Sains Malaysiana 55(1)(2026): 1-17

http://doi.org/10.17576/jsm-2026-5501-01

 

Enhanced Expression of Recombinant Cephalosporin Acylase in Escherichia coli via Autoinduction Medium

(Pengekspresan Sefalosporin Asilase Rekombinan yang Dipertingkatkan dalam Escherichia coli melalui Medium Autoinduksi)

 

Ahmad Wibisana^1,8,*, Sasmito Wulyoadi¹, Rudiyono¹, Efrida Martius², Afada Naafi’u Fariha³, Fahroziah Assyifa³, Dihan Laziba³, Niknik Nurhayati⁴, Anna Safarida⁵, Uli Julia Nasution Mail⁶& Catur Sriherwanto⁷

 

¹Research Centre for Pharmaceutical Ingredients and Traditional Medicine, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia

²Research Centre for Vaccines and Drugs, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia

³Directorate of Laboratory Management, Research Facilities, and Science and Technology Area, National Research and Innovation Agency of Indonesia, B.J. Habibie Building, Jl. M.H. Thamrin No. 8, Central Jakarta, Indonesia

⁴Research Centre for Genetic Engineering, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia

⁵Research Centre for Applied Botany, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia

⁶Bureau of Organization and Human Resources, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia

⁷Research Centre for Applied Microbiology, National Research and Innovation Agency of Indonesia, Cibinong, Bogor, Indonesia

⁸Chemical Engineering, Pamulang University, Indonesia. Jl. Witana Harja 18b, Pamulang, South Tangerang, Indonesia

 

Diserahkan: 13 September 2024/Diterima: 6 Oktober 2025

 

Abstract

The large-scale synthesis of β-lactam antibiotics, particularly cephalosporins, relies heavily on the industrial production of recombinant cephalosporin acylase (CCA). This study aimed to identify the most suitable Escherichia coli host strain for CCA production. The selected strain was then used for optimizing medium components and culture conditions using an autoinduction medium in shaking flask fermentation. Initially, three E. coli strains were evaluated: BL21(DE3), BL21(DE3) RIPL, and B(DE3) Origami, to determine the most efficient host for recombinant CCA expression. Among the tested conditions, the autoinduction medium proved particularly effective, contributing to a significant improvement in enzyme yield and serving as the basis for subsequent optimization experiments. To identify the key factors influencing CCA production, a Plackett-Burman design was employed to screen medium components and fermentation parameters, with E. coli BL21(DE3) as the most optimal expression host. The significant variables were then optimized using a Central Composite Design, which resulted in a maximum enzyme activity of 53.3 U/mL under the following conditions: 0.78 g/L glucose monohydrate, 1.12 g/L glycerol, 6.21 mL/L trace elements, and an enzyme expression time of 19.48 h. The optimized conditions led to a 3.8-fold increase in CCA activity compared to unoptimized conditions. These findings provide valuable insights into efficient recombinant CCA production and have important implications for its industrial application in antibiotic synthesis.

Keywords: Central composite design; enzyme characterization; enzyme kinetics; enzyme purification; optimization

 

Abstrak

Sintesis berskala besar antibiotik β-laktam, terutamanya sefalosporin sangat bergantung kepada penghasilan industri sefalosporin asilase (CCA) rekombinan. Kajian ini bertujuan untuk mengenal pasti strain perumah Escherichia coli yang paling sesuai untuk pengeluaran CCA. Strain terpilih kemudiannya digunakan untuk mengoptimumkan komponen medium dan keadaan kultur menggunakan medium autoinduksi dalam penapaian kelalang goncang. Pada mulanya, tiga strain E. coli telah dinilai: BL21(DE3), BL21(DE3) RIPL dan B(DE3) Origami, untuk menentukan hos yang paling berkesan untuk pengekspresan CCA rekombinan. Antara keadaan yang diuji, medium autoinduksi terbukti sangat berkesan, menyumbang kepada peningkatan ketara dalam hasil enzim dan berfungsi sebagai asas untuk uji kaji pengoptimuman seterusnya. Untuk mengenal pasti faktor utama yang mempengaruhi pengeluaran CCA, reka bentuk Plackett-Burman telah digunakan untuk menapis komponen sederhana dan parameter fermentasi dengan E. coli BL21(DE3) sebagai hos pengekspresan yang paling optimum. Pemboleh ubah ketara kemudiannya dioptimumkan menggunakan reka bentuk komposit pusat yang menghasilkan aktiviti enzim maksimum 53.3 U/mL di bawah keadaan berikut: 0.78 g/L glukosa monohidrat, 1.12 g/L gliserol, 6.21 mL/L unsur surih dan masa pengekspresan enzim selama 19.48 jam. Keadaan yang dioptimumkan membawa kepada peningkatan 3.8 kali ganda dalam aktiviti CCA berbanding keadaan yang tidak dioptimumkan. Penemuan ini memberikan pandangan berharga tentang pengeluaran CCA rekombinan yang cekap dan mempunyai implikasi penting dalam aplikasi industri, terutamanya sintesis antibiotik.

Kata kunci: Kinetik enzim; pencirian enzim; pengoptimuman; penulenan enzim; reka bentuk komposit pusat

 

RUJUKAN

Briand, L., Marcion, G., Kriznik, A., Heydel, J.M., Artur, Y., Garrido, C., Seigneuric, R. & Neiers, F. 2016. A self-inducible heterologous protein expression system in Escherichia coli. Scientific Reports 6: 33037.

Bui, T., Patel, P. & Preuss, C.V. 2024. Cephalosporins. In StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK551517/ (Accessed on 21st May 2025).

Conti, G., Pollegioni, L., Molla, G. & Rosini, E. 2014. Strategic manipulation of an industrial biocatalyst - Evolution of a cephalosporin C acylase. FEBS Journal 281: 2443-2455.

Correddu, D., Montaño López, J.D.J., Angermayr, S.A., Middleditch, M.J., Payne, L.S. & Leung, I.K. 2020. Effect of consecutive rare codons on the recombinant production of human proteins in Escherichia coli. IUBMB Life 72(2): 266-274.

de Marco, A. 2009. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microbial Cell Factories 8: 1-18.

Faizullabhoy, M. & Wani, G. 2024. Cephalosporin drugs market - by generation (first, second), indication (respiratory, UTI, STI, gastrointestinal infection), route of administration (oral, parenteral, topical), type (generic, branded), distribution channel, global forecast 2024–2032. Global Market Insights Report ID: GMI8194. https://www.gminsights.com/industry-analysis/cephalosporin-drugs-market (Accessed on 21st May 2025).

Falak, S., Sajed, M. & Rashid, N. 2022. Strategies to enhance soluble production of heterologous proteins in Escherichia coli. Biologia 77: 893-905.

Gaurav, K., Kundu, K. & Kundu, S. 2010. Biosynthesis of cephalosporin-C acylase enzyme: Optimal media design, purification, and characterization. Artificial Cells, Blood Substitutes, and Biotechnology 38(5): 277-283.

Isakova, A., Artykov, A., Vorontsova, Y., Dolgikh, D., Kirpichnikov, M., Gasparian, M. & Yagolovich, A. 2023. Application of an autoinduction strategy to optimize the heterologous production of an antitumor bispecific fusion protein based on the TRAIL receptor-selective mutant variant in Escherichia coli. Molecular Biotechnology 65(4): 581-589.

Jobanputra, A.H. & Vasait, R.D. 2015. Cephalosporin C acylase from Pseudomonas species: Production and enhancement of its activity by optimization of process parameters. Biocatalysis and Agricultural Biotechnology 4(4): 465-470.

Julkipli, J., Syamsu, K. & Wibisana, A. 2022. Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique. Bionatura 7(1): 9.

Kataoka, K. & Takasu, A. 2020. IPTG-independent autoinduction of extracellular matrix proteins using recombinant E. coli as the expression host. Polymer Journal 53(2): 385-391.

Khani, M. & Bagheri, M. 2020. Protein expression and purification skimmed milk as an alternative for IPTG in induction of recombinant protein expression. Protein Expression and Purification 170: 105593.

Kim, S. & Kim, Y. 2001. Active site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification. Journal of Biological Chemistry 276(51): 48376-48381.

Kim, Y., Kim, S., Earnest, T.N. & Hol, W.G. 2002. Precursor structure of cephalosporin acylase: Insights into autoproteolytic activation in a new N-terminal hydrolase family. Journal of Biological Chemistry 277(4): 2823-2829.

Li, Z., Kessler, W., Van Den Heuvel, J. & Rinas, U. 2011. Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems. Applied Microbiology and Biotechnology 91(4): 1203-1213.

Li, X., Wang, J., Su, W., Li, C., Qu, G., Yuan, B. & Sun, Z. 2023. Characterization and engineering of cephalosporin C acylases to produce 7-Aminocephalosporanic acid. Molecular Catalysis 550: 113595.

Martius, E., Wibisana, A. & Ardiyani, Y. 2018. The optimization of soluble cephalosporin C acylase expression in E. coli. IJES 7: 29-34.

Mühlmann, M., Forsten, E., Noack, S. & Büchs, J. 2017. Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microbial Cell Factories 16(1): 220.

Nikolova, G., Georgieva, Y., Atanasova, A., Radulova, G. & Kapogianni, A. 2021. Autoinduction as means for optimization of the heterologous expression of recombinant single ‑ chain Fv (scFv) antibodies. Molecular Biotechnology 63(11): 1049-1056.

Niwa, M., Saito, Y., Sasaki, H. & Ishii, Y. 1994. Cephalosporin C acylase. U.S. Patent No. 5,336,613. Washington, DC: U.S. Patent and Trademark Office.

Pollegioni, L., Rosini, E. & Molla, G. 2013. Cephalosporin C acylase: Dream and(/or) reality. Applied Microbiology and Biotechnology 97: 2341-2355.

Rasyidah, M.A., Sismindari, S. & Purwanto, P. 2024. Cephalosporin C acylase: Important role, obstacles, and strategies to optimize expression in E. coli. Journal of Applied Pharmaceutical Science 14(10): 15-24.

Shahzadi, I. 2021. Scale-up fermentation of Escherichia coli for the production of recombinant endoglucanase from Clostridium thermocellum. Scientific Reports 11(1): 7145.

Shin, Y.C., Jeon, J.Y., Jung, K.H., Park, M.R. & Kim, Y. 2009. Cephalosporin C acylase mutant and method for preparing 7-ACA using same. U.S. Patent No. 7,592,168. Washington, DC: U.S. Patent and Trademark Office.

Sun, H., Liu, T., Luo, H., Nie, Z., Chang, Y., Yu, H. & Zhongyao, S. 2021. Optimization of cephalosporin C acylase expression in Escherichia coli by high-throughput screening a constitutive promoter mutant library. Applied Biochemistry and Biotechnology 193: 1056-1071.

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. Journal of Bioscience and Bioengineering 113(1): 36-41.

Zhang, W., Liu, Y., Zheng, H., Yang, S. & Jiang, W. 2005. Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis. Appl. Environ. Microbiol. 71(9): 5290-5296.

 

*Pengarang untuk surat-menyurat; email: ahma016@brin.go.id