Purification and refolding protocol for cold-active recombinant esterase Aa SGNH1 from Aphanizomenon flos-aquae expressed as insoluble inclusion bodies

• Published in: Preparative biochemistry & biotechnology 2022-04, Vol.52 (4), p.394-403
• Main Authors: Knepp, Zachary J, Ghaner, Ashlea, Root, Kyle T
• Format: Article
• Language: English
• Subjects: Aphanizomenon; Cloning, Molecular; Detergents - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Esterases - metabolism; Inclusion Bodies - chemistry; Recombinant Proteins; Renal Dialysis
• ISSN: 1082-6068; 1532-2297
• DOI: 10.1080/10826068.2021.1952601

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene ( SGNH1), originating from the cyanobacterium , was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB . Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of -nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5-15 °C) and under alkaline conditions (pH 8-10). It was found that the kinetic properties of SGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.