Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

Effect of Different Kinds of Substrates on the Growth and Yield Performance of Pleurotus sapidus (Oyster Mushroom)

Cultivation of edible fungi (mushrooms) is a useful method for bio-conservation in the agriculture industry. For the better production of mushrooms, we used the agricultural waste material as a substrate for the cultivation of mushrooms. After mushroom harvesting, these substrates are available as an excellent source of soil conditioner. In this study, we used the sawdust of a simbal tree (Bombax ceiba L.), office scrap paper, and poultry manure as a substrate to cultivate the Pleurotus sapidus (oyster mushroom). Data recorded from the inoculation of spawn to the harvesting of fruiting bodies by using different parameters. The results revealed that the Treatment-T4 (sawdust of simbal tree 50% + poultry manure 50%) required the least number of days (16 days) for 100% spawn-running, development of pinhead (6.5 days), and fruiting bodies (5.5 days), produced the maximum number of fruiting bodies (24.25), maximum yield (388.40 g), and highest biological efficacy (77.68%). The Treatment-T5 (office scrap paper 50% + poultry manure 50%) took the maximum number of days (32 days) for 100% spawn-running, development of pinhead (18 days) and fruiting bodies (17 days), produced least number of fruiting bodies (9.5), minimum yield (224.50 g), and lowest biological efficacy (44.90%). Therefore, the cultivation of oyster mushrooms is an excellent or environment-friendly technique because the substrate used for mushroom cultivation is economical and readily available.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of β-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.