Optimizing agro-waste substrates for enhanced enzymatic production in ethnomedicinal mushroom Lentinus squarrosulus Mont.

The ethnomedicinal subtropical mushroom Lentinus squarrosulus Mont. is a polypore with high wood-degrading potential. This study aimed to collect L. squarrosulus isolates, analyze lignocellulolytic enzymes - Laccase (Lac), Lignin peroxidase (LiP) and Manganese peroxidase (MnP) and evaluate substrates for mass production. Sporophores of Lentinus sp. (Isolate L1 to L5) were collected from different regions of Tamil Nadu and characterized morphologically and molecularly. Qualitative screening tests with guaiacol substrate demonstrated the production of laccase by all the isolates; however, isolate L3 showed maximum reddish-brown zonation. Additionally, isolate L3 produced the highest green colouration on the ABTS (2,2’-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid) substrate. This confirmed the oxidation of ABTS to ABTS-azine in the presence of laccase, outperforming the other L. squarrosulus isolates. All the isolates tested with Azure-B agar for LiP and MnP showed faint discolouration, suggesting lower secretion of peroxidase enzymes. Quantitative analysis using a lignolysis basal medium also confirmed that isolate L3 secreted Lac predominantly, followed by LiP and MnP enzymes extracellularly. All isolates showed peak Lac activity at pH 4.5, while LiP and MnP production were highest at pH 5.0. The optimal temperature for all lignolytic enzymes was 28±2ºC. Maximum laccase secretion occurred between the 5th - 7th day after inoculation (DAI), with LiP and MnP peaking at 7 DAI. Nine agro-waste substrates were evaluated, with isolate L3 showing the highest biological efficiency on Paddy straw (99.62%), followed by Arecanut sheath (97.43%) and Groundnut shell (95.15%). This study enhances our understanding of L. squarrosulus enzymatic capabilities. It provides insights for mass production, which has strong potential for industrial bioremediation and waste management due to its high laccase production.