Enzyme activity screening methods for fungal solid-state fermentation

Abstract

Solid state fermentation is a technique where the fermentation is performed in moist conditions of a solid substrate with little to no free water present. Solid state fermentation can be performed especially well with fungi. The conditions of solid-state fermentation mimic the natural growth environment of filamentous fungi. Fungal solid-state fermentations can be utilized in production of enzymes. Enzymes have a significant market and are used in various applications from pulp and paper processes to pharmaceuticals. Enzymes are catalysts that lower the reaction’s activation energy. Their activity depends on multiple environmental factors such as temperature and pH. When conducting enzyme activity assays these factors must also be considered. The aim of the study was to provide a starting point for enzymatic screening methods for filamentous fungi strains that were cultivated in solid-state fermentation. These methods included API ZYM assay in addition to glutaminase and protease enzyme activity assays. Solid state fermentations were conducted in trays with substrate sizes of 200 g and 250 g with two substrate alternatives of pea side stream with oat flakes and brewers spent grain with faba bean ground. Fermentation samples were used in enzyme activity assay development and in API ZYM assays. Three substrates, casein, hemoglobin and azocasein, were experimented with in protease method development. While glutaminase method was conducted with glutamine substrate. Enzyme activities were determined with appropriate kits. Protease activity assay revealed to be time consuming and challenging. Inconclusive results were observed with casein substrate, while hemoglobin was only briefly experimented with due to dissolution issues. However, azocasein was found to be a functional substrate for protease assay. It was further assessed with positive protease control samples to determine a functional reaction concentration simultaneously to reduce formation of solid particles observed from the reaction. Glutaminase assay was found to be functional from the beginning and it was able to provide preliminary results of glutaminase activities. Glutaminase activities were observed to increase throughout fermentation, specifically from hour 17 to 42 with few exceptions. However, it was discovered that the glutamine reaction concentration was too low for the activity assay and in few assays nearly all substrate was hydrolyzed. Overall, the study was able to provide a starting point for enzyme activity method development and clear path for further development. In future, the reaction concentration of both assays must be further assessed, for azocasein method the protein concentration of extraction solution needs to be accounted for and overall notable increase in needed for the glutaminase method.