| The productivity of polar ocean is influenced by the polar sea ice. Pseudoalteromonas sp. SM495(Ps. sp. SM495) is a cold-adapted bacterium isolated from Arctic sea ice. According to our research results, the strain Ps. sp. SM495secretes abundant proteases. Among these proteases, E495was found to have three mature forms, which may be a strategy of this strain to efficiently degrade the organic nitrogen in sea ice. At the same time, Ps. sp. SM495secretes large amounts of enzymes related to carbon cycling. In this article we studied the interactions between Ps. sp. SM495and the diatom Fragilaria sp. strain04, and we also studied the characateristics of carbon hydrolase from Arctic sea ice, thus illuminating the ecological effect of Ps. sp. SM495.Diatom is the main producers in sea ice environment. Through microscopic observation, measuring the exopolysaccharides and the spectrometry, we found that when adding extracellular enzymes of Ps. sp. SM495to the diatom cultures, the expolysaccharides of diatom cultures could be degraded, resulting the decreased algal viscosity and the better-distributed diatom cultures. The color of the diatom changed from brown to green observed under light microscope.. In combination with the absorption spectra, it could be deduced that the extracellular enzymes could have an algae-lyric effect to certain extent.We selected the extracellular carbon hydrolase genes from Ps. sp. SM495: alginate lyases, endoglucanases and pectin lyases. The expression level of these genes at the medium containing diatoms were determined by Real-time quantitative PCR. These results showed diatom cultures had certain inducing effect on the expression of these genes. The expression level of endoglucanase increased most obviously. Then we found the inducing effect existed in the enzyme activity by measuring the activity. This suggests that a large number of endoglucanases may be related to the degradation of the diatom exopolysaccharides. This claim supports our previous observation at the level of transcription and biochemistry.And we purified the endoglucanase which couled be induced by diatom cultures. This enzyme contains a signal peptide, a catalytic domain (CD), one linker and a cellulose binding motif (CBM), which we named Ce1495. The wild enzyme was purified from the fermented liquid, which only contained the catalytic domain. And then the holoenzyme was obtained by heterologous expression, which we called recombinase. The optimal substrate of wild enzyme and recombinase are both sodium carboxymethyl cellulose (CMC-Na) and the optimal pH is7.0. The wild enzyme has a maxium activity at45℃, and is stable at40℃. And the recombinase has a maxium activity at35℃, and is stable at30℃. This suggested that linker of Ce1495was also flexible, which resulting the recombinase more cold-adapted. We studied the effect of metal ion and EDTA on the activity. The results suggested that some metal ions could be a promoters (Mn2+、Mg2+、Ca2+) or inhibitors (Cu2+、Zn2+、EDTA) for Ce1495. The different effects of ions on wild enzyme and recombinase implied that the linker and CBM of Ce1495could perhaps affect the binding of ions and enzymes. The influence of NaCl on enzyme activity shows that Ce1495is a salt-tolerant enzyme, which remains70%enzyme activity under4M NaCl. The recombinase has a higher activity than wild enzyme under0.5M NaCl (140%vs120%).By studying the interaction between Ps. sp. SM495and diatom Fragilaria sp. strain04, and the function of extracellular enzymes in the carbon degradation, we elucidated the ecology function of Ps. sp. SM495, and this work was also useful to clarify the mechanism of organic carbon degradation at Arctic sea ice environment. |
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