (13.70 days) and paddy straw (3.10 days) when used as spawn and bed substrates, respectively. DMRX-2160 took the minimum time for pin head formation (4.10 days) and first harvest (15.33 days), with maximum yield (11.29 g/Kg) and biological efficiency (1.13%), when paddy straw was used as bed substrate. The pinheads of S. commune strains were formed 3-6 days after spawning and the sporocarps took an average of 12 days from the day of pinhead formation to complete maturity. When the mushroom attained full maturity the basidiocarp became differentiated into pilear region, gills on underside were split into midregion and the cap was multistratous. Its stipe became rudimentary or even absent. Quantification of laccase and peroxidase activity during solid state cultivation revealed that DMRX-2160 showed the maximum intracellular laccase (36.39±0.78 U/g) and peroxidase activitiy (605.67±40.82 U/g) at mycelial stage however, the activities declined over the subsequent stages. DMRX-2160 showed the maximum extracellular laccase (15.16±2.59 U/ml) and peroxidase (121.13±5.40 U/ml) activities at young fruiting body stage. Thus, DMRX-2160 was identified as the best S. commune strain, in terms of maximum extracellular enzyme activities during submerged cultivation and solid-state cultivation, as well as maximum yield. The change in lignolytic activities over the growth stages starting from mycelial run to sporophore formation was further analyzed by gene expression studies of DMRX-2160 cultivated in paddy straw using Real time PCR. Laccase and peroxidase genes were found maximally expressed in the mycelium of DMRX- 2160 (42.52-fold for laccase and 4.86-fold for peroxidase than young fruiting body stage). The expression of these genes showed downregulation over the subsequent developmental stages with its lowest expression in young fruiting body stage. Gene expression data showed a similar trend to enzyme assays. Thus, the present study identified S. commune strain DMRX-2160 as a promising strain which can utilise paddy straw as substrate for fruiting body production with maximum biological efficiency, as well as a prospective enzyme source for the extraction of hydrolytic enzymes having biotechnological and industrial applications.