Screening, enzyme optimization and identification of ligninolytic fungi: A focus on Cladosporium tenuissimum

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Lignin-modifying enzymes (LMEs) produced by fungi, play a crucial role in the degradation of lignocellulosic biomass and have considerable potential in biotechnological applications. The present study aimed to isolate and characterize a fungal strain capable of producing ligninolytic enzymes and to determine the optimal conditions for their production. Fungal isolates were screened for ligninolytic activity using qualitative plate assays with ABTS, methylene blue, and azure B. The most promising isolate was further evaluated for enzyme production under different temperatures, pH levels, and incubation conditions, followed by molecular identification using ITS-PCR amplification, sequencing, and phylogenetic analysis. Qualitative assays confirmed the production of laccase, LiP, and MnP by one isolate. Quantitative analysis revealed that the optimal temperature for enzyme production was 30 °C. Maximum laccase activity was observed at pH 6 (90.85 ± 0.12 IU mL-¹), whereas LiP and MnP showed highest activities at pH 4 (105.55 ± 0.07 IU mL-¹) and pH 5 (70.69 ± 0.20 IU mL-¹), respectively. Peak production of laccase and LiP occurred on the sixth day of incubation (108.55 ± 0.12 IU mL-¹ and 163.10 ± 0.11 IU mL-¹), while MnP activity reached its maximum on the eighth day (100.67 ± 0.18 IU mL-¹). ITS rDNA sequencing identified the isolate F18 as Cladosporium tenuissimum (accession number PV687687), which clustered closely with reference strains in the phylogenetic analysis.These findings highlight the potential of C. tenuissimum as a promising source of ligninolytic enzymes and provide insights into the optimal conditions for their production for lignocellulosic biomass degradation and related biotechnological applications.

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