EXPRESSION OF bglp15.2 GENE CARRYING INULINASE SIGNAL PEPTIDE IN Saccharomyces cerevisiae BY4741 TO PRODUCE EXTRACELLULARLY β-GLUCOSIDASE

• Main Author: BADI'ATUL FITRI (NIM: 21115003), ARMAYA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/21254
• Institution: Institut Teknologi Bandung

One of the important enzyme in cellulase complex is β-glucosidase. In this research, adding peptide signal of inulinase gene from Kluyveromyces marxianus, cloning, and expressing of bglp15.2 gene in S. cerevisiae BY4741 has been done. Gene of bglp15.2 encoding β-glucosidase has been isolated from metagenome of water and sediment sample from Kawio Island deep sea, North Sulawesi. The bglp15.2 gene has 90% identity to nucleotide sequence of Shewanella frigidimarina NCIMB 400 (Acc.: CP000447.1) bacteria. Adding peptide signal was aimed to secrete β-glucosidase and has been done with PCR (Polymerase Chain Reaction) method. The bglp15.2 and bglp15.2INU gene (the bglp15.2 gene that has peptide signal nucleotide sequence) cloned in Escherichia coli DH5α with pGEM-T-Easy vector and pBEVY-GL shuttle vector. The genes were transfered from pGEM-T-Easy vector to pBEVY-GL vector by restriction and ligation method. The pBEVY-GL shuttle vector was used for transforming S. cerevisiae BY4741 with bglp15.2 and bglp15.2INU. Transformation of S. cerevisiae BY4741 has been done with lithium asetat and PEG (Polyethylene Glycol) 4000 without DNA carrier method. The result of each transformation steps were confirmed by qolony PCR which was done with two pairs of primer, that were vector universal primers and gene spesific primers. From the each of qolony PCR steps, the amplicon with expected size was obtained. The recombinant S. cerevisiae BY4741 that grown in 48 hours (OD600 3) have extracellularly β-glucosidase enzyme activity of 0,0178 U/ml and the intracellularly activity was 0,0181 U/ml. The Bglp15.2 enzyme without peptide signal didn’t secreted and has intracellularly activity of 0,0079 U/ml after 10 hours induction (OD600 1,5). With K. marxianus inulinase signal peptide, Bglp15.2INU protein couldn’t be secreted entirely.