Yasemin Alan

Anaerobic fungi play a key role in microbial communities driving the anaerobic digestion process for biogas production. Biogas, as a versatile and sustainable technology, addresses waste management and meets energy demands, serving as an eco-friendly alternative to natural gas.

Anaerobic fungi enzymes contribute to breaking down complex organics, and understanding these enzymes aids in optimizing the anaerobic digestion process, improving substrate breakdown, and enhancing overall biogas production efficiency.

This study aims to investigate the recombinant expression and characterization of literature-known enzymes from various anaerobic fungi. To achieve this, six specific enzymes were identified from the literature and procured as synthetic genes, intended for subsequent recombinant expression in E. coli. These enzymes, renowned for their capability to degrade complex polysaccharides present in plant cell walls, hold significant value in the efficient degradation of agricultural residues. Following the cloning and expression studies of the genes encoding these specific enzymes, the purification step was executed, followed by conducting

enzyme activity assays. In the course of enzyme characterization and optimization studies, the impact of varying substrate concentrations, metal ions, pH, and temperature on enzyme activity was investigated. The findings revealed that xylose isomerase enzyme from Piromyces sp. strain E2 exhibited a peak activity at a pH of 6.85 (0.0047 μmol/min) and a temperature of 60°C (0.0029 ± 0.0002 μmol/min). In the metal ion effect test, the enzyme exhibited sufficient activity under the tested conditions in the presence of two out of three divalent metal ions (Co²⁺, Mg²⁺,

Mn²⁺). Additionally, the lichenase enzyme from Orpinomyces sp. strain PC-2 demonstrated an optimal pH range of 5.0 to 6.0, with an average activity of 0.0413 μmol/min. These findings highlight the optimal conditions for these enzymes, contributing to the improvement of biogas production within industrial processes.