IAL   21557
INSTITUTO DE AGROBIOTECNOLOGIA DEL LITORAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Characterization of two glycoside phosphorylases from Ruminococcus albus 8
Autor/es:
STORANI A; GUERRERO SA; IGLESIAS AA
Reunión:
Congreso; SAIB-SAMIGE Joint Meeting 2020 on line; 2020
Institución organizadora:
Sociedad Argentina de Investigaciones Bioquímicas
Resumen:
Ruminococcus albus is one of the main ruminal bacteria that produce extracellular cellulolytic enzymes. Cello-oligosaccharides are predominant end-products of the degradation of cellulose and are crucial carbon sources for these bacteria. The phosphorolysis of this cello-oligosaccharides is believed to be the more important process for R. albus. It metabolizes cello-oligosaccharides through phosphorolysis, catalyzed by cellobiose phosphorylase (CBP) (EC 2.4.1.20) and cellodextrin phosphorylase (CDP) (EC 2.4.1.49). According to the sequence-based classification system of carbohydrate-active enzymes (CAZy), CBP and CDP are members of glycoside hydrolase (GH) family 94, together with N,N′-diacetylchitobiose phosphorylase (ChBP) (EC 2.4.1.280), and laminaribiose phosphorylase (EC 2.4.1.31). We identified two genes (CUS_6564 and CUS_7967) from the genome of R. albus 8 encoding putative glycoside phosphorylases. Both proteins belong to GH 94 family but its amino acid sequences differ considerably between each other (32 % identity). First one, present 60 % identity with characterized CBPs but the second one, present only 33 % identity with others CBPs and 35 % identity with CDPs and ChBPs. We hypothesized that CUS_6564 and CUS_7967 codify for CBP and CDP, respectively. To determinate it, both were expressed in E. coli and recombinant proteins were purified and biochemically characterized. Its quaternary structures were determined, and both correspond to a homodimer. Each protein was then investigated for its phosphorolytic capacity on different substrates as cellobiose, laminaribiose, lactose, cellulose, carboxymethyl cellulose (CMC) and RASC (resuspended acid swallowed cellulose), xylans, and chitosan. CBP present activity only with disaccharides cellobiose and lactose (kcat 152 and 0.03 s-1, respectively) while CDP in addition to cellobiose (kcat 19.7 s-1) present activity with cellulose, CMC, and RASC (kcat 0.010, 0.012, 0.015 s-1, respectively). We also tested their capacity to synthesize different oligosaccharides using D-glucose 1-phosphate as the donor and different acceptors like glucose, xylose, galactose, glucosamine, and cellobiose. CBP showed higher activity using glucose as acceptor over cellobiose (kcat 178 vs. 0.04 s 1). In contrast, CDP showed higher activity with cellobiose as acceptor (kcat 300 vs. 5 s-1). In addition, CDP showed the capacity to synthesize cello-oligosaccharides from cellobiose and Glc-1P (10 and 50 mM, respectively). This capacity was absent in CBP even when the reaction was carried out for 4 hours. These results indicate CUS_6564 and CUS_7967 as CBP and CDP, respectively. Our goal is to complete a detailed characterization of these enzymes, which would allow us to use them for different processes. While synthetic glycoside phosphorylase reactions can be used for the synthesis of high-value carbohydrates, phosphorolysis reactions can be used for polymer degradation in waste recycling and biofuel production.