PEMURNIAN ENZIM SEFALOSPORIN-C ASILASE DAN OPTIMASI PROSES KROMATOGRAFI PENUKAR ION

Uli Julia Nasution, Silvia Melinda Wijaya, Ahmad Wibisana, Anna Safarrida, Indra Rachmawati, Dian Japany Puspitasari, Sidrotun Naim, Anis Herliyanti Mahsunah, Sasmito Wulyoadi, Suyanto Suyanto

Abstract


Purification of Cephalosporin-C Acylase and Its Optimization of Ion-Exchange Chromatography


ABSTRACT

Cephalosporin-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

 

ABSTRAK

Sefalosporin-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


Keywords


7-ACA; aktivitas spesifik; CCA; purifikasi protein; sefalosporin C

Full Text:

PDF

References


 Block H, Maertens B, Spriestersbach A, Brinker N, Kubicek J, Fabis R, Labahn J, Schafer F (2009) Immobilized-metal affinity chromatography (IMAC): A review. Methods Enzymol 463:439-473. doi:10.1016/S0076-6879(09)63027-5

Cheung RC, Wong JH, Ng TB (2012) Immobilized metal ion affinity chromatography: A review on its applications. Appl Microbiol Biotechnol 96:1411-1420. doi: 10.1007/s00253-012-4507-0

Conti G, Pollegioni L, Molla G, Rosini E (2014) Strategic manipulation of an industrial biocatalyst – Evolution of a cephalosporin C acylase. FEBS J 281:2443-2455. doi: 10.1111/febs.12798

Duong-Ly KC, Gabelli SB (2014) Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol 541:85-94. doi: 10.1016/B978-0-12-420119-4.00007-0

Fechtig B, Peter H, Bickel H, Wischer E (1968) Concerning the preparation of 7-amino-cephalosporanic acid. Helv Chim Acta 51:1108-1119. doi: 10.1002/hlca.19680510513

Gaurav K, Kundu K, Kundu S (2010) Biosynthesis of Cephalosporin-C Acylase Enzyme: Optimal Media Design, Purification, and Characterization. Artif Cells Blood Substit Immobil Biotechnol 38:277-283. doi: 10.3109/10731199.2010.482036

He H, Luo H, Li X, Wang X, Liang C, Wei YY, Shen Z (2015) Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules. Biotechnol Prog 31:387-395. doi: 10.1002/btpr.2044

Jobanputra AH, Vasait R (2015) Cephalosporin C acylase from Pseudomonas species: Production and enhancement of its activity by optimization of process parameters. Biocatal Agric Biotechnol 4:465-470. doi: 10.1016/j.bcab.2015.06.009

Kong K-F, Schneper L, Mathee K (2009) Beta-lactam antibiotics: from antibiosis to resistance and bacteriology. APMIS 118:1-36. doi: 10.1111/j.1600-0463.2009.02563.x

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. doi: 10.1038/227680a0

Ma X, Su E, Deng S, Xie Y, Wei D (2014) An efficient method for recovering recombinant Cephalosporin C deacetylase from the cytoplasm of E. coli cells. Chemical and Biochemical Engineering Quarterly 28:349-355. doi: 10.15255/CABEQ.2013.1904

Martius E, Wibisana A, Ardiyani Y (2018) The optimization of soluble cephalosporin C acylase expression in E. coli. IJES 7:29-34. doi:10.9790/1813-0703012934

Morin R, Jackson B, Flynn E, Roseske R, Andrews S (1969) Chemistry of cephalosporin antibiotics XIV: reaction of cephalosporin C with nitrosyl chloride. J Am Chem Soc 91:1396-1400. doi: 10.1021/ja01034a022

Parmar A, Kumar H, Marwaha S, Kenedy J (1998) Recent trends in enzymatic conversion of Cephalosporin C to 7-Aminocephalosporanic Acid (7-ACA). Crit Rev Biotechnol 18:1-12. doi: 10.1080/0738-859891224194

Pollegioni L, Rosini E, Molla G (2013) Cephalosporin C acylase: dream and(/or) reality. Appl Microbiol Biotechnol 97:2341-2355. doi: 10.1007/s00253-013-4741-0

Robinson PK (2015) Enzymes: principle and biotechnological applications. Essays Biochem 59:1-41. doi:10.1042/bse0590001

Rosano G, Ceccarelli E (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. doi: 10.3389/fmicb.2014.00172

Shin YC, Jeon J, Jung KH, Park MR, Kim Y. (2009) Cephalosporin C acylase mutant and method for preparing 7-ACA using same. United States of America Paten No. US 7,592,168 B2.

Tan SC, Yiap BC (2009) DNA, RNA, and protein extraction: The past and the present. J Biomed Biotechnol. 2009:1-10. doi: 10.1155/2009/574398

Tripathi N (2016) Production and purification of recombinant proteins from Escherichia coli. ChemBioEng Reviews 3:116-133. doi: 10.1002/cben.201600002

Wang Y, Yu H, Song W, An M, Zhang J, Luo H, Shen Z (2012) Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel. J Biosci Bioeng 113:36-41. doi: 10.1016/j.jbiosc.2011.08.027

Xiao Y, Huo X, Qian Y, Zhang Y, Chen G, Ouyang P, Lin Z (2014) Engineering of a CPC acylase using a facile pH indicator assay. J Ind Microbiol Biotechnol 41:1617-1625. doi: 10.1007/s10295-014-1501-9




DOI: http://dx.doi.org/10.29122/jbbi.v5i2.2902

Refbacks

  • There are currently no refbacks.


© Copyright: BY-NC-SA