Glucansucrase approaches in novel homopolysaccharide producer lactic acid bacteria isolated from Argentinean fruits

LOBO, R.E:; CATALDO, P.G.; HEBERT, E.M.; TORINO, M.I.

Virtual

Congreso; Third Meeting & First Workshop of the Argentine Network of Enzymatic Technology (TEz Network); 2021

Certainlactic acid bacteria (LAB) are capable of producing an extracellular α?D?glucan homopolysaccharide (HoPS)formed by glucose units when growing in sucrose-enriched media. Theirstructural diversity is linked to their vast commercial value and applicationsin the food, cosmetic, medical, and biotechnology fields, reaching muchattention in recent years. In this sense, this HoPS type is synthesized inlarge amounts by extracellular free or cell-anchored enzymes calledglucansucrases (GSs: dextransucrase, mutansucrase, alternansucrase, andreuteransucrase) belonging to the GH70 family. Usually, these GSs use theglycosidic bond energy from sucrose to extend the polymerization of glucosylunits. Previously, we reported the bioprospection of thirty-one HoPS producerLAB strains, isolated from wild fruits of northern Argentina and belonging tothe Weisella (W.) and Leuconostoc (Ln.) genera in a sucrose- enriched MRSmedium (MRS-S). In addition, the HoPS from the selected producer strains (W.cibaria FMy 2-21-1, W. confusa CRL951, Ln. pseudomesenteroides F-G2-22, Ln.citreum F-Cq1-496 and Ln. dextranicum CRL983) were purified and subsequentlycharacterized, turning out to be all dextrans (α?glucan type). Thereby, we aimed to identifythe GS encoding genes in the aforementioned five LAB strains and evaluate theGS activity in both extracellular contexts (supernatant and cell pellet) ofMRS-S cultures. Finally, we selected one strain with the highest GS activityfor further studies: morphology (TEM), polymer production (PAS reaction),bacterial growth (CFU.mL-1), sugar consumption (HPLC), and acidifying activity:pH and organic acids production (HPLC). Total DNA was extracted following theprotocol described by Pospiech and Neumann for Gram positive bacteria and PCRreactions were performed using oligonucleotides designed to amplify regionswithin specific GS genes. The resulting amplicons were sequenced and used asquery the BLAST tool in the NCBI database (https://www.ncbi.nlm.nih.gov/). Thescreening for putative GS genes revealed a positive signal for all HoPS+strains that possess high sequence identity with GS genes from LAB strains ofthe same species. For example, the sequence analysis for FMy 2-21-1 and CRL983strains showed 92.77% and 95.77% similarity to the gene coding for a GS of W.cibaria LBAE-K39 (GU237484.3) and Ln. dextranicum strain DSM 20484(CP012009.1), respectively. Extracellular GS activity, either in the culturesupernatant or cell pellets, were determined in an enzymatic reaction mixturewith 5% sucrose through the Somogyi - Nelson assay. The extracellularactivities showed large variation among the strains, from 5.24 U/Log(CFU.mL-1)to 10.66 U/Log(CFU.mL-1) corresponding to CRL983 and F-Cq1-496 strains,respectively. By contrast, only Ln. citreum F-Cq1-496 exhibited activity incell pellet, which was higher than in supernatant [32.55 U/Log(CFU.mL-1) and10.66 U/Log(CFU.mL-1), respectively]. Therefore, we selected the F-Cq1-496strain to study their metabolic activity in MRS-S culture at 30ºC (0, 3, 6, 10and 16 h). At the end of fermentation, the LAB strain grew more than threelogarithmic units of CFU.mL-1 (3.33 Log(CFU.mL-1) and reduced the pH of theculture medium by two pH units (2.16). The increase in acidity was associatedwith the production of lactic acid (3.33 g.L-1) and acetic acid (2.17 g.L-1).Moreover, the polysaccharide production was 609 mg.L-1 in concomitance with adrastic decrease of sucrose. Finally, mannitol production (>1.5 g.L-1 at 24h) was observed associated to decreasing levels of fructose released fromsucrose. Therefore, fructose can either be used as a carbon source to obtainATP or as an electron acceptor to synthesize mannitol and regenerate NAD+ viathe mannitol dehydrogenase reaction. TEM micrograph showed a strong interactionbetween the LAB strain and the polymer matrix generated. In conclusion, GSgenes were detected in all studied LAB strains and though the extracellularenzyme activities were varied, only Ln. citreum F-Cq1-496 exhibited GS activityin their cellular pellet. Therefore, Ln. citreum strain was selected toevaluate different fermentation parameters, exhibiting the highest polymerproduction with effective performance in the studied sucrose-enriched medium