• Siti Fatimah Zaharah Mohamad Fuzi ᵃFaculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia, 84600, Pagoh, Muar, Johor, Malaysia ᵇFuture Food Research Innovation, FAST, UTHM, 84600, Pagoh, Muar, Johor, Malaysia
  • Nur Atiqah Lyana Nor Ashikin Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia, 84600, Pagoh, Muar, Johor, Malaysia
  • Low Kheng Oon Malaysia Genome Institute & Vaccine, National Institutes of Biotechnology, Jalan Bangi, 43000 Kajang, Selangor, Malaysia



Immobilized E. coli, recombinant xylanase, graphene oxide, induction time, cultural conditions


Escherichia coli is the most prevalent host organism for the production of recombinant en-zymes. This was feasible due to the possibility of genetic modification and the availability of multiple E. coli strains as recombinant systems. The primary disadvantage of using E. coli as a host, however, is bacterial cell lysis due to tension build-up in the periplasmic space caused by the overexpression of the recombinant enzyme. Therefore, immobilization is preferable to cytoplasmic excretion for directing the expression of recombinant enzymes into the culture medium. This research investigated the effect of graphene oxide (GO) on the xylanase and β-galactosidase activity of immobilized recombinant E. coli. The effect of culture conditions (expression medium, IPTG, post induction temperature, post induction duration, agitation rate, and pH) on xylanase excretion and cell survival of an immobilized cell was studied using the one factor at a time (OFAT) method. After 24 hours of induction, using terrific broth (TB) as a medium increased xylanase excretion to 0.060 U/ml and resulted in decreased β-galactosidase activity (1.218 U/ml). Apart from that, a lower concentration of isopropyl -D-1-thiogalactopyranoside (IPTG) at 0.01 mM, a lower post-induction temperature (25°C), a 5-hour post-induction time, neutral pH, and 150 rpm significantly increased the xylanase excretion of immobilized cells with low β-galactosidase activity. This study established that immobilizing recombinant E. coli on GO may be advantageous for the excretion of recombinant proteins with a high cell viability.


Man, R. C., Ismail, A. F., Ghazali, N. F., Fuzi, S. F. Z. M., & Illias, R. M. 2015. Effects of the Immobilization of Recombinant Escherichia coli on Cyclodextrin Glucanotransferase (CGTase) Excretion and Cell Viability. Biochemical Engineering Journal. 98: 91-98. Doi: 10.1016/j.bej.2015.02.013.

Yoon, S. H., Kim, S. K., & Kim, J. F. 2010. Secretory Production of Recombinant Proteins in Escherichia coli. Recent Patents on Biotechnology. 4(1): 23-29. Doi: 10.2174/187220810790069550.

Arnau, J., Yaver, D., Hjort, C. M. 2019. Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories. Grand Challenges in Fungal Biotechnology. 27: 179-210. Doi: 10.1007/978-3-030-29541-7_7.

Chukwuma, O. B., Rafatullah, M., Tajarudin, H. A., Ismail, N. 2022. Lignocellulolytic Enzymes in Biotechnological and Industrial Processes: A Review. Sustainability. 12: 7282. Doi: 10.3390/su12187282.

Chandra, P., Enespa, Singh, R., Arora P. K. 2020. Microbial Lipases and Their Industrial Applications: A Comprehensive Review. Microbial Cell Factories. 19: 169. Doi: 10.1186/s12934-020-01428-8.

Choi, J. H., & Lee, S. Y. 2004. Secretory and Extracellular Production of Proteins Using Escherichia coli. Applied Microbiology and Biotechnology. 64(5): 625-635. Doi: 10.1007/s00253-004-1559-9.

Wyre, C., & Overton., T. W. 2014. Use of a Stress‑Minimisation Paradigm in High Cell Density Fed‑Batch Escherichia coli Fermentations to Optimise Recombinant Protein Production. Journal of Microbiology Biotechnology. 41: 1391-1404. Doi: 10.1007/s10295-014-1489-1.

Ramchuran, S. O., Olle, H., & Nordberg, K. 2005. Effect of Post Induction Nutrient Feed Composition and Use of Lactose as Inducer During Production of Thermostable Xylanase in Escherichia Coli Glucose-limited Fed-batch Cultivations. Journal of Bioscience and Bioengineering. 99(5): 477-84. Doi: 10.1263/jbb.99.477.

Morowvat, M. H., Babaeipour, V., Memari, H. R., & Vahidi, H. 2015. Optimization of Fermentation Conditions for Recombinant Human Interferon Beta Production by Escherichia coli Using the Response Surface Methodology. Jundishapur Journal of Microbiology. 8(4): 1-11. Doi: 10.5812/jjm.8(4)2015.16236.

Nor Ashikin, N. A. L., Fuzi, S. F. Z. M., Abdul Manaf, S. A., Abdul Manas, N. H., Md Shaarani@ Md Nawi, S., Md Illias, R. 2022. Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability. Arabian Journal of Chemistry,15: 6,103803. Doi: 10.1016/j.arabjc.2022.103803.

Ghoshoon, M. B., Raee, M. J., Shabanpoor, M. R., Dehghani, Z., Ebrahimi, N., Berenjian, A., Negahdaripour, M., Hemmati, S., Sadeghian, I., Ghasemi, Y. 2022. Whole cell immobilization of Recombinant E. coli Cells by Calcium Alginate Beads; Evaluation of Plasmid Stability and Production of Extracellular L-asparaginase. Separation Science and Technology. 57(17): 2836-2842. Doi: 10.1080/01496395.2021.1962910.

Tripathi, N. K., Shrivastava, A. 2019. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Frontiers In Bioengineering & Biotechnology. 20(7): 420. Doi: 10.3389/fbioe.2019.00420.

Rosano, G. L., Morales, E. S., Ceccarelli, E. A. 2019. New Tools for Recombinant Protein Production in Escherichia coli: A 5-Year Update. Protein Science. 28: 1412–1422. 10.1002/pro.3668.

Nor Ashikin, N. A. L., Wahab, M. K. H. A., Illias, R. M., Fuzi, S. F. Z. M. 2017. Comparison of Thermostable Xylanase Production by Escherichia coli Immobilised onto Different Nanoparticles. Chemical Engineering Transactions. 56: 1825-1830. Doi: 10.3303/CET1756305.

Gao, Y., Zheng, H., Hu, N., Hao, M., Wu, Z. 2018. Technology of Fermentation Coupling with Foam Separation for Improving the Production of Nisin using a κ-carrageenan with Loofa Sponges Matrix and an Hourglass-shaped Column. Biochemical Engineering Journal. 133: 140-148. Doi: 10.1016/j.bej.2018.02.008.

Wang, C., Chen, X., Jiang, X., Li, N., Zhu, P., Xu, H. 2022. Facile and Green Synthesis of Reduced Graphene Oxide/loofah Sponge for Streptomyces Albulus Immobilization and ε-poly-l-lysine Production. Bioresource Technology. 349: 126534. Doi: 10.1016/j.biortech.2021.126534.

Wang, L., Yin, Y., Zhang, S., Wu, D., Lv, Y., Hu, Y., Wei, Q, Yuan, Q., Wang, J. 2019. A Rapid Microwave-assisted Phosphoric-Acid Treatment on Carbon Fiber Surface For Enhanced Cell Immobilization In Xylitol Fermentation. Colloids and Surfaces B: Biointerfaces. 175: 697-702. Doi: 10.1016/j.colsurfb.2018.12.045.

Zhang, J., Zhang, J., Zhang, F., Yang, H., Huang, X., Liu, H., & Guo, S. 2010. Graphene Oxide as a Matrix for Enzyme Immobilization. Langmuir. 26(9): 6083-6085. Doi:10.1021/la904014z.

Ammar, A., Al, A. M., Alali, M., & Karim, A. 2016. Influence of Graphene Oxide on Mechanical, Morphological, Barrier, and Electrical Properties of Polymer Membranes. Arabian Journal of Chemistry. 9(2): 274-286. Doi: 10.1016/j.arabjc.2015.07.006.

Zajkoska, P., Martin, R., & Michal, R. 2013. Biocatalysis with Immobilized Escherichia coli. Applied Microbiology and Biotechnology. 97: 1441-1455. Doi: 10.1007/s00253-012-4651-6.

Saifuddin, N., Raziah, A. Z., & Junizah, A. R. 2013. Carbon Nanotubes: A Review on Structure and Their Interaction with Proteins. Journal of Chemistry. 1-16. Doi: 10.1155/2013/676815.

Hakimi, M. K., Anuar M., & Illias, R. M. 2016. Thermostability Enhancement of Xylanase Aspergillus fumigatus RT-1. Journal of Molecular Catalysis B: Enzymatic. 134(A): 154-163.

Fuzi, S. F. Z. M., Farah, D. A. B., Abdul, M. A. M., & Rosli, M. I. 2011. Development and Validation of a Medium for Recombinant Endo- Β -1, 4-Xylanase Production by Kluyveromyces lactis. Annals of Microbiology. 62(1). Doi: 10.1007/s13213-011-0258-x.

Man, R. C., Ismail, A. F., Fuzi, S. F. Z. M., Ghazali, N. F., Md Illias, R. 2016. Effects of Culture Conditions of Immobilized Recombinant Escherichia coli on Cyclodextrin Glucanotransferase (CGTase) Excretion and Cell Stability. Process Biochemistry. 98: 91-98. Doi: 10.1016/j.bej.2015.02.013.

Afzali, N., Bahareh, R. Z., & Nahidhagh, N. 2016. Optimization of Trichoderma reesei Medium for Increasing Xylanase Enzyme Production. Advances in Bioresearch. 7: 94-99. Doi: 10.15515/abr.0976-4585.7.3.9499.

Papaneophytou, C. P., & Kontopidis, G. 2014. Statistical Approaches to Maximize Recombinant Protein Expression in Escherichia coli: A General Review. Protein Expression and Purification. 94: 22-32.

Joseph, B. C., Suthakaran P., Sankaranarayanan, S., & Musti, M. 2015. An Overview of the Parameters for Recombinant Protein Expression in Escherichia coli. Cell Science & Therapy. 6(5): 1-7. Doi: 10.4172/2157-7013.1000221.

Ramírez-Nuñez, J., Romero-Medrano, R., Nevárez-Moorillón, G. V., & Gutiérrez-Méndez, N. 2011. Effect of pH and Salt Gradient on the Autolysis of Lactococcus lactis Strains. Brazilian Journal of Microbiology. 52: 1495-1499. Doi: 10.1590/S1517-83822011000400036.

Norsyahida, A., Rahmah, N., & Ahmad, R. M. Y. 2009. Effects of Feeding and Induction Strategy on the Production of Bmr1 Antigen in Recombinant E. Coli. Applied Microbiology. 49: 544-50. Doi: 10.1111/j.1472-765X.2009.02694.x.

Nkohla, A., Okaiyeto, K., Olaniran, A. & Nwodo, U. 2017. Optimization of Growth Parameters for Cellulase and Xylanase Production by Bacillus Species Isolated from Decaying Biomass. Journal of Biotech Research. 8: 33-47.

Fatokun, E. N., Uchechukwu, U. N., Ademola, O. O., & Anthony, I. 2017. Optimization of Process Conditions for the Production of Holocellulase by a Bacillus Species Isolated from Nahoon Beach Sediments. American Journal of Biochemistry and Biotechnology. 13(2): 70-80. Doi: 10.3844/ajbbsp.2017.70.80.

Khusro, A., Kannan, B., Valan, M. & Agastian, P. 2016. Statistical Optimization of Thermo-Alkali Stable Xylanase Production from Bacillus tequilensis Strain. Electronic Journal of Biotechnology. 22: 16-25. Doi: 10.1016/j.ejbt.2016.04.002.

Shang, T., Si, D., Zhang, D. & Liu, X. 2017. Enhancement of Thermoalkaliphilic Xylanase Production by Pichia Pastoris Through Novel Fed-Batch Strategy in High Cell-Density Fermentation. BMC Biotechnology. 17(55): 1-10. Doi: 10.1186/s12896-017-0361-6.

Behnam, S., Karimi, K., Khanahmadi, M. & Salimian, Z. 2016. Optimization of Xylanase Production by Mucor indicus, Mucor hiemalis, and Rhizopus oryzae through Solid State Fermentation. Biological Journal of Microorganism. 4(16): 1-10. Doi: 10.1515_tjb-2016-0036.

Patel, H., & Gupte, A., G. 2016. Optimization of Different Culture Conditions for Enhanced Laccase Production and Its Purification from Tricholoma giganteum AGHP. Bioresourcing and Bioprocessing. 3(11): 1-10. Doi: 10.1186/s40643-016-0088-6.

Nor, S., Azaman, A., Ramanan, R., & Tan, J. 2010. Screening for the Optimal Induction Parameters for Periplasmic Producing Interferon-Α2b in Escherichia coli. African Journal of Biotechnology. 9(38): 6345-6354. Doi: 10.5897/AJB10.556.

Krause, M., Ukkonen, K., Haataja, T., & Ruottinen, M. 2010. A Novel Fed-batch Based Cultivation Method Provides High Cell-Density and Improves Yield of Soluble Recombinant Proteins in Shaken Cultures. Microbiology Cell Factories, 9(11): 1-11. Doi: 10.1186/1475-2859-9-11.

Jiang, Y. M., Wang, Y. T. & Dong, Z. W. 2007. Effects of Induction Starting Time and Ca2+ on Expression of Active Penicillin G Acylase in Escherichia coli. Biotechnology Progress. 23(5): 1031-1037. Doi: 10.1021/bp070100.

Huang, R., Chao, Z., Hui, T., & Ruming, Z. 2013. Effect of Culture Conditions on Production of Xylanase by Trichoderma reesei. Advanced Materials Research. 784: 856-860. Doi: 10.4028/

Silva, L. C. A., Honorato, T. L., Cavalcante, R. S., Franco, T. T., & Rodrigues, S. 2016. Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced under Solid Stated Fermentation by Trichoderma spp. Indian Journal of Microbiology. 52(1): 60-65. Doi:10.1007/s12088.

Cavalieri, N., Guimaraes, D. A., Carvalho, S. D., & Nogueira, P. 2013. Effect of Xylanase from Aspergillus niger and Aspergillus flavus on Pulp Biobleaching and Enzyme Production using Agroindustrial Residues as Substract. Bioprocess and Biotechnology. 2-9. Doi: 10.1186/2193.

Andrew, G. M., Keith, F. T. 2022. Parameter Reliability and Understanding Enzyme Function. Molecules. 27(1): 263. Doi:10.3390/molecules27010263.






Science and Engineering

How to Cite