Agarase Gene Mining Of Flammeovirga Sp. OC4 And Prebiotics Function Of Its Produced Oligosaccharides

Fujian province is the main planting area of Gracilaria lemaneiformis in China.The main economic value of Gracilaria lemaneiformis was to produce agar.Oligosaccharides made from agar had more physiological activity and higher value.Through the preparation of oligosaccharides,high value utilization of the industrial chain of Gracilaria lemaneiformis could be realized.But the preparation of oligosaccharides was confronted with two difficulties:one was a lot of strong acids and alkalis needed,and useless by-product algal residue produced in the extraction process,which brought great pollution to the environment;the other was that if oligosaccharide was further produced by acid hydrolysis method,another strong acid was needed and the products were uneven,which impaired subsequent applications.Flammeovirga sp.OC4,which can degrade Gracilaria lemaneiformis,was isolated from sea water by in situ enrichment in deep sea.Oligosaccharides were prepared by one-step method quickly.The key agarase genes of Flammeovirga sp.OC4,obtained by gene mining,were expressed through heterologous vector,by which oligosaccharide monomers were prepared.Mechanism of promoting lactic acid bacteria growth with the purified monomers was studied.The main research contents and achievements were as follows:（1）The small training cabin designed for enrichment was put in the South China Sea under the depth of 2000 meters for 1 year,and 4 strains with ability of degrading agar were obtained from the cabin,among which Flammeovirga sp.OC4,identified by morphological observation and 16S r RNA,was selected for its high enzyme activity.The optimum temperature and p H of agarase produced by Flammeovirga sp.OC4 was50℃and p H 8.More than 60%of the maximum activity of the enzyme was kept after placing 60 min under p H 5.0-10.0.Both Mn²⁺and Ba²⁺could enhance the enzyme activity.（2）One-step method of preparing oligosaccharides through fermenting Gracilaria lemaneiformis by Flammeovirga sp.OC4 was established.The parameters were optimized using Plackett-Burman design and response surface methodology.The results showed that oligosaccharides yielded from Gracilaria lemaneiformis was 3.09g/L in the case of optimizing parameters in a 250 m L fermenter with a working liquid volume of 44.8 m L,30 g/L Gracilaria lemaneiformis powder,4.84 g/L（NH4）₂SO₄ at36.7℃and a shaking speed of 200×g for 42 h,presenting an overall 36.1%increase in oligosaccharides yield compared with the original culture medium.The yield of oligosaccharides was 3.05 g/L,2.98 g/L and 2.89 g/L respectively when the fermentation scale was enlarged to 5 L,50 L and 200 L.Through TLC and ion chromatography identification,these oligosaccharides were proved to be consist of neoagarobiose,agarotriose,neoagarotetraose,agaropentaose,neoagarohexaose.（3）The whole genome of Flammeovirga sp.OC4 was sequenced,and six agarase genes were analyzed with phylogenetic tree,three of which belonged to GH86 family,while two GH16 family and one GH118 family.Two engineering strains with agarase activity were obtained by constructing E.coli-p Cold II expression system.Agarases Aga3027 and Aga4436,produced by the two strains,could hydrolyse agar into neoagarotetraose and neoagarohexaose.Aga3027 was an alkali-resistant agarase with the optimum p H and temperature 9 and 40℃,respectively.Aga4436 was a thermostable agarase with its optimum p H 9 and optimum temperature 55℃.（4）A mixture of neoagarotetraose and neoagarohexaose was prepared through degrading agarose by Aga3027,which was seperated by gel chromatographic column G-10 and P-2.The NA4 and NA6 monomers were identified by TLC and ion chromatography.The optimum concentration of promoting Lactobacillus plantarum growth was determined by automatic growth curve analyzer,which was 0.4 g/L and 0.8g/L when it came to GLC and NAOS,respectively.For NA4,NA6,FOS,XOS,the optimum concentration was 0.6 g/L.Compared in each optimum concentration,the ranking of promoting effect of Lactobacillus plantarum growth was NA4,FOS,NA6,NAOS,XOS,GLC from high to low.（5）Through comparing the transcriptome of Lactobacillus plantarum under different concentrations of NA4 and NA6,the differentially expressed genes（DEGs）were found to be similar in GO annotation results.In cellular component branch,the most relevant items annotated in each group were“cell,cell part”and“membrane”.In biological process branch,the most relevant items annotated in each group were“cellular process”,“metabolic process”,“single-organism process”.In molecular function branch,when under the optimum concentration of NA4 and NA6,the most relevant items annotated in each group were“catalytic activity”,“binding”and“transporter activity”,but the most relevant items were changed into“binding”,“catalytic activity”and“structural molecule activity”under low concentration.It could be inferred that NA4 and NA6 possessed similar mechanisms of promoting the growth of Lactobacillus plantarum,and when the concentration of NA4 and NA6 increased,they had higher influence on“transporter activity”.According to the KEGG and COG annotations of DEGs in each group,there was a great similarity among high concentration NA4,high concentration NA6 and low concentration NA4.They were significantly enriched in the three KEGG pathways:“purine metabolism”,“pyrimidine metabolism”,“alanine,aspartate and glutamate metabolism”.It’s interesting that DEGs annotated to COG classifications most were“nucleotide transport and metabolism”and“amino acid transport and metabolism”,which further indicated that the promoting effect of NA4 and NA6 might be related to the transport and metabolism of nucleic acid and amino acid.Meanwhile,the effect between low concentration NA4 and NA6possessed some similarities,their DEGs were both enriched in the“ribosome”KEGG pathway.This indicated that the dominant influence factors of NA4 and NA6 at low and high concentrations were not the same,but at low concentrations,NA4 could produce similar effects of the optimal concentration,which was corresponded with the previous founding that NA4 had better promoting effect on Lactobacillus plantarum.