Caracterizacion de la sintesis del (1,3)(1,2)-b-d-glucano y de las bacterias lacticas productoras
- Autores: María Laura Werning Hernández
- Directores de la Tesis: Paloma López García (dir. tes.), Pilar Fernández de Palencia Delgado (codir. tes.)
- Lectura: En la Universidad Autónoma de Madrid ( España ) en 2010
- Idioma: español
- Tribunal Calificador de la Tesis: José Berenguer Carlos (presid.), Miguel Ángel de Pedro (secret.), Monica Amblar Esteban (voc.), Ana Irastorza (voc.), Maria Teresa Dueñas Chasco (voc.), Ernesto García López (voc.), Nieves García (voc.)
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- Resumen
Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented foods, and among them, some ß-glucans have immunomodulating properties.
Pediococcus parvulus 2.6 and other lactic acid bacteria (LAB) isolated from infected alcoholic beverages produce an identical (1,3)(1,2)-ß-D-glucan. The characterization of the synthesis of this EPS is the main objective of this work. The study was performed with a combination of genetic engineering, molecular microbiological, biochemical and biophysical approaches. The results obtained as well as their interpretation are detailed below.
We have identified and determine the nucleotide sequence of 6 glycosyltransferase (gtf) genes from LAB belonging to the Pediococcus, Lactococcus and Oenococus genera. Their genetic location in P. parvulus 2.6, L. diolivorans G77 and O. oeni I4 has been established. The gene is carried by two different plasmids of 35 kb and 5,5 kb in the first two hosts, whereas in O. oeni the gene is chromosomally located.
Bioinformatic analysis of the gtf gene product indicated that it is an enzyme that belongs to the GT-2 membrane-bond glycosyltransferase family. Moreover, the topological prediction of the GTF protein indicates that a cytosolic glycosyl transferase domain is flanked by two and four trans-membrane segments. Detection of glycosyltransferase activity in membrane preparations of GTF overexpressor strains strongly supports the bioinformatic predictions for the enzyme activity and location.
In addition, the heterologous expression of the P. parvulus 2.6R gtf in Streptococcus pneumoniae and in Lactoccus lactis enable these strains to synthesize a ß-glucan immunologically indistinguishable from that of S. pneumoniae serotype 37. The overproduction of the P. parvulus 2.6 GTF glucosyltransferase in L. lactis resulted in synthesis and secretion of 300 mg/l of the (1,3)(1,2)-ß-D-glucan in this host. The method used to purify the EPS allowed an 80% recovery of pure ß-glucan with a high molecular mass (6,5 x 106 Da) similar to that of the biopolymer synthesized by P. parvulus (9,6 x 106 Da).
An immunological method for detection of (1,3)(1,2)-ß-D-glucans has been standardized and validated in this work. The ELISA method is based in the use of anti-serotype 37 pneumococcal antibodies and allows direct quantification of the exopolysaccharide in the supernatant of its producing bacteria grown in complex media, which contain mono- and polysaccharides with a threshold of 18 ng/ml. The method is substantially specific for this ß-Dglucan, only detecting (1,3)(1,6)-ß-glucan at high concentrations (>10 ¿g/ml) and not interacting with linear (1,3)-ß-D-glucan or heteropolysaccharides.
The above described method has been also used for the in vitro enzymatic characterization of the GTF glucosyltransferase associated with membrane vesicles of L. lactis NZ9000. The results showed that the activity is optimal at 37ºC in the pH range of 6,5 to 7, it displays typical Michaelis Menten kinetics with respect to UDP-glucose (Km = 0,125 mM) and has a Vmax = 0,018 ¿g EPS/mg prot /min. Moreover, ß-glucan synthesis proceeds linearly with time up to 4 h, and catalysis is inhibited in the presence of UTP, UDP or EDTA. Finally, the chemical characterization of the reaction product revealed that indeed it is the (1,3)(1,2)-ß-D-glucan.
Finally, a comparative analysis has been performed of the potential probiotic properties of the ß-D-glucan producer P. parvulus 2.6R and its isogenic EPS-non-producer 2.6RN strain.
After exposure to gastrointestinal stress, both strains showed the same pattern of resistance, indicating that the ability to produce EPS does not protect P. parvulus in this sense. By contrast, the adhesion of 2.6R to Caco-2 cells was twenty four-fold higher than that of 2.6NR strain.
Moreover, the high adherence of the EPS producer (6%) to Caco-2 cells was partially abolished (decrease to 1.8%) after EPS removal by washing. Both strains induce the production of the anti-inflammatory cytokine IL-10 by polarized macrophages, however the levels of proinflammatory cytokines (TNF-¿ and IL-8) are higher in response to the 2.6NR strain, suggesting that EPS could be considered a beneficial immunomodulator.
