Janaina Marques Almeida

We determined the effect of the His-tag on the structure, activity, stability and immobilization of LipC12, a highly active lipase from a metagenomic library. We purified LipC12 with a N-terminal His-tag and then removed the tag using tobacco etch virus (TEV) protease. Circular dichroism analysis showed that the overall structure of LipC12 was largely unaffected by His-tag removal. The specific hydrolytic activities against natural and artificial substrates were significantly increased by the removal of the His-tag. On the other hand, His-tagged LipC12 was significantly more active and stable in the presence of polar organic solvents than untagged LipC12. The immobilization efficiency on Immobead 150 was 100% for both forms of LipC12 and protein desorption studies confirmed that the His-tag does not participate in the covalent binding of the enzyme. In the case of immobilized LipC12, the His-tag negatively influenced the hydrolytic activity, as it had for the free lipase, however, it positively influenced the esterification activity. These results raise the possibility of tailoring recombinant lipases for different applications, where the His-tag may be retained or removed, as appropriate for the desired activity.

β-Glucosidases are important enzymes with significant prospects in the industrial biotechnology, including their use in biomass hydrolysis for bioethanol production. In this study, the use of canola meal as carbon source for β-glucosidase production by a Trichoderma viride strain in submerged fermentation was evaluated by applying central composite design and response surface methodology to optimize the production process. This statistical approach was also used to improve the passion fruit peel hydrolysis by T. viride crude extract. The model developed 3.6-fold increased β-glucosidase activity. The culture conditions that resulted in the highest β-glucosidase levels were a substrate concentration of 2.9 %, pH of medium 4.2 and cultivation time of 206 h. The β-glucosidases produced under optimal conditions showed attractive properties for industrial applications, such as activity at high temperatures and stability at 55 °C and over a wide pH range. In addition, the enzymatic hydrolysis of passion fruit peel by T. viride crude extract was very promising, resulting in glucose yields of 66.4 %. This study, therefore, presents canola meal as an inexpensive and attractive substrate for the production of microbial β-glucosidases.