Identification and characterization of TeGA, a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus

WAYLLACE, NATAEL; HEDÍN, NICOLAS; BUSI, MARIA VICTORIA; DIEGO FABIAN GOMEZ CASATI

Virtual

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

SAIB - SAMIGE

Glucoamylases (GAs) are exo-amylases that hydrolyzeα-1,4 glycosidic linkages by the successive removal of glucose units from thenon-reducing end of starch and related substrates, releasing β-D-glucose. Theyare classified into the GH15 family of glycoside hydrolases (www.cazy.org).These enzymes also hydrolyze α-1,6 and α-1,3 glycosidic bonds but at a lowerrate. GAs play an important role in starch degradation, particularly inprocesses that involve its hydrolysis, such as food manufacturing, but also inthe pharmaceutical, textile and biofuel industries. The main application of GAs(sometimes together with α-amylases and pullulanases) occurs in the process ofsaccharification of partially processed starch or dextrins to obtain glucose.The preference for this type of enzymes in these industries is mainly due totheir high thermostability. Because of this, there is great interest inisolating new GAs suitable for new industrial applications. We identified anovel glucoamylase (TeGA) from Thermoanaerobacterethanolicus, a thermophilic anaerobic bacterium. The protein is composedmainly of an N-terminal GH15_N domain linked to a C-terminal catalytic domain,found in the GH15 family of glycosyl hydrolases. TeGA was expressed in E. coli (BL21) cells and its expressionwas optimized in order to obtain the highest amount of soluble protein. PurifiedTeGA showed a high optimum temperature (75°C), one of the highest specificactivities for a bacterial glucoamylase (75.3 U/mg) and it also remained stablein a wide pH range (from pH 3.0 to 10.0). Although the enzyme was active preferentiallywith small substrates such as maltose, it was also capable of hydrolyzingsoluble starch from potato, corn or rice. TeGA showed a high thermostability upto around 70°C, which was increased in the presence of PEG 8000, and alsoshowed to be stable in the presence of moderate concentrations of ethanol. We proposethat this novel GA could be suitable for use in different industrial processesthat require enzymes that act at high temperatures, such as the production ofbioethanol.