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All Journal International Journal of Advances in Applied Sciences HAYATI Journal of Biosciences Jurnal Bioteknologi & Biosains Indonesia (JBBI)
Mahsunah, Anis Herliyati
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Earth & Planetary Sciences Environmental Science Immunology & microbiology Materials Science & Nanotechnology Mathematics Physics

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PENGARUH VARIASI KONSENTRASI METANOL DAN LAMA INDUKSI TERHADAP EKSPRESI PROINSULIN OLEH Pichia pastoris SECARA INTRASELULER Martius, Efrida; Triyadi, Andree; Sofia, Dewi Yustika; Mahsunah, Anis Herliyati
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 6 No. 1 (2019): June 2019
Publisher : Balai Bioteknologi, Badan Pengkajian dan Penerapan Teknologi (BPPT)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1800.186 KB) | DOI: 10.29122/jbbi.v6i1.3176

Abstract

The Effects of Variation in Methanol Concentration and Induction Time on Intracellular Proinsulin Expression by Pichia pastoris ABSTRACTDiabetes is a metabolic disorder characterized by hyperglycemia. There were 215 million diabetic patients in 2014 and the number is expected to rise in 2040. Generally, insulin is used to treat diabetic patients. Insulin production by recombinant technology has been done, though still inefficient, by using E. coli and S. cerevisiae expression system. Another alternative expression system is methylotrophic yeast Pichia pastoris. In this research, proinsulin has been expressed by P. pastoris intracellularly. P. pastoris strains used in this research were X33, GS115, and KM71H. All recombinant strains were MutS. Best cultivation media was BMGY. Proinsulin expression was observed at 25°C. Pichia pastoris strain that expressed proinsulin best was GS115-PI. It was supported by PCR in which the strain GS115-PI gave 504 bp-sized bands. Based on proinsulin formation time, the final methanol concentration of 0.5% in 72 hours was found to be the best treatment.Keywords: BMGY, methanol, phenotype, Pichia pastoris, proinsulin ABSTRAKDiabetes melitus merupakan kelainan yang ditandai dengan hiperglikemia. Penderita diabetes pada tahun 2014 di dunia mencapai 215 juta dan diperkirakan akan meningkat pada tahun 2040. Umumnya penderita diabetes diberi pengobatan insulin sehingga menunjukkan akan ada peningkatan kebutuhan insulin. Produksi insulin dengan teknologi DNA rekombinan telah dilakukan dengan menggunakan sistem ekspresi E. coli dan S. cerevisiae namun masih belum efisien. Sistem alternatif lain adalah ragi metilotropik Pichia pastoris. Dalam penelitian ini dilakukan ekspresi proinsulin dari P. pastoris secara intraseluler. Galur P. pastoris yang digunakan dalam penelitian ini adalah X33, GS115, dan KM71H. Semua galur rekombinan adalah MutS. Media tumbuh terbaik adalah BMGY. Ekspresi proinsulin terlihat pada suhu 25°C. Hasil PCR menunjukkan bahwa galur GS115-PI yang dapat menghasilkan pita amplikon berukuran 504 bp. Hasil PCR ini dibuktikan oleh hasil seleksi galur yang menunjukkan bahwa galur GS115-PI dapat mengekspresi proinsulin dibandingkan galur lainnya. Berdasarkan kecepatan pembentukan pita protein proinsulin, variasi konsentrasi akhir metanol 0,5% dengan lama induksi 72 jam merupakan perlakuan terbaik.Kata Kunci: BMGY, fenotipe, metanol, Pichia pastoris, proinsulin
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.
Double step method in lipid extraction from biomass Aurantiochytrium sp powder Sulistiawati, Endah; Suhendra, Suhendra; Setyawan, Martomo; Mahsunah, Anis Herliyati
International Journal of Advances in Applied Sciences Vol 12, No 4: December 2023
Publisher : Institute of Advanced Engineering and Science

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.11591/ijaas.v12.i4.pp376-383

Abstract

Aurantiochytrium sp is a marine microalgae that is rich in lipids. Extraction of lipids from microalgae requires effort to select a suitable solvent and extraction method. This research used a double-step extraction method to study a mixture of n-hexane and methanol as a solvent. The variables studied were stirring time, the n-hexane to methanol (H/M) mixture ratio, and the solvent-to-biomass ratio (S/B). This research concluded that an optimum stirring time was 30 min, and a mixture of n-hexane and methanol solvents with a volume ratio of 1:1 is optimum. The optimum solvent-to-biomass ratio was S/B=20 mL/g dry microalgae. Under these conditions, the yield of oil was 83.88%. Double-step extraction can increase the yield by 10-40%.
Co-Authors Anja Meryandini DINI NURDIANI Efrida Martius Endah Sulistiawati Martomo Setyawan Puspitasari, Dian Japany Rika Indri Astuti Sofia, Dewi Yustika Suhendra Suhendra Triyadi, Andree