HETEROLOGOUS EXPRESSION OF SERINE HYDROXYMETHYLTRANSFERASE ON PICHIA PASTORIS

• Main Author: Wulandari Nasution, Marina
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/59591
• Institution: Institut Teknologi Bandung

Serine hydroxymethyltransferase (SHMT; E.C. 2.1.2.1) is an enzyme that requires pyridoxal-5’-phosphate (PLP) as a cofactor and the SHMT enzyme functions to catalyze the interconversion of glycine to serine using 5,10 methylene- tetrahydrofolate (mTHF). This study aims to obtain the pPICZ?A expression vector which has been inserted with the SHMT gene, and to obtain Pichia pastoris transformant containing the recombinant plasmid pPICZ?A-SHMT with Mut+ phenotypic properties, and the transforman will be expressed and hopefully have activity of crude extract enzyme SHMT. To obtain crude extract enzymes, a subcloning approach is used on the cloned SHMT gene, then cut and inserted into pPICZ?A expression vector, then used in Pichia pastoris transformation process using the recombinant plasmid, the results of the transformation were used for SHMT enzyme expression. The cloned SHMT gene was amplified using a primer designed with restriction recognition sites restriction enzymes EcoRI and XbaI and an electropherogram was generated with SHMT gene fragment with a size of ~1,3 kb. The amplified SHMT gene then ligated with the pJET 1.2/blunt cloned plasmid to produce the pJET-SHMT recombinant plasmid and used in the transformation process of Escherichia coli TOP10F’. The transformants possessing the pJET- SHMT recombinant plasmid were the inoculated and the plasmid was isolated. The recombinant pJET-SHMT plasmid was cut by restriction enzymes EcoRI and XbaI. The restriction product is ligated with the expression vector pPICZ?A which had been cut with the same restriction enzyme and produced the recombinant plasmid pPICZ?A-SHMT. The recombinant plasmid pPICZ?A-SHMT is linearized using the restriction enzyme SacI and used in the transformation of Pichia pastoris GS115 and resulted in a transformant with the recombinant plasmid pPICZ?A-SHMT. The phenotypic properties of the Pichia pastoris transformants were determined using the Polymerase Chain Reaction (PCR) and produced 2 bands on the electropherogram which explained that the Pichia pastoris GS115 transformant had Mut+ phenotypic properties. Expression of the SHMT enzyme using a transformant containing the recombinant plasmid pPICZ?A-SHMT resulted in a crude extract of the SHMT enzyme which had activity on the sixth day. The specific activity of the crude extract of the enzyme was highest on induction with 2% methanol, which was about 15,23 U/mg. The specific activity of the crude extract of the enzyme can indicate that the SHMT enzyme can be expressed using Pichia pastoris host cells, but when it’s compared with the activity of the SHMT enzyme in Escherichia coli which has an activity of about 118.16 U/mg, the SHMT enzyme in Pichia pastoris has a lower activity. However, with a yeast as a host cell the possibility of the enzyme being endotoxin is lower because Pichia pastoris has been verified as Generally Recognized as Safe that Pichia pastoris is safe for the environment.