Sucrose degradation in oxigenic photosynthetic organisms by amylosucrase

M. PEREZ-CENCI; G.L. SALERNO

Rosario

Congreso; VIII Congreso Argentino de Microbiología General; 2013

SAMIGE

Sucrose (Suc) metabolism occurs mainly in oxygenic photosynthetic organisms (plants andcyanobacteria). The disaccharide breakdown is carried out by the action of acid or neutral invertases that catalyze the irreversible hydrolysis of Suc into hexoses, or by Suc synthase (SuS), a freely reversible that provides sugar nucleotide for polysaccharide synthesis. In cyanobacteria, Suc was reported as an osmoprotectant molecule. On the other hand, there are only few reports on Suc metabolism in other prokaryotes. It has been found in halotolerant methylotrophic bacteria, where it accumulates in response to salinity. Suc degradation in these bacteria is achieved by amylosucrases (AS). The aim of this work was to study an AS present in an oxygenic photosynthetic organism and the functional characterization of its encoding gene in a euryhaline cyanobacterium strain. For functional characterization, the 1,986-bp orf sequence was PCR-amplified, cloned, and expressed in E. coli using the pRSET system. Since AS catalyze not only Suc hydrolysis but also its isomerization and polymer synthesis, we measured the three activities in crude cell extract prepared from recombinant E. coli BL21 cells after incubation with Suc at three different temperatures. The optimum temperature for hydrolysis and transglycosylation was found to be around 30ºC. The purified His6::AS recombinant protein had a maximum activity at pH between 6.5 and 7.0. The apparent molecular mass of the recombinant polypeptide was calculated to be about 78 kDa by SDS-PAGE, which is in accordance with the predicted deduced amino-acid sequence. Expression analysis by RT-PCR shows that AS transcripts increase after a 2, 6, and 24 h sodium chloride addition. Interestingly, homologous sequences were retrieved from 3 among 63 cyanobacterial sequenced genomes. This is the first report on the cleavage of Suc by an AS bacterial-type in an oxygenic photosynthetic organism. Taking together, our results led us to conclude that cyanobacterial AS might have been acquired by lateral gene transfer from  Non-photsynthetic bacteria.