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Highlights: [•] A novel gene encoding an α-galactosidase was cloned from deep sea bacterium. [•] Detailed enzymatic characteristics of the enzyme were studied. [•] Directed evolution of the enzyme was carried out by site-directed mutagenesis. [•] Two mutants with improved catalytic efficiency were obtained. [ABSTRACT FROM AUTHOR]

Adrenocorticotropic hormone (ACTH) is an old medicine derived from porcine pituitary gland that has been marketed for more than 60 years. In this study, we present a recombinant approach to produce ACTH in Escherichia coli (E. coli). The SUMO-tagged fusion protein was cloned and expressed after induction with isopropyl-β- d -thiogalactopyranoside (IPTG) at 25 °C for 8 h. The fusion protein was extracted and purified by anion exchange chromatography, and the SUMO tag was subsequently removed by digestion with ubiquitin-like protease 1 (ULP1). Approximately 95.3 mg of recombinant ACTH with 94.2% purity was obtained after cation exchange purification performed on a 5 mL column, from 286 mL fermentation broth based on the amount of pellets homogenized. The molecular mass of the recombinant ACTH was confirmed by mass spectrometry to equal 4567.32 Da. • ACTH fused with SUMO tag was cloned and expressed for the first time. • After parameter optimization, the yield of high-density fermentation of ACTH fusion protein reached 1.8 g/L. • The purity of ACTH prepared by chromatographic purification was more than 94%. • The analytical HPLC method for determining the purity of ACTH was disclosed for the first time. • This study developed an alternative route for large-scale preparation of ACTH. [ABSTRACT FROM AUTHOR]