Solubilization of Cephalosporin Acylase from Escherichia coli BL21 (DE3) Inclusion Bodies

2nd International Conference on Biosciences

Hardianto, D.^1 ; Isdiyono, B.W.^2 ; Royani, J.I.^1

Center of Technology for Pharmaceutical and Medical, Agency for the Assessment and Application of Technology, LAPTIAB Building, Puspiptek Area, Tangerang Selatan

15314, Indonesia^1

Faculty of Life Sciences, Surya University, Unity Tower Building, Boulevard Gading Serpong, Kav. M5 No. 21 Street, Serpong

15810, Indonesia^2

关键词: 7-aminocephalosporanic acid (7-ACA);    Acylase;    Cephalosporin antibiotics;    Enzymatic conversions;    Escherichia coli bl21;    Heterologous expression;    Inclusion bodies;    solubilization;   

Others  :  https://iopscience.iop.org/article/10.1088/1755-1315/197/1/012040/pdf
DOI  :  10.1088/1755-1315/197/1/012040

来源: IOP

【 摘 要 】

The 7-aminocephalosporanic acid (7-ACA) is a precursor for semisynthetic cephalosporin antibiotics. The enzymatic conversion of cephalosporin C (CPC) to 7-ACA is cephalosporin acylase (CA). Fragment DNA of CA gene was inserted in pET21a(+) and expressed in Escherichia coli BL21(DE3) (E. coli). Heterologous expression of foreign genes can produce about 30 % of soluble protein and 70% of insoluble protein as inclusion bodies (IBs) in E. coli. IBs are cytoplasmic aggregates of inactive protein and mostly consist of recombinant protein. The formation of IBs in E. coli is a challenge for recovery of recombinant protein in industrial scale. Solubilization and refolding are crucial process to obtain active recombinant protein from IBs. The purpose of this research was optimization of solubilization process to increase soluble CA from IBs. The cells of E. coli were disrupted by sonication. The pellet of IBs was washed by triton X-100 and solubilized by urea or guanidine HCl (GdnHCl). The transformant of E. coli containing pET21a(+)-acyII expressed CA as soluble and insoluble protein (IBs). Several methods were used for solubilization IBs pellet. The two-step denaturing (2DR) was the best method for solubilization IBs pellet.