| As the largest water environment covering more than 70%of the earth's surface area,the ocean is rich in biological resources.Algae plays an important role as a producer in the ocean.Brown algae,which is one of the macroalgae,is a source of nutrition for many marine microorganisms.The main ingredient is alginate,a kind of polysaccharide.Alginate and its oligosaccharides are widely used in food,medicine,agriculture,cosmetics and other fields.Therefore,alginate has great application prospects and economic markets,but there is still no efficient alginate lyase for industrial production and utilization.The purpose of this study is to screen and isolate strains with high alginate degradation activity,to exogenously express the alginate lyase gene,and to analyze of carbohydrate active enzyme genes in the algae surface microbial genome.The findings are as follows:1.Genome sequencing and annotation of 52 strains of algae epiphytic bacteria,and based on 16S rDNA sequence and CAZy database comparison,analysis of the diversity of microorganisms and the diversity of carbohydrate active enzymes.According to the 16S rDNA gene sequence analysis,the distribution of 52 strains from the surface of the algae was in the phylum Proteobacteria(51.92%),Bacteroides(38.46%),Firmicutes(7.69%)and Actinobacteria(1.92%);The dominant order was the Flavobacteriales and the dominant family is Flavobacterium.On the taxonomic level of the genus,Aquimarina and Roseobacter are abundant.The analysis of CAZy annotation showed that there were 5033 GTs,4696 GHs,2700 CBMs,2319 CEs,444 PLs,305 AAs and a total of 15497 carbohydrate activity enzymes.Functional gene annotation found 165 genes related to alginate cleavage,of which 68 genes were distributed in Proteobacteria,including MBOAT family,PL7,PL6,PL17 family,LytT family,ALGX family,Heparinase ?/?-like protein Family and mannuronic acid specific substrate alginate lyase;there were 97 genes related to alginate cleavage were annotated in Bacteroides,in addition to the above families,Alginate export family and Pgu1 family were found2.The alginate degradation activity verification of 52 strains of algae epiphytic bacteria,screening for alginate-secreting strains from three algae samples,obtention several strains with potential alginate-degrading activity and identification of one potential new specie.Through the active spot plate experiment,22 strains out of 52 strains have algin degrading activity.A further preliminary screening of the three algae surfaces resulted in a total of 71 potential alginate-degrading strains,of which 17 strains were duplicated with the strains isolated in the previous laboratory.All strains were distributed in 4 phyla,23 genera,including Bacteroides,Proteobacteria,Firmicutes,and Actinobacteria.According to the standard of 16S rRNA gene homology<98%,the strains obtained from the prescreening included 19 potential new species.Then by measuring the relative enzyme activity of the crude enzyme solution of 71 strains and active strains isolated in the laboratory,rescreening obtained 10 strains with strong degradation ability,of which the strain with the highest enzyme activity was strain M269,followed by strain M341,and the enzyme activity reached more than 94%of the strain M269.Heterogeneous taxonomic identification of strain M341 was determined from the aspects of genetics,physiological,biochemical characteristics and chemical characteristics.The results showed that strain M341 is a gram-negative,facultative anaerobic,flagella-free bacilli,the main fatty acids are iso-C15:0,iso-C15:0 G and summed feature 3.The main respiratory quinone is MK-6.The main components of polar lipids are phosphatidylethanolamine,an unknown glycolipid and aminolipid.Combining with genetic characteristics,the strain M341 is determined to represent a new species of the genus Tenacibaculum,the family Flavobacteriaceae,the order Flavobacteriales,the class Flavobacteriia in the phylum Bacteroidetes.3.Exogenous expression of 9 alginate lyase genes in strains M269 and M341,and optimization the induced expression conditions of protein AL341-1.The five alginate lyases in strain M269 and the two alginate lyases in strain M341 were successfully exogenously expressed,and after the induction,the expressed proteins of al269-],al269-6 and al269-9 were in precipitation fragment.The alginate lyase activity plate test found that the precipitated components were active;the expression product of al269-7 was located in the supernatant component,but no clear degradation circle appeared in the activity test;the expression products of al269-8 and al341-1 were located in the supernatant component.The activity test results showed that the supernatant were active,indicating that the two enzymes were soluble proteins;neither al341-2 nor al341-3 were successfully induced expression.The optimal conditions for induced expression of recombinant alginate lyase al341-1 were:initial bacterial density of OD600=0.8,inducer IPTG concentration=1.0 mmol/L,induction time=9 h,and induction temperature=20?. |
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