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All Journal HAYATI Journal of Biosciences Jurnal Bioteknologi & Biosains Indonesia (JBBI) Indonesian Journal of Science and Education
Puspitasari, Dian Japany
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemistry Earth & Planetary Sciences Immunology & microbiology Materials Science & Nanotechnology 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
Influence of Co-feeding Methanol-sorbitol Ratio on Production of Human Insulin Precursor Expressed by Mut+ Pichia pastoris Puspitasari, Dian Japany; Mahsunah, Anis Herliyati; Nurdiani, Dini; Astuti, Rika Indri; Meryandini, Anja
HAYATI Journal of Biosciences Vol. 32 No. 3 (2025): May 2025
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.32.3.581-588

Abstract

An increasing number of diabetic patients and the demand for insulin encourage the development of recombinant insulin production on a large scale. Human insulin precursor (HIP) expressed by Mut+ Pichia pastoris using methanol as an inducer was developed. However, methanol above 5% (w/v) or 1.56 M is toxic for the host. Sorbitol was introduced as a co-substrate with methanol. To our knowledge, the study of methanol/sorbitol co-feeding on human insulin precursor (HIP) expression by Mut+ Pichia pastoris in a bioreactor has yet to be reported. This study aimed to investigate the influence of the methanol-sorbitol co-feeding ratio on the expression of HIP expressed by P. pastoris X33 Mut+. The study was conducted by comparing the cultivation of P. pastoris/pD902-IP Mut+ in a 10-liter bioreactor under three conditions: feeding 100% methanol, mass ratio of MeOH:sorbitol 12:1 and 3:1. The oxygen consumption of methanol/sorbitol is less than the methanol feeding. The mass ratio of MeOH:sorbitol 12:1 produced the highest HIP titer (1326.5 mg/L), 1.5 times higher than methanol feeding, the lowest specific growth rate, but the highest specific productivity at the induction phase. MeOH:sorbitol mass ratio 3:1 produced the highest dry cell weight (DCW) amount (96 g/L). These results suggested that an appropriate ratio of sorbitol-methanol can be a choice to replace methanol feeding in a Mut+ P. pastoris.
The Development of human insulin precursor expressed from P.pastoris-X33 in a 10L-fermenter Puspitasari, Dian Japany
Indonesian Journal of Science and Education Vol. 7 No. 1 (2023): Indonesian Journal of Science and Education
Publisher : Universitas Tidar

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.31002/ijose.v7i1.212

Abstract

Human insulin is a hormone that regulate blood glucose in human. Due to the vital role of insulin and the trend of diabetic case globally, insulin demand has been rise in the world. Indonesia as a country that had diabetic patient more than 10 million people in 2017 is still imported raw material of human insulin. The development of production process of human insulin is needed in order to support government objective to be independence in medicine aspect. Human insulin precursor (HIP) expressed from a Pichia pastoris X33/pD902-IP had been developed and optimised in a small scale. However, the scaling up in fermenter 10L has not been studied. Using a 10L-fermenter the fermentation system of P.pastoris X33/pD902-IP was developed. Fermentation done in 120 hours using a basal salt medium (half concentration) for the vegetative and induction media. To induce HIP expression, methanol is fed by pulse strategy with a gradient concentration 1-3% for 48 hours. The dry cell weight (DCW) and HIP titer were 72 g/L and 192 mg/L, respectively. This development is the first fermentation process of HIP in fermenter 10L in Indonesia.
Co-Authors Ahmad Wibisana, Ahmad Anis Herliyanti Mahsunah, Anis Herliyanti Anja Meryandini Anna Safarrida, Anna DINI NURDIANI INDRA RACHMAWATI Mahsunah, Anis Herliyati Naim, Sidrotun Nasution, Uli Julia Rika Indri Astuti Suyanto Suyanto Wijaya, Silvia Melinda Wulyoadi, Sasmito