| As a major aldehyde pollutant that was existed widely in industry and our daily life, acetaldehyde is threating human health. The excess accumulation of acetaldehyde in body, especially arising from intemperance, will seriously damage our body in many ways. Deep sea microorganism is abundant and distinctive in heredity, physiology and ecological functions. Therefore, the development of acetaldehyde-degrading bacteria from deep sea provides a new method to harness acetaldehyde pollutant, prevent and treat alcoholic diseases and relieve discomfort caused by drinking.Acetaldehyde-degrading bacteria in the deep sea water of the South China Sea were enriched in situ and in the laboratory respectively, and their diversity were then studied by construction of16S rRNA gene clone library and sequencing. The bacterial groups and their quantity were basically the same between these two samples, including Proteobacteria, Actinobacteria, Firmicutes, Cyanobacteria and one unclassified bacteria. Four subgroups, α,γ,δ and ε,were found in Proteobacteria group. The γ-Proteobacteria was dominant (occupied80%clones). The species belong to γ-Proteobacteria and their proportion was nearly identical between two enrichment samples, and Vibrio was the predominant genus (55%), followed by Halomonas and Marinobacter (about10%).The ALDH (aldehyde dehydrogenase, ALDH) diversity in the east Pacific deep sea sediment sample were analyzed using a pair of primers that was designed by comparing the sequences of ALDHs in GenBank. A total of35partial sequences of ALDH gene, which belong to γ-Proteobacteria, Firmicutes, and Bacteroidetes, were obtained from constructed gene clone library. Among these three groups, the ALDH genes from γ-Proteobacteria were dominant (occupied74.7%,23gene types), and the ratio of ALDH gene from Firmicutes and Bacteroidetes were15.9%and9.4%, respectively. ALDH genes from Vibrio was most abundant in clone library (21.2%), followed by Halomonas (15.9%).A total of25acetaldehyde-degrading strains were isolated from three samples, including in-situ-enriched and lab-enriched samples of deep sea water from South China Sea, and lab-enriched sample of deep sea sediment from east Pacific, using acetaldehyde as the sole carbon source. Among them,21strains belong to γ-Proteobacteria, including Vibrio, Halomonas, Pseudoalteromonas, Pseudomonas, Psychrobacter and Marinobacter,4strains belong to Firmicutes, including Enterococcus, Planomicrobium and Bacillus. The result indicated that y-Proteobacteria play an important role in aldehyde metabolism in different types of deep sea environmental sample, and acetaldehyde-degrading bacteria belong to Vibrio and Halomonas existed widely in deep sea environment, whatever using culture-dependent or uncultured-dependent method. Five strains could tolerate1.5g/L acetaldehyde and degrade350mg/L acetaldehyde within24hours, including Halomonas sp. ACH-L-5, Halomonas sp. ACH-L-8, Vibrio sp. ACH-S-12, Psychrobacter sp. ACH-DSS-1C and ACH-DSS-10C2. They were worth for further study.A a-amylase gene amy608from Flammeovirga sp. Wpaga001was expressed in E.coli BL21(DE3) by using expression vector pCold I. The amy608consists of1875bp and encodes an enzyme protein of624amino acides whose predicted molecular mass was71414Da. The Amy608a-amylase has three conserved amino acid residues, Asp336, Glu365, Asp445, constituting the conserved catalytic site. The optimum temperature and pH for this enzyme were40℃and7.0, respectively. The thermal stability of this enzyme was poor. The Ca2+largely enhanced its activity. The Amy608a-amylase hydrolysed soluble starch, generated glucose and a series of maltooligosaccharides, including maltose, maltotriose, maltotetraose and the maltooligosaccharides who were higher degree of polymerization. |
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