Cloning, Expression And Character Determination Of The Hydrolase Catalysis Domain From A Thermophilic Esterase APE1547

Esterase (EC 3. 1 .1. 1) is widely expressed in animals, plants and microbes, and it becomes one of the most useful enzymes for its activity both in water and nonaqueous. Generally, enzymes that catalytic substrate only in normal temperature cannot meet the need of the industry, which limited the improvement of using of the enzymes. So far, thermophilic esterase becomes a hot spot in researching for its stable activity under high temperature, organic solvent and denaturing agent.Thermophilic esterase APE 1547 coming from Aeropyrum pernix K1 has two domains, it containsα/βhydrolytic domain in the C-terminal, and a seven blades formedβ-propeller domain in the N-terminal, 1-23 amino acid of which is also belongs to part of the catalytic domain. Previous research has reported that APE 1547 has an activity of both peptidase and esterase. After compared the catalytic domain of APE 1547 to other esterase and lipase, we decided to express the catalytic domain of APE 1547.In this paper, we linked a 23 amino acid of the N-terminal to the catalytic domain of the C-terminal by PCR and then linked to pET11a and pET15b vector to express the complete catalytic domain in the form of inclusion. We decided the optimized expression conditions that the catalytic domain is expressed best in 2YT culture at the IPTG concentration of 0.5 mM for 6 hours at 37℃. The inclusion is been refolded after washing the supersonic precipitation. Both the dialysed and chromatographic method can get activity esterase. The best refolding way is by gel filtration chromatography S-300, and the specific activity is 2394mU/mg, nearly 20% of that of the wild enzyme.The characterization of enzyme suggests that catalytic activity of the constructed enzyme decreased after losing the propeller domain. And also, it loses the high temperature stable activity from 90℃to 52℃, Arrhenius free energy degrade from 33.3 kJ/mol to 15.47 kJ/mol, and the optimized pH changes from 8.0 to 8.5. The optimized substrate is still pNPC8, but Km value increased from 14μM to 76μM and Kcat decreased from 805.8s-1 to 503s-1. However, the stability of the constructed enzyme decreased greatly when the concentration of the protein is 0.1mg/ml, the enzyme half-life is 90 min at 50℃, and the activity disappeared after 15 min at 60℃.The function of the enzyme closely involves to its structure. To identify the function of each domain, we eliminated the propeller domain of APE1547. The result shows the propeller domain plays an important role in substrate control and thermophilic stability, the catalytic domain mainly plays key role in catalysis. This work provides a good example for the molecular modification based on a nature enzyme.