화학공학소재연구정보센터
Process Biochemistry, Vol.103, 71-77, 2021
Extracellular expression of natural cytosolic nitrilase from Rhodococcus zopfii through constructing a transmembrane tunnel structure in Escherichia coli cells
Nitrilases are a class of industrially important hydrolases that convert nitriles to carboxylic acids. As a natural cytosolic enzyme, production of nitrilase suffer from inclusion body and cumbersome purification procedure. Extracellular expression of recombinant proteins offers advantages including improved protein folding and simplified downstream bioprocesses, which is more suitable for industrial production. Herein, extracellular expression of nitrilase (RzNIT) from Rhodococcus zopfii in Escherichia coli cells was investigated using active and passive transport strategies. The active transport of RzNIT mediated by N-terminal fused signal peptides and secretory proteins had negative effects on both solubility and activity. For the passive transport, PhiX174 protein E was co-expressed to form a 40-70 nm transmembrane tunnel structure in cell membranes. RzNIT was spon-taneously released from cytoplasm into extracellular medium through the constructed hole. Under the optimum conditions, the extracellular RzNIT activity reached 7.6 U/mL (86 % of its total activity) toward 3-cyanopyridine. The calculated concentration of extracellular RzNIT reached 0.13 mg/mL. The obtained results provide a novel and efficient strategy for extracellular production of recombinant nitrilases.