Characterization of a type I pullulanase from seawater and production of the recombinant enzyme

CASTILLO JULIETA DE LAS MERCEDES; CAMINATA LANDRIEL SOLEDAD; COSTA HERNÁN

Rosario

Workshop; V Encuentro & II Workshop de la Red Argentina de Tecnología Enzimática; 2023

Red Argentina de Tecnología Enzimática (RedTez)

Enzymes involved in processes of synthesis, degradation, and modification of glycoside and glycoconjugates are called CAZymes. Within this group of enzymes, glycoside hydrolases (GH) catalyze the hydrolysis and/or rearrangement of glycosidic bonds. GHs are classified into 180 families based on sequence similarities. Family 13 is the most complex because it presents more than 30 different types of enzymes including α-amylases, pullulanases, glycosyltransferases, and maltooligosaccharide-forming amylases.Pullulanases hydrolyze α-1,6 bonds of pullulan and act as starch debranchers, generating products with important applications in different industries. They are found mainly in bacteria isolated from different habitats like marine environments, polar regions, hypersaline lakes, and thermal waters. These enzymes are classified into type I and II pullulanases. Type I pullulanases specifically hydrolyze α-1,6 bonds of branched polysaccharides, whereas type II pullulanases also act on α-1,4 bonds. In this work, we characterized a type I pullulanase from an alkalophilic bacterial strain isolated from seawater. The purified type I pullulanase was stable for 1 h at pH 6-9, retaining 90 % of its enzymatic activity, whereas it retained 50 % activity at pH 12. The enzyme showed thermal stability when it was incubated at 20-40 °C for 1 h (90 % activity). In turn, it proved 50 % activity at NaCl concentrations more than twice observed in seawater. Moreover, the enzyme maintained its activity in presence of Tween 20 and it was partially inhibited by SDS. In contrast, the pullulanase was inhibited by the presence of multiple cations and EDTA. The kinetic parameters and activation energy of this enzyme were similar to other type I pullulanases.Complete genome sequencing of the bacterial strain allowed its identification as Exiguobacterium alkaliphilum strain AM5. In silico prediction of the primary structure of the type I pullulanase showed the structural domains described previously for these enzymes. The recombinant enzyme was produced in Escherichia coli and purified by FPLC ion exchange chromatography. The purified recombinant pullulanase was incubated with 1 % pullulan, and the hydrolysis products were analyzed by HPLC. Our results show that the recombinant enzyme depolymerizes pullulan as the wild-type enzyme.The type I pullulanase isolated from E. alkaliphilum strain AM5 characterized in this work could be potentially applied in starch transformation bioprocesses under alkaline conditions such as in detergent industry and wastewater treatment.