Investigation of the effect of different carbon sources and media pH values on recombinant protein production of Pichia pastoris yeast

Fruits are high-value products that are favored by consumers due to their pleasant taste, aroma, and color. Fruits continue to respire and undergo biochemical reactions during post-harvest period wich lead to rapid spoilage and decay. Therefore frits should be processed into value-added products quickly. Among these products, fruit juice and concentrate production are the most prominent. Various cell wall- degrading enzymes are used in fruit juice production to increase juice yield during the pressing stage and to produce clear fruit juice. One of the proteins accompanying cell wall-degrading enzymes is the expansin protein. Expansin is a protein found in plants. As the expansin protein present in small amounts in plants, its extraction through classical methods requires increased cost and time. To produce expansin protein using modern biotechnological methods recombinant Pichia pastoris harboring the tomato LeExp1 gene was used in this investigation. This study aimed to determine the optimal fermentation conditions of the methylotrophic yeast P. pastoris. For this purpose, 16 experimental designs were established with glycerol, sorbitol, and mannitol as substitutes for methanol as the carbon source, and with medium pH values of 4.0, 5.0, 6.0, and 7.0, while maintaining a fermentation temperature of 30ºC for all designs. The results were compared to the fermentation conditions where methanol was used as the carbon source and the medium pH value was 6.0. To ensure the traceability and verifiability of the research results, biomass quantity, total protein concentration, RNA transcription, and Western Blot analyses were performed. As a result of the study, the biomass concentration was measured at 21,3 mg/mL and 34,7 mg/mL, and the total protein concentration was measured at 30,35 mg/mL and 30,00 mg/mL, respectively, when using sorbitol or glycerol with methanol as the carbon source and a medium pH value of 7.0 for recombinant expansin protein production by P. pastoris. RNA Transcription analysis revealed the presence of the LeExp1 gene on agarose gel at all incubation stages (24-48-72-96 hours). The RNA Transcription analysis confirmed the successful transcription of the LeExp1 gene into the host organism P. pastoris. The proteins secreted by P. pastoris were separated on a gel using SDS Page analysis, and the presence of the expansin protein with a size of 30 kDa was detected. The SDS Page gel was blotted onto a PVDF membrane using Western Blot analysis, and the presence of the expansin protein was detected with a specific antibody.