| China has a large ocean area.The microbial resources in seawater are abundant,and marine bacteria are the most abundant ones.Due to the complexity and particularity of the living environment,marine bacteria can secrete extracellular polysaccharides?EPSs?with novel structure and high biological activity.EPSs play an important role in the self-protection of marine bacteria.The problem of ocean acidification and warming caused by environmental pollution is increasing,forcing marine bacteria to respond to changes in these environmental conditions by regulating the metabolism of intracellular and extracellular substances.At present,there are few reports on the environmental conditions affecting the structure and biological activity of marine bacterial extracellular polysaccharides.In this study,P.agarivorans Hao 2018 isolated from the surface of the abalone seedlings was used as the research object to analyze the changes of the primary structure and biological activity of the EPSs under different environmental conditions.The effect of environmental factors on the structure-activity relationship of EPSs was analyzed.Through the genome and transcriptome sequencing,the complete genome map of the strain was drawn and the metabolic regulation of carbohydrates was analyzed.The results obtained are as follows:The extracellular polysaccharide of P.agarivorans Hao2018 under different environmental conditions?temperature,pH and sea salt concentration?was isolated.The crude EPSs were extracted by water extraction and alcohol precipitation.The free protein was removed by sevage method.The purification of EPSs through anion exchange column and dextran gel column.After dialysis and desalting,the pure EPSs were obtained by lyophilization.The monosaccharide composition,glycosidic bond configuration,major glycocyclic form and degree of methylation of the EPSs were analyzed.The monosaccharide composition of the extracellular polysaccharide is glucose,mannose and rhamnose,and the monosaccharide ratio changes under different environmental conditions.Compared with the optimized fermentation conditions?25°C,pH8,sea salt concentration of 4.5%?,the change of environmental factors caused the increase of mannose ratio and the decrease of glucose ratio in different degrees.The change of pH caused the biggest change of monosaccharide composition ratio.The mannose ratio increased from 4.5%?pH 8?to 35.96%?pH 9?,and the glucose ratio decreased from 92.90%?pH 8?to 59.36%?pH 9?.The results of infrared spectroscopy indicated that the changes of temperature and pH increased the methylation degree of extracellular polysaccharides.With the increase of temperature,the glycosidic bond configuration showed a change of???+???.The change of the composition of EPSs in different pH and sea salt concentration conditions is similar to the temperature;the main sugar ring form has a transition process of pyran type?15°C??pyran type?25°C??furan type?35°C?with increasing temperature.With the increase of pH,the transition process of furan type?pH7??pyran type?pH8??furan type?pH9?appeared,and furan type?3.5%??pyran type?4.5%??pyran type?5.5%?is a conversion process with the increase of sea salt concentration.In the study of antioxidant activity,the EPSs were found to have higher clearing ability of OH·and O2·-,and total reducing power was lower than ascorbic acid.The change of temperature can improve the ability of EPSs to scavenge OH·and O2·-,and the scavenging ability of EPS-35°C is the highest,reaching 22.13%?OH·?and 36.78%?O2·-?;the pH change is also improved the EPSs'ability to scavenge OH·and O2·-,the scavenging ability of EPS-pH9 reached 28.63%?OH·?and 40.41%?O2·-?;the EPSs isolated from different sea salt concentrations had the similar ability to remove free radicals.The removal ability of OH·was around 18%,while the removal ability of O2·-was between 29%and 32%.Further structure-activity relationship analysis showed that the increase of mannose ratio can enhance the capture ability of extracellular polysaccharides to OH·and O2·-.Methylation is beneficial to the increase of antioxidant activity of EPSs.The antioxidant activity of the EPSs with the glycosidic bond of?is superior to that of?+?.The antioxidant activity of EPSs with the main sugar ring form of furan-type is superior to that of pyran type.The genome of P.agarivorans Hao2018 consists of two circular chromosomes and one plasmid.Transcriptome analysis showed that 1189 genes were up-regulated and 1088genes were down-regulated in the pre-stationary period?24 h?of the strain compared with the pre-logarithmic growth phase?2 h?.A large number of genes related to glucose metabolism are up-regulated,and genes associated with the conversion and metabolism of mannose are down-regulated,which is echoed with the composition of monosaccharides?glucose:mannose:rhamnose=92.90:4.50:2.60?under optimized conditions.This result provides a new theoretical basis for further study of the metabolic regulation mechanism of the bacteria in response to environmental factors. |
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