Improvement of recombinant L-Asparaginase production in Pichia pastoris

Pichia pastoris is a successful expression system that is frequently preferred in the secretion of proteins for both basic research and industrial purposes. In this study, recombinant Rhizomucor miehei (RmASNase) L-asparaginase was produced in Pichia pastoris. The impact of gene copy number on increasing protein production was examined with six clones harboring various gene copy numbers (1-5 and 5 +). The results demonstrated that the clone with three copies of the expression cassette integrated had the highest production level. Also, biochemical characterization of the enzyme was performed. It was determined that the optimum pH and temperature values of the purified enzyme were pH 7.0 and 50 °C, respectively. Stability analyses of the enzyme showed that it maintains its activity of 80% in the pH range of 5-9 and 67% in the temperature range of 20-50 °C. Ca⁺² and Mn⁺² ions increased the enzyme activity to 121% and 138%, respectively. In future studies, it is also possible to improve the activity and stability values of the enzyme with advanced molecular techniques and to increase production efficiency by producing at fermenter scale and under optimum conditions.