1 Volume 7 / Issue 1 / 9 / http://dx.doi.org/10.21931/RB/2022.07.01.9 Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique Julkipli Julkipli 1 , Khaswar Syamsu 1 and Ahmad Wibisana 2 * Abstract: Cephalosporin C acylase (CCA) is an essential enzyme for the one-step conversion of cephalosporin C into 7-aminocephalosporanic acid (7-ACA), an intermediate compound used to synthesize various semi-synthetic cephalosporin antibiotics. The industrial process prefers to use enzymes in immobilized form rather than soluble. A crosslinked enzyme aggregate (CLEAs) is a potential matrix-less enzyme immobilization technique to produce stable immobilized enzymes with high activity and low production costs. This study aimed to optimize the CCA immobilization using the CLEAs technique with Chitosan as a co-aggregate. The CCA lysate was obtained from harvesting CCA fermentation broth using a mutant strain of Escherichia coli through cell separation and lysis steps. Partially purified CCA by ammonium sulfate addition was conducted to obtain an active fraction of 20-60% saturation, followed by co-aggregation with Chitosan to form physical CCA aggregates. The aggregates were then immobilized by a crosslinking technique using glutaraldehyde to form CLEAs-CCA. Optimization of the immobilization process was carried out by Response Surface Methodology in three steps, (i) screening of the influencing factors, (ii) determining the level of the significant factors, and (iii) optimizing the immobilization condition. The CLEAs-CCA activity was used as a response parameter. Under optimum conditions, CLEAs-CCA activity obtained was 85.91 Ug -1 . Key words: 7-aminocephalosporanic acid, cephalosporin C acylase, Chitosan, crosslinked enzyme aggregates, response surface. methodology. ARTICLE / INVESTIGACIÓN Introduction Cephalosporins are antibiotics with a broad antibacterial spectrum and almost cover 50% of β-lactam antibiotics hu- mans use 1,2 . Most of them are derived from 7-aminocephalos- poranic acid (7-ACA), a precursor compound generated from hydrolysis of cephalosporin C (CPC), a natural antibacterial obtained from Acremonium chrysogenum fermentation 3 . The- refore, it is essential to produce 7-ACA efficiently. The industrial demands for efficient and environmentally friendly 7-ACA production have shifted from chemical to en- zymatic processes. Initially, a two-step bioconversion using D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase was developed 4 . Recently, one-step bioconversion using cephalosporin C acylase (CCA, EC 3.5.1.11) has paid the attention of researchers because it is more efficient 5 . The industrial application of enzymes is preferred in the immobilized rather than soluble form due to the more efficient bioconversion process 6 . Immobilization of enzymes on a matrix is a popular method. However, a dilution of enzyme up to 99% occurred, resulting in decreased activity 7 . In addition, the ma- trix used causes an increase in the enzyme cost and generates a waste matrix generated from the inactive biocatalyst 8 . To overcome these problems, enzyme immobilization by cross- linked enzyme aggregates (CLEAs) technique, a matrix-less immobilization approach, is a potential method to be applied 7 . It showed a simple, fast, and efficient method to immobilize various enzymes 8-10 . To the best of our knowledge, there is no publication on the application of CLEA for CCA immobilization. The CLEAs formation involves two critical steps, i.e., en- zyme precipitation to form aggregates and crosslinking of the aggregates 7 . Ammonium sulfate and glutaraldehyde are often used as precipitation and crosslinker agents, respectively 9,11,12 . However, crosslinking the enzyme with low superficial lysine residues with glutaraldehyde may cause weak enzyme bin- ding, making it quickly released from its CLEAs 13 . The addition of co-aggregates, such as bovine serum albumin, poly-ethyle- neimine, and poly-lysine, could form larger aggregates size and higher stability 9,12,13 . Chitosan is a relatively inexpensive biopolymer (polyaminosaccharides, copolymers of N-ace- tyl-D-glucosamine and N-amino-D-glucosamine) with high availability rich in reactive amino groups, potentials to mediate the formation crosslinks between the aggregates 14,15 . In this research, optimization of the CCA immobilization by CLEAs technique using Chitosan as a co-aggregate was stu- died. Optimization was conducted with Response Surface Me- thodology (RSM), as it is an effective and widely used method for optimization in bioprocesses, including enzyme immobili- zation 16-18 . CLEAs-CCA (Ug-1) activity was used as a response. The experiments were carried out in three steps, namely, (i) screening of the influencing factors using 2 n-2 fractional fac- torial design (FFD), (ii) determining the level of significant fac- 1 Biotechnology Study Program, Graduate School, IPB University, IPB Dramaga Campus, Bogor, Indonesia. 2 The National Research and Innovation Agency (BRIN), Tangerang Selatan, Banten, Indonesia and Chemical Engineering Study Program, Pamulang University, Witana Campus, Tangerang Selatan, Banten, Indonesia. Corresponding author: ahmad.wibisana@brin.go.id DOI. 10.21931/RB/2022.07.01.9 Citation: Julkipli J, Syamsu K and Wibisana A. Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique. Revis Bionatura 2022;7(1). 9. http://dx.doi.org/10.21931/RB/2022.07.01.9 Received: 13 June2021 / Accepted: 10 August 2021 / Published: 15 February 2022 http://www.revistabionatura.com Publisher’s Note: Bionatura stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).