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All Journal Jurnal Bioteknologi & Biosains Indonesia (JBBI) Journal of Multidisciplinary Applied Natural Science
Nasution, Uli Julia
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Energy Environmental Science Immunology & microbiology Materials Science & Nanotechnology Mathematics Physics

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PEMURNIAN ENZIM SEFALOSPORIN-C ASILASE DAN OPTIMASI PROSES KROMATOGRAFI PENUKAR ION Nasution, Uli Julia; Wijaya, Silvia Melinda; Wibisana, Ahmad; Safarrida, Anna; Rachmawati, Indra; Puspitasari, Dian Japany; Naim, Sidrotun; Mahsunah, Anis Herliyanti; Wulyoadi, Sasmito; Suyanto, Suyanto
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 5 No. 2 (2018): December 2018
Publisher : Balai Bioteknologi, Badan Pengkajian dan Penerapan Teknologi (BPPT)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1567.447 KB) | DOI: 10.29122/jbbi.v5i2.2902

Abstract

Purification of Cephalosporin-C Acylase and Its Optimization of Ion-Exchange ChromatographyABSTRACTCephalosporin-C acylase (CCA) has an important role in the one-step conversion of cephalosporin-C into 7-ACA. Purification process aims to increase specific activity of CCA enzyme. Purification began with cell lysis, ammonium sulphate precipitation, dialysis, ion exchange chromatography (IEC) and size exclusion chromatography. IEC optimization of elution step was also done to compare gradient and isocratic elusion. Purification was capable to increase the enzyme purity upto 33.66 fold, with specific activity of 3.00 U/mg and the yield reached 41.41%. Optimization of elusion during IEC showed that isocratic protein elusion was more efficient (taking shorter time, 3 column volume (CV) only) than that of gradient batch (up to 9 CV). SDS-PAGE analysis demonstrated that the recombinant CCA enzyme existed in two types, active enzyme containing α-subunit (25 kDa) and β-subunit (58 kDa), and inactive enzyme (83 kDa) as precursor. Furthermore, 30% ammonium sulphate saturated precipitation was able to precipitate this inactive CCA.Keywords: 7-ACA,CCA, cephalosporin C, protein purification, specific activity ABSTRAKSefalosporin-C asilase (CCA) merupakan enzim yang berperan penting dalam konversi satu tahap sefalosporin-C menjadi 7-ACA. Proses purifikasi merupakan salah satu cara untuk meningkatkan aktivitas spesifik enzim CCA. Proses purifikasi dimulai dari memecah sel, diikuti dengan tahap presipitasi menggunakan amonium sulfat, dialisis, kromatografi penukar ion (IEC) dan kromatografi eksklusi. Optimasi proses IEC pada tahap elusi juga dilakukan untuk membandingkan elusi enzim CCA secara gradien dan isokratik. Proses purifikasi pada penelitian ini mampu meningkatkan kemurnian enzim hingga 33,66 kali, dengan aktivitas spesifik sebesar 3,00 U/mg dan perolehan enzim sebesar 41,41%. Hasil optimasi IEC pada proses elusi secara isokratik lebih efisien dari segi waktu (hanya membutuhkan 3 kolom volume (CV) dibandingkan dengan secara gradien (sampai 9 CV). Hasil SDS-PAGE menunjukkan bahwa CCA rekombinan merupakan enzim dengan 2 macam bentuk yaitu enzim aktif, yang terdiri dari subunit α (25 kDa) dan β (58 kDa), dan enzim tidak aktif berupa prekursor (83 kDa). Proses presipitasi menggunakan amonium sulfat 30% tersaturasi dapat mengendapkan prekursor CCA.Kata Kunci: 7-ACA, aktivitas spesifik, CCA, purifikasi protein, sefalosporin C
Modification of a Gene Cassette to Express Proglargine in Pichia pastoris: Elimination of Glu-Ala Repeats, Spacer and C-peptide Sequences Nasution, Uli Julia; Astuti, Rika Indri; Wahyudi, Aris Tri; Hardianto, Dudi; Martius, Efrida
Journal of Multidisciplinary Applied Natural Science Articles in Press
Publisher : Pandawa Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.47352/jmans.2774-3047.368

Abstract

The growing global prevalence of diabetes mellitus has sharply increased the demand for insulin and its analogues. Pichia pastoris is a well-established system for recombinant pro-insulin and its analogues production. However, conventional gene constructs often include additional sequences, such as Glu-Ala repeats, spacer peptides, and c-peptides that complicate downstream processing and reduce efficiency. This study aimed to construct and express a proglargine (PG) gene cassette lacking the Glu-Ala repeats, spacer, and c-peptide in P. pastoris GS115 to obtain a uniform PG protein. The recombinant vector propagated in Escherichia coli TOP10F’, then expressed in P. pastorisGS115. Selected transformants were cultivated in YPG medium, then induced with 1% and 2% methanol daily in BMMY. The optimum methanol concentration further evaluated in ½ BSM induction medium. The result demonstrated that optimal PG expression was achieved with 2% methanol induction in BMMY, producing higher levels than those with ½ BSM. Among the transformants, PG.c2 produced the highest PG protein in BMMY medium induced with 2% methanol. Dot-blot analysis confirmed the secretion of PG, and LC-HRMS analysis demonstrated 100% amino acid sequence coverage of PG, confirming the integrity and completeness of the expressed protein. This study presented a newly modified PG gene cassette, inserted into pPICZαA vector, to express uniform secreted PG in P. pastoris GS115. By simplifying the precursor structure, a more homogeneous precursor product can be obtained, which is expected to simplify purification and also the downstream enzymatic process of PG into mature insulin glargine.
Co-Authors Ahmad Wibisana, Ahmad Anis Herliyanti Mahsunah, Anis Herliyanti Anna Safarrida, Anna Aris Tri Wahyudi Dudi Hardianto, Dudi Efrida Martius INDRA RACHMAWATI Naim, Sidrotun Puspitasari, Dian Japany Rika Indri Astuti Suyanto Suyanto Wijaya, Silvia Melinda Wulyoadi, Sasmito