dc.contributor.advisor | Ambarsari, Laksmi | |
dc.contributor.advisor | Kurniatin, Popi Asri | |
dc.contributor.advisor | Fuad, Asrul Muhamad | |
dc.contributor.author | Aryani, Hanifah | |
dc.date.accessioned | 2024-08-18T23:47:06Z | |
dc.date.available | 2024-08-18T23:47:06Z | |
dc.date.issued | 2024 | |
dc.identifier.uri | http://repository.ipb.ac.id/handle/123456789/157813 | |
dc.description.abstract | Glukosa oksidase (GOX, EC1.1.3.4) adalah enzim yang mengkatalisis oksidasi ß-D-glukosa menjadi d-glukonolakton dan hidrogen-peroksida (H2O2), dimana d-glukonolakton yang terhidrolisis berubah menjadi asam glukonat yang bermanfaat dalam bidang kesehatan. Sumber utama GOX industri dihasilkan oleh jamur dari kelompok Aspergillus dan Penicilium. Pada penelitian terdahulu diketahui bahwa GOX dari isolat A. niger IPBCC-08.160 menunjukkan aktivitas intraseluler yang lebih tinggi daripada ekstraselulernya. Penelitian ini bertujuan untuk meningkatkan produksi GOX ekstraseluler dengan teknologi DNA rekombinan pada sistem ekspresi Pichia pastoris. Gen GOX asal A. niger IPBCC-08.160 dimodifikasi dengan menghilangkan sinyal sekresi naturalnya, menambahkan situs restriksi XhoI di kedua ujungnya, spacer glu-ala-glu-ala di ujung 5', dan situs XbaI di ujung 3'. Gen yang dimodifikasi ini, dinamai GOX-Xho (1797 pb), telah berhasil diklon ke dalam plasmid kloning pTA2 dan dikarakterisasi melalui analisis sekuen DNA. Selanjutnya gen GOX-Xho berhasil disubklon ke dalam vektor ekspresi terinduksi pPICZaB pada situs XhoI dan menghasilkan plasmid rekombinan dengan orientasi gen yang benar. Gen tersebut telah terfusi dengan peptida sinyal mating factor-? (MF-?) untuk ekspresi protein ekstraseluler pada sel inang. Plasmid ini berhasil ditransformasi ke dalam sel P. pastoris BG11 melalui metode elektroporasi dan menghasilkan beberapa koloni ragi rekombinan. Selanjutnya koloni tersebut diseleksi pada media YPD dengan konsentrasi zeocin 100, 200, 1000 dan 2000 µg/ml untuk memperoleh koloni transforman yang stabil secara genetik. Sekitar 20% koloni tumbuh baik pada media dengan konsentrasi zeocin 1000 µg/mL dan sebagian koloni tersebut digunakan dalam uji ekspresi protein. Analisis ekspresi protein menunjukkan bahwa beberapa galur berhasil mengekspresikan GOX rekombinan (±60 kDa) secara ekstraseluler. Selain itu, variasi ekspresi protein juga teramati dari beberapa galur rekombinan yang diujikan. | |
dc.description.abstract | Glucose oxidase (GOX, EC1.1.3.4) is an enzyme that catalyzes the oxidation of ß-D-glucose to d-gluconolactone and hydrogen-peroxide (H2O2), where the hydrolyzed d-gluconolactone turns into gluconic acid which is useful in the health sector. The main industrial source of GOX is produced by fungi from the Aspergillus and Penicillium groups. In previous research, it was known that GOX from the A. niger isolate IPBCC 08.160 showed higher intracellular activity than extracellular activity. This research aims to increase extracellular GOX production using recombinant DNA technology in the Pichia pastoris expression system. The GOX gene from A. niger IPBCC 08.160 was modified by removing the natural secretion signal, adding XhoI restriction sites at both ends, a glu-ala-glu-ala spacer at the 5'-end, and an XbaI site at the 3'-end. This modified gene, named GOX-Xho (1797 bp), was successfully cloned into the pTA2 cloning plasmid and characterized through DNA sequence analysis. Next, the GOX-Xho gene was successfully subcloned into the pPICZaB inducible expression vector at the XhoI site and produced a recombinant plasmid with the correct gene orientation. The gene has been fused with the mating factor-? (MF-?) signal peptide for extracellular protein expression in host cells. This plasmid was successfully transformed into P. pastoris BG11 cells via the electroporation method and produced several recombinant yeast colonies. Next, those colonies were further selected on YPD media with zeocin concentrations of 100, 200, 1000, and 2000 µg/ml to obtain genetically stable transformant colonies. Approximately 20% of colonies grew well on media with 1000 µg/mL of zeocin and some of those colonies were used for protein expression assay. Protein expression analysis showed that some strains successfully expressed recombinant GOX (±60 kDa) extracellularly. In addition, variations in protein expression were also observed from several recombinant strains tested. | |
dc.description.sponsorship | | |
dc.language.iso | id | |
dc.publisher | IPB University | id |
dc.title | Rekonstruksi, Subkloning Gen Glukosa Oksidase (GOX-Xho) ke dalam Plasmid pPICZaB, dan Ekspresinya pada Pichia pastoris | id |
dc.title.alternative | Reconstruction, Subcloning of Glucose Oxidase Gene (GOX-Xho) into Plasmid pPICZaB, and its Expression in Pichia pastoris. | |
dc.type | Tesis | |
dc.subject.keyword | kloning gen | id |
dc.subject.keyword | Aspergillus niger IPBCC 08.610 | id |
dc.subject.keyword | enzim rekombinan | id |
dc.subject.keyword | glukosa oksidase (GOX) | id |
dc.subject.keyword | Pichia pastoris | id |
dc.subject.keyword | vektor induksi | id |