title: Extracellular expression of natural cytosolic arginine deiminase from Pseudomonas putida and its application in the production of L-citrulline.
abstract: The Pseudomonas putida arginine deiminase (ADI), a natural cytosolic enzyme, and Thermobifida fusca cutinase were co-expressed in Escherichia coli, and the optimized cutinase gene was used for increasing its expression level. 90.9% of the total ADI protein was released into culture medium probably through a nonspecific leaking mechanism caused by the co-expressed cutinase. The enzymatic properties of the extracellular ADI were found to be similar to those of ADI prepared by conventional cytosolic expression. Extracellular production of ADI was further scaled up in a 3-L fermentor. When the protein expression was induced by IPTG (25.0μM) and lactose (0.1gL(-1)h(-1)) at 30°C, the extracellular ADI activity reached 101.2UmL(-1), which represented the highest ADI production ever reported. In addition, the enzymatic synthesis of l-citrulline was performed using the extracellularly expressed ADI, and the conversion rate reached 100% with high substrate concentration at 650gL(-1).
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Lingqia
authors.initials: L
authors.lastname: Su
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: jingwu@jiangnan.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000192:Actinomycetales / Q000235:genetics; D001120:Arginine / Q000378:metabolism; D019149:Bioreactors; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli; D005285:Fermentation; D006867:Hydrolases / Q000378:metabolism*; D007785:Lactose; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics
chemical_list: D003470:Culture Media; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; C019723:arginine deiminase; D007785:Lactose
keywords: Arginine deiminase; Extracellular expression; Fermentation optimization; Pseudomonas putida; l-Citrulline