J Appl Biomed 13:189-194, 2015 | DOI: 10.1016/j.jab.2014.11.003

The expression, purification and activity analysis of Francisella tularensis citrulline ureidase in Escherichia coli

Tingheng Zhu^a,*, Sanyu Fang^a, Weixia Wang^b, Kun Wang^a, Zhifeng Cui^a, Changchun Wang^c,*

^a College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China

^b China National Rice Research Institute, Hangzhou 310006, China

^c College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China

Citrulline ureidase (CTU, EC3.5.1.20) degrades citrulline into ornithine, carbon dioxide, and ammonia. Here, we present the report on expression of recombinant CTU in Escherichia coli. The soluble and active recombinant CTU was expressed in the periplasmic space with the vector pET-22b and the His-tagged CTU was purified with Ni-Affinity Chromatography. The yield of soluble recombinant protein was significantly increased when 1% sorbitol was supplemented in medium. By using phenylisothiocyanate (PITC) pre-column derivatization HPLC, the enzyme activity of recombinant CTU was determined via measuring of the substrate citrulline and the corresponding products. Our results could be useful in the study of CTU biochemical characteristics, enzymatic preparation of ornithine and development of an enzymatic detection method of citrulline.

Keywords: Citrulline ureidase; Ornithine; Citrulline; Francisella tularensis; Disease diagnosis

Received: August 25, 2014; Revised: November 20, 2014; Accepted: November 21, 2014; Published: July 1, 2015  Show citation