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All Journal Microbiology Indonesia Jurnal Bioteknologi & Biosains Indonesia (JBBI)
INDRA RACHMAWATI
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Immunology & microbiology Medicine & Pharmacology

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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 : 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
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
Isolation and Identification of Endophytic Fungi from Srikaya Plants (Annona squamosa) Having Potential Secondary Metabolites as Anti-Breast Cancer Activity PRASETYAWAN YUNIANTO; SYOFI ROSMALAWATI; INDRA RACHMAWATI; WAHYUDI PRIYONO SUWARSO; WAHONO SUMARYONO
Microbiology Indonesia Vol. 6 No. 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (580.415 KB) | DOI: 10.5454/mi.6.1.4

Abstract

Annonaceous acetogenin was extracted from Annona squamosa (Srikaya) seeds. It has cytotoxic activity against cancer cells and lower toxicity compared to other cancer drugs. Endophyte from Annonaceae is expected to have similar extracted metabolites to the host, thus increasing the economic value. This research is a preliminary study to obtain active compounds with potential as anti-cancer agents from endophytic fungi of Srikaya plants. Four endophytic fungal strains were isolated from Srikaya plants (Annona squamosa) and identified based on 28S rDNA sequence. The isolates are SKY II.3.1, SKY I.1.2, SKY II.3.2, and SKY III.3.1, and have similarity with Fusarium sp Vega760, Fusarium sp NRRL 22354 NRRL223, Nectria rigidiuscula, and Fusarium sp BOL35, respectively. The identified isolates were fermented in liquid media for three weeks. The liquid and mycelium were extracted using ethyl acetate. Whole extract of each fermented isolate was partitioned and evaporated to obtain ethyl acetate extract. Cytotoxicity assay of ethyl acetate extract was carried out at level 100 ppm by Methyl Thyazole Tetrazolium (MTT) viability test towards MCF-7 (breast cancer cell). The result indicated that each ethyl acetate extract could inhibit the viability of cell MCF-7 with 11.34, 99.78, 91.48, and 96.84%, for SKY II.3.1, SKY I.1.2, SKY II.3.2, and SKY III.3.1, respectively. Based on the results of cytotoxicity assay on MCF-7 breast cancer cells, endophytic fungi isolates SKY I.1.2, SKY II.3.2, and SKY III.3.1 are potential as sources of anti-breast cancer compounds.
Co-Authors Ahmad Wibisana, Ahmad Anis Herliyanti Mahsunah, Anis Herliyanti Anna Safarrida, Anna Naim, Sidrotun Naim, Sidrotun Nasution, Uli Julia Nasution, Uli Julia PRASETYAWAN YUNIANTO Puspitasari, Dian Japany Puspitasari, Dian Japany Suyanto Suyanto SYOFI ROSMALAWATI Wahono Sumaryono Wahyudi Priyono Suwarso Wijaya, Silvia Melinda Wijaya, Silvia Melinda Wulyoadi, Sasmito Wulyoadi, Sasmito