Bima Wedana Isdiyono, Dudi Hardianto, Fransiskus Xaverius Ivan


Production of Cephalosporin Acylase Recombinant as Biocatalyst for 7-Aminocephalosporanic Acid Production

7-aminocephalosporanic acid (7-ACA) is a precursor for the production of semisynthetic cephalosporin derivatives. The enzymatic 7-ACA production can use two-stage and one-step enzymatic methods. Two-stage enzymatic method uses D-amino acid oxidase (DAAO) enzyme to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA) in the first stage and glutaryl-7-aminocephalosporanic acid acylase to produce 7-ACA in the second stage. The one-stage enzymatic method using cephalosporin acylase (CPC acylase) converts the CPC to 7-ACA directly. The aim of this research was to produce recombinant CPC acylase in Escherichia coli BL21(DE3). Transformantion culture E. coli BL21(DE3) was induced with concentrations of IPTG 0; 0.25; 0.5; 0.75; 1; 2 mM for 5 hours. The induction time of IPTG was determined at 0, 1, 2, 3, 4, and 5 hours. The results showed that CPC acylase produced by E. coli BL21(DE3) with optimum condition of CPC acylase production was 0.5 mM IPTG and optimal induction time of IPTG was 5 hours.

Keywords: Cephalosporin, cephalosporin acylase, 7-ACA, protein expression, Escherichia coli BL21(DE3)



Asam 7-aminosefalosporanat (7-ACA) merupakan prekursor untuk produksi turunan sefalosporin semisintetik. Produksi 7-ACA secara enzimatik dapat menggunakan metode dua tahap dan satu tahap enzimatik. Metode enzimatik secara dua tahap menggunakan enzim asam D-amino oksidase (DAAO) untuk menghasilkan asam glutaril-7-aminosefalosporinat (GL-7-ACA) pada tahap pertama dan menggunakan asam glutaril-7-aminosefalosporinat asilase untuk menghasilkan 7-ACA pada tahap kedua. Metode enzimatik satu tahap dengan sefalosporin asilase (CPC asilase) mengubah CPC menjadi 7-ACA secara langsung. Tujuan penelitian adalah memproduksi rekombinan CPC asilase di dalam sel Escherichia coli BL21(DE3). Kultur Transforman E. coli BL21(DE3) diinduksi dengan konsentrasi IPTG 0; 0,25; 0,5; 0,75; 1; 2 mM selama 5 jam. Waktu induksi IPTG ditentukan pada 0, 1, 2, 3, 4 dan 5 jam. Hasil penelitian menunjukan bahwa CPC asilase diproduksi oleh E. coli BL21(DE3) dengan kondisi optimal produksi CPC asilase adalah konsentrasi IPTG 0,5 mM dan waktu induksi IPTG optimal adalah 5 jam.


Sefalosporin; sefalosporin asilase; 7-ACA; rekayasa genetik; Escherichia coli BL21(DE3)

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Cho KJ, Kim JK, Lee JH, Shin HJ, Park SS, Kim KH (2009) Structural features of cephalosporin acylase reveal the basis of autocatalytic activation. Biochem Biophys Res Commun 390:342-348. doi: 10.1016/j.bbrc.2009.09.134

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

Egorov AM, Kurochkina VB, Sklyarenko AV, Nys PS (2000) Enzymatic transformation of betalactam antibiotics: Trend of development and approaches to practical implementation. Biocatalysis Fundament Appl 41:43-46

Elander RP (2003) Industrial production of β-lactam antibiotic. Appl Microbiol Biotechnol 61:385-392. doi: 10.1007/s00253-003-1274-y

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

Hardianto D, Royani JI, Safarrida A (2016a) Cephalosporin C acylase from microbes for one-step enzymatic transformation of cephalosporin C to 7-aminocephalosporanic acid. J Pure Appl Microbiol 10:2495-2499. doi: 10.22207/JPAM.10.4.03

Hardianto D, Isdiyono BW, Ivan FX (2016b) Biokonversi sefalosporin C menjadi asam 7-aminosefalosporanat dengan sefalosporin asilase. J Bioteknol Biosains Indones 3(2): 89-95

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

Khare P, Raj V, Chandra S, Agarwal S (2014) Quantitative and qualitative assessment of DNA extracted from saliva for its use in forensic identification. J Forensic Dent Sci 6:81-85. doi: 10.4103/0975-1475.132529

Larentis AL, Nicolau JF, Esteves Gdos S, Vareschini DT, de Almeida FV, dos Reis MG, Galler R, Medeiros MA (2014) Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Res Notes 7:671. doi: 10.1186/1756-0500-7-671

Li Q, Huang X, Zhu Y (2014) Evaluation of active designs of cephalosporin C acylase by molecular dynamics simulation and molecular docking. J Mol Model 20:2314. doi: 10.1007/s00894-014-2314-5

Margawati ET, Ridwan M (2009). Expression and characterization of recombinant protein of JSU pGEX eiher by single or double cell lysis. Biota 14:166-171

Oh B, Kim M, Yoon J, Chung K, Shin Y, Lee D, Kim Y (2003) Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues. Biochem Biophys Res Commun 310:19-27. PMID: 14511642

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

Ren Y, Lei Y, Zhu Y (2014) Site-directed mutagenesis of cephalosporin C acylase and enzymatic conversion of cephalosporin C to 7-amino-cephalosporanic acid. Turk J Biochem 39:51-56. doi: 10.5505/tjb.2014.48569

Sambrook J, Russell DW (2001) Molecular cloning: A laboratory manual. Cold Spring Harbor Lab Press, New York

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

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 acyalse using a facile pH indicator assay. J Ind Microbiol Biotechnol 41:1617-1625. doi: 10.1007/s10295-014-1501-9

Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, Lan W, Wang J, Wu H, Cao C, Wang X (2011) Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS One 6:e22981. doi: 10.1371/journal.pone.0022981

Zhang J, Yu H, Wang Y, Luo H, Shen Z (2014) Determination of the second autoproteolytic cleavage site of cephalosporin C acylase and the effect deleting its flanking residues in the α-C-terminal region. J Biotechnol 184:138-145. doi: 10.1016/j.jbiotec.2014.05.016




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