Studies on the regulation, structure and function of a glucoamylase from Sacharophagus degradans.

WAYLLACE, N.; HEDIN, N; BUSI, M. V.; DIEGO FABIAN GOMEZ CASATI

Parana

Congreso; LIV Reunión de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Saccharophagusdegradans is a gram negative marine bacterium. It is the most versatile bacterium, in terms of the degradation of complexpolymers (CP) found to date. It is capable to degrade at least 10 complex polymers such as agar, alginate, chitin, cellulose, fucoidan, laminarin,pectin, pullulan, starch and xylan. The high rate of degradation of these polysaccharides makes this organism an excellent candidate to identifyand investigate the biological properties of the degradative enzymes. Thus, we identify a gene coding for a putative glucoamylase fromSaccharophagusdegradans (sdGA, accession number: ABD79864). The sdGA gene was synthesized by Genscript (USA) with codonoptimization for the expression in E. coli cells and cloned into pRSF-DUET vector (named pRSFDsdGA). The plasmid pRSFDSdGA wassuccessfully expressed in Escherichia coli BL21 Rosetta cells. Recombinant sd_GA is fused to a histidine tag at the N-terminal region of the protein. The full-length gene codes for a 803-amino acid polypeptide. The mature protein is a monomer with the molecular mass of 88 kDa.Recombinant sd_GA was purified using a nickel column. The recombinant enzyme showed maximum activity using maltose as substrate at 39°C and pH 6.0. Kinetic studies revealed that the Vmax and Km values of sd_GA are 0.0061 μmol/μg.min and 11,5 mM, respectively. Studies on the thermal stability of sd_GA and the effect of different metabolites on enzyme regulation were also performed. Our results show that both glyceroland acarbose are good stabilizers for sdGA.