OPTIMIZATION OF PRODUCTION OF PROINSULIN ASPART IN PICHIA PASTORIS KM71 ASP.RWR USING FERMENTOR WITH FED-BATCH METHOD

• Main Author: Assegaf, Faisal
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/64925
• Institution: Institut Teknologi Bandung

Diabetes mellitus is a metabolic disease characterized by increased blood sugar levels (hyperglycemia) due to decreased insulin levels and insulin activity. The high incidence of diabetes mellitus in Indonesia is at risk of causing death, so comprehensive treatment is needed. The use of external insulin is an alternative to diabetes treatment, but there are some side effects such as hyperglycemia and hypoglycemia due to long-term use of insulin therapy. Therefore, to reduce these side effects, insulin analogues were developed. Insulin aspart is an analog insulin that has a proline residue substitution on the B chain (B28) to aspartate. The purpose of this study was to optimize the production of proinsulin aspart in Pichia pastoris KM71 Asp.RWR carrying the gene encoding proinsulin aspart using a 2 L scale fermenter using the fed batch cultivation method. The clone of P. pastoris KM71 Asp.RWR was cultivated in a 2 L fermenter by optimizing the amount of addition of glycerol and histidine, as well as the technique of giving histidine gradually and directly, testing the consistency of proinsulin aspart expression, and characterizing proinsulin aspart produced using SDS-PAGE, western blot method, and peptide mapping. The best insulin aspart production was obtained at the ratio of glycerol and histidine 1:5 with gradual histidine feeding technique. Expression of proinsulin aspart in P. pastoris KM71 Asp.RWR by fed-batch method showed consistent results based on SDS-PAGE analysis and yield of proinsulin aspart. SDS-PAGE analysis showed proinsulin aspart has a molecular mass of ~6 kDa and the results of western blot and peptide mapping confirmed the production of proinsulin aspart. A semiquantitative analysis using densitometry and BCA obtained an estimate of the mass of proinsulin protein aspart in three batches, namely 1.27 g, 0.45 g, and 1.26 g with yields of 5.97 mg/OD, 4.01 mg/OD, and 8.95 g/OD. The results of this study indicate that proinsulin aspart produced in P. pastoris KM71 Asp.RWW with a fermenter scale of 2 L has a potential to be a candidate for therapy for people with diabetes mellitus.