Clonagem e expressão da enzima β-glucuronidase obtida de uma biblioteca metagenômica de terra preta de índio

Abstract

Enzymes are present in all living cells, where they act as biological catalysts in the metabolic control of all living organisms. Microorganisms are increasingly highlighted as a producer of these biocatalyst molecules. The metagenomic technique using molecular and bioinformatics tools makes it possible to discover new enzymes from any environmental sample. Black Earth the Indian is found in some regions of the Amazon, with a high content of organic matter due to the high biological activity in this environment. From a metagenomic library of Black earth the indian built and deposited at the Federal University of Amazonas, it was possible to identify a group of hydrolases enzymes. Within this group, the enzyme β-glucuronidase was screened, which plays a fundamental role in bacterial metabolism, as a reporter gene in plant transformation, in an anti-doping test and acts in the biotransformation of the glycyrrhizin molecule into derivatives that adds greater economic value to the pharmaceutical and cosmetic industry. β-glucuronidase was cloned and expressed in E coli. BL21 (DE3) pLysS using the plasmid pET28a (+) Promega as vector. Its molecular mass was estimated at 61 kDa. Its catalytic activity in 100mM sodium phosphate buffer using X-gal (20 mg / mL) (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) as substrate was better at 40 0C and pH 7. With kinetic parameters Km and Vmax 4.08 mg and 0.45 mn.min-1 respectively. The results of this project will contribute to the better study of the catalytic capacity, to compare its hydrolytic capacity with other commercial β-glucuronidases and thus make it more suitable for application in different biotechnological branches.