Influence of His-Tag on activitu, stability and immobilization of the lipase LipC12

ROTUNO MOURE V.; TONETTO G.; SANCHEZ D.; MITCHELL D.; FERREIRA M.; KRIEGER N.

Florianopolis

Congreso; XIII ENZITEC - Seminário Brasileiro de Tecnologia Enzimática; 2018

Universidade Federal de Santa Catarina y Universidade Federal da Fronteira Sul

LipC12 is a recombinant lipase from metagenomic library that shows activity and stability as high as many commercial lipases. LipC12 is expressed with a His-tag to facilitate its purification, but the presence of the His-tag can affect the immobilization procedure and the performance of the enzyme. In order to assess the influence of His-tag on activity, stability and immobilization of LipC12, we purified LipC12 with a His-tag at N-terminus (HisLipC12) and then removed the His-tag using tobacco etch virus (TEV) protease. As judged by circular dichroism analysis, the overall structure of LipC12 was largelly unaffected by His-tag removal. The specific hydrolysis activity of LipC12 (without His-tag) against olive oil was 4590 ± 176 U mg-1, approximately 80% higher than HisLipC12 (2555 ± 29 U mg-1). HisLipC12 showed significantly higher activity in the presence of acetone, DMF e DMSO and was also more stable than LipC12 in acetone, acetonitrile, DMF, DMSO, methanol, propanol, isopropanol and ethanol. The immobilization of HisLipC12 and LipC12 was performed with 100% efficiency. Again, the hydrolytic activity of immobilized LipC12 (123 ± 4 U g-1) was approximately 128% higher than HisLipC12 (54 ± 1 U g-1). On the other hand, the esterification activity for HisLipC12 was 9.21 ± 0.01 U g-1, while for LipC12 it was 5.26 ± 0.03 U g-1. After the esterification reaction, there was a loss of hydrolysis activity against olive oil of 46% for HisLipC12 and 76% for LipC12, suggesting that the removal of His-tag decreased lipase stability in n-hexane. In conclusion, the removal of His-tag altered activity and stability of the LipC12, with opposite effects in water and in organic media. The results presented provided new insights into the effect of His-tags on lipase activity and stability and show that, depending on the reaction medium, the presence of His-tag can be advantageous.