Screening of glucansucrase activity and molecular aspects of novel homopolysaccharide producer lactic acid bacteria in sucrose?enriched media

LOBO, RENE EMANUEL; CATALDO, PABLO GABRIEL; HEBERT, ELVIRA MARIA; TORINO, MARÍA INÉS

Virtual

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

The Argentine Network of Enzymatic Technology (TEz Network)

Certain lactic acid bacteria (LAB) are capable of producing an extracelular α?D?glucan homopolysaccharide (HoPS) formed by glucose units when growing in sucrose-enriched media. Their structural diversity ia reponsible for their vast commercial value and application potential in the food, cosmetic, medical, and biotechnology fields, garnering much attention in recent years. In this sense, this HoPS type is synthesized in large amounts by extracellular free or LAB-anchored enzymes called glucansucrases (GSs: dextransucrase, mutansucrase, alternansucrase, and reuteransucrase) belonging to the GH70 family. Usually, these GSs use the glycosidic bond energy from sucrose to extend the polymerization of glucosyl units. Previously, we reported the bioprospection of thirty-one HoPS producer LAB belonging to the Weisella (W.) and Leuconostoc (Ln.) genera in a sucrose- enriched MRS medium (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. mesenteroides subsp. dextranicum CRL983) were purified and subsequently characterized, turning out to be all dextrans (α?glucan type). Thereby, we aimed to identify the GS encoding genes in the aforementioned five LAB strains and to evaluate the GS activity in different extracellular contexts (supernatant and cell wall-asociated) in MRS-S. Finally, we selected one strain with the highest GS activity for further studies: morphology (TEM), polymer production (PAS kit), bacterial growth (CFU.mL-1), sugar consumption (HPLC), and acidifying activity: pH and organic acids production (HPLC). Total DNA was extracted from the five LAB strains following the Pospiech and Neumann protocol for Gram positive bacteria. PCR were performed using oligonucleotides designed to amplify regions within specific GS genes. The resulting amplicons were sequenced and used as query the BLAST tool within the NCBI database (https://www.ncbi.nlm.nih.gov/). The screening for putative GS genes revealed a positive signal for all HoPS+ strains that possess high sequence identity with GS genes from other LAB strains of the same species. For example, the sequence analysis for FMy 2-21-1 and CRL983 strains showed 92.77% and 95.77% similarity to the gene coding for a dextransucrase of W. cibaria LBAE-K39 (GU237484.3) and Ln. mesenteroides subsp. dextranicum strain DSM 20484 (CP012009.1), respectively. Extracellular glucansucrase activity, either in the culture supernatant or associated with the cell pellets from the Ln., and W. strains, were determined in a enzymatic reaction mixture with 5% sucrose through the Somogyi and Nelson assay. The extracellular activities 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 in cell pellet which was higher than its supernatant cell-free activity (32.55 U/ Log(CFU.mL-1) and 10.66 U/Log(CFU.mL-1), respectively). This activity values could be due to higher expression of cell-wall associated GS, or a higher enzyme activity. Therefore, we selected the F-Cq1-496 strain to study their metabolic activity in the MRS-S culture medium (0, 3, 6, 10 and 16 h at 30°C). At the end of the fermentation, the LAB strain grew more than three logarithmic units of CFU.mL-1 (3.33 Log(CFU.mL-1) and reduced the pH of the culture medium by two pH units (2.16). The increase in acidity was associated with 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 a drastic decrease of sucrose in the culture medium at the end of the fermentation. Finally, mannitol production (>1.5 g.L-1 at 24 h) was observed during the fermentation and was associated with decreasing levels of fructose, which was hydrolyzed from sucrose. Therefore, fructose can either be used as a carbon source to obtain ATP or as an electron acceptor to synthesize mannitol and regenerate NAD via the mannitol dehydrogenase reaction. On the other hand, the TEM micrograph showed a strong interaction between the LAB strain and the polymer matrix generated. In conclusion, GS genes were detected in all studied LAB strains and though the extracellular enzyme activities were varied, only Ln. citreum F-Cq1-496 exhibited GS activity in their cellular pellet. Therefore, Ln. citreum strain was selected to evaluate different fermentation parameters, exhibiting the highest polymer production with effective performance in the studied sucrose-enriched medium.