ANALISIS SEKUEN DAN EKSPRESI GEN PAC SINTETIK PENYANDI ENZIM PENISILIN G ASILASE (PGA) DARI ESCHERICHIA COLI PADA INANG E. COLI BL21(DE3) DAN E. COLI HB101

ACHMAD MAKIN AMIN, Prof. Dr. apt. Sismindari, S.U.; Dr. apt. Purwanto, M.Sc.

2022 | Tesis | MAGISTER BIOTEKNOLOGI

Kasus kejadian infeksi akibat bakteri yang tinggi di Indonesia perlu diimbangi dengan kemandirian dalam produksi bahan baku obat antibiotik, sehingga tidak ketergantungan impor dari luar negeri. Salah satu, antibiotik yang banyak digunakan yaitu golongan beta-lactam seperti penisilin dan turunannya. Produksi antibiotik turunan penisilin membutuhkan katalis enzim Penisilin G Asilase (PGA). Enzim PGA dapat diproduksi dengan teknik rekayasa rekombinan. Pada penelitian terdahulu telah dilakukan ekspresi gen pac sintetik, pengkode enzim PGA pada E. coli BL21(DE3), dengan hasil aktivitas enzim PGA yang rendah (0,01754 U/mg). Sehingga, pada penelitian saat ini dilakukan ekspresi gen pac sintetik pada inang yang berbeda dengan menggunakan tiga jenis inducer berbeda. Tujuan yang ingin dicapai dalam penelitian ini, untuk mendapatkan produksi protein rekombinan PGA dengan aktivitas enzim yang tinggi. Penelitian kali ini dilakukan transformasi dan sekuensing utuh gen pac sintetik dalam konstruk plasmid rekombinan pET22b-pgaEc pada inang E. coli HB101 dan E. coli BL21(DE3) untuk mengetahui urutan basa nitrogen setelah ditransformasi. Inang dengan plasmid rekombinan mengandung gen pac sintetik tanpa adanya mutasi kemudian diinduksi ekspresi protein rekombinan PGA dengan tiga inducer yang berbeda IPTG 0,05 mM, laktosa 0,2%, dan arabinosa 1,5% dengan suhu inkubasi 20°C, pada kecepatan agitasi 150 rpm selama 17 jam. Protein rekombinan PGA diisolasi dengan metode sonikasi serta dilakukan treatment Freeze-Thawing untuk mendapatkan kembali protein rekombinan PGA yang aktif secara biologis. Verifikasi protein rekombinan dilakukan dengan elektroforesis SDS-PAGE 12%. Protein rekombinan PGA dilakukan uji aktivitas enzim dengan menggunakan pDAB. Hasil dari penelitian ini, sekuensing gen pac sintetik dari inang E. coli HB101 dan E. coli BL21(DE3) tidak ada perubahan urutan basa nitrogen. E. coli HB101 mampu menghasilkan ekspresi protein rekombinan PGA dengan aktivitas tertinggi (10,17 U/mg) dibanding dengan inang E. coli BL21(DE3) (6,67 U/mg). Jenis inducer yang paling optimum untuk induksi ekspresi protein rekombinan PGA dengan aktivitas enzim yang tinggi yaitu menggunakan arabinosa 1,5%.

The high incidence of bacterial infections in Indonesia needs to be balanced with independence in the production of raw materials for antibiotic drugs so that it does not depend on imports from other countries. One of the most widely used antibiotics is the beta-lactam group, such as penicillin and its derivatives. The production of penicillin-derived antibiotics requires the enzyme Penicillin G acylase (PGA) as a catalyst. PGA enzymes can be produced by recombinant engineering techniques. In a previous study, the expression of the synthetic pac gene, encoding the PGA enzyme in E. coli BL21(DE3), was carried out, with the result that the PGA enzyme activity was low (0.01754 U/mg). Thus, in the current study, the expression of the synthetic pac gene was carried out in different hosts using three different inducers. The aim of this research is to obtain the production of PGA recombinant protein with high enzyme activity. In this study, the complete transformation and sequencing of the synthetic pac gene in the pET22b-pgaEc recombinant plasmid construct in the host E. coli HB101 and E. coli BL21(DE3) was carried out to determine the sequence of nitrogen bases after transformation. The host whose recombinant plasmid contained the synthetic pac gene without any mutations was then induced to express PGA recombinant protein with three different inducers IPTG 0.05 mM, lactose 0.2%, and arabinose 1.5% with an incubation temperature of 20°C, at agitation speed. 150 rpm for 17 hours. PGA recombinant protein was isolated by sonication method and Freeze-Thawing treatment was performed to recover biologically active PGA recombinant protein. Verification of recombinant protein was carried out by SDS-PAGE 12% electrophoresis. PGA recombinant protein was tested for enzyme activity using pDAB. The results of this study, sequencing of the synthetic pac gene from the host E. coli HB101 and E. coli BL21(DE3) did not change the nitrogen base sequence. E. coli HB101 was able to produce PGA recombinant protein expression with the highest activity (10.17 U/mg) compared to the host E. coli BL21(DE3) (6.67 U/mg). The most optimum type of inducer for the induction of PGA recombinant protein expression with high enzyme activity was using 1,5% arabinose

Kata Kunci : Gen pac Sintetik, PGA, E. coli BL21(DE3), E. coli HB101