Characterization of a new type I pullulanase isolated from Exiguobacterium alkaliphylum

CASTILLO DE LAS MERCEDES, JULIETA; BERTONERI, FLORENCIA AGOSTINA; CAMINATA LANDRIEL, SOLEDAD; COSTA, HERNÁN

Workshop; Tercer encuentro de la Red Argentina de Tecnología Enzimática - Primer workshop RedTEz; 2021

Red Argentina de Tecnología Enzimática (RedTEz)

Microorganisms are essential in production processes that involve enzymes due to their high production capacity, low cost, and potential of genetic manipulation. There is strong biotechnological interest in microbial enzymes applicable to different areas including food processing, agriculture, pharmaceuticals, and molecular biology. Pullulan is a polysaccharide consisting mainly of maltotriose units linked by α-1,6 glycosidic bonds. Pullulan degrading enzymes belong to Family 13 of glycoside hydrolases and are widely distributed in nature. Pullulanases are extracellular debranching enzymes and show specificity of action on α-1,6 linkages of pullulan, generating maltotriose. According to their mode of action and the hydrolysis products they generate on different α-glucans, they are classified into type I and II pullulanases. Type I enzymes specifically hydrolyze α-1,6 glycosidic linkages of branched α-glucans such as amylopectin, whereas type II act on the -1,4 and -1,6 bonds of different α-glucans. These enzymes generate various bioactive oligosaccharides by bioconversion of starch. Pullulanases are used as additives in detergents for washing dishes and clothes in alkaline conditions. Type I pullulanases are of interest for the food industry to produce maltose and maltotriose syrups and to obtain high purity fructose and glucose.By bioprospecting from different niches, we previously isolated several aerobic bacteria that produce amylolytic enzymes. A strain called AM5 obtained from a seawater sample was of interest because it produces pullulanase and can grow under alkaline conditions (pH 10) at 30 °C. We identified this isolate as Exiguobacterium alkaliphylum through biochemical tests and molecular studies of the 16S rRNA gene. Bacterial growth and enzyme production were evaluated for 5 days in minimal saline medium at pH 10 using 1% soluble starch or pullulan as a carbon source. The highest production of pullulanase was observed at 48 hours using pullulan as a substrate. Enzyme purification was carried out from the cell-free culture supernatant using pullulan-derivatized sepharose 6B column affinity chromatography. Furthermore, the crude extract was also purified by FPLC with a mono Q 5/50 GL ion exchange column. The purity of the fractions was analyzed by SDS-PAGE with silver staining and a single polypeptide chain with an apparent molecular weight of 105 kDa was detected. The enzyme was classified as type I pullulanase by determination of its activity on different α-glucans and analysis of tryptic digestion by mass spectrometry. The optimal conditions for enzyme activity were within a pH range of 7 to 9 and at 50 °C.Overall, the type I pullulanase isolated from Exiguobacterium alkaliphylum is of interest due to its potential use in bioprocesses of transformation of starch under alkaline conditions.