Study On Alginate Lyase Of Agarivorans Albus B2Z047

Macroalgae occupy huge biomass in the ocean system.Large seaweeds and terrestrial biomass constitute the most abundant and sustainable renewable resources on the earth.The degradation and transformation of terrestrial biomass has been studied in depth,while the research on the degradation and transformation of marine biomass has just started.Therefore,how to make full use of seaweed biological resources has become an important part of biomass conversion.As a large seaweed,the cell wall of brown algae is composed of a variety of polysaccharide components,of which alginate is the main component.The transformation and utilization of alginate is firstly the degradation of alginate.The degradation of alginate by the biological enzymatic method has the characteristics of non-polluting,mild action conditions,and has great potential application value.Marine bacteria are the main source of selection for alginate lyase.This research subject is to study the alginate lyase produced by a strain of bacteria B2Z047,which is selected from the intestine of abalone,which can efficiently degrade alginate.The main results of this research are as follows:1.The isolated alginate-degrading bacterium-B2Z047 was classified into multiple phases,and the strain B2Z047 was identified as Agarivorans albus.The 16S rRNA gene similarity between strain B2Z047 and Agarivorans albus MKT 106T was 98.35%,ANI was 97.1%,and dDDH was 75%.Among them,ANI and dDDH are larger than the boundaries of new species,so the strain B2Z047 belongs to Proteobacteria,Gammaproteobacteria,Alteromonadales,Alteromonadaceae,Agarivora ns.According to the phylogenetic tree of 16S rRNA gene and the comparison of genome data,the strain B2Z047 can be identified as Agarivorans albus.The bacterial colony is white with a diameter of about 1.0-1.5 mm.The optimum growth temperature of the bacteria is 30℃,the optimum growth pH is 7.0-8.0,and the optimum growth salinity is 3%.The bacteria is motile,the main respiratory quinone is Q-8,the main fatty acids（>10%）are C16:0,summed feature 2（C12:0 aldehyde or unknown ECL 10.9525）and summed feature 3（C16:1ω7c/C16:1ω6c）,The main polar lipids are phosphatidylethanolamine,two unknown phospholipids,two unknown lipids and one amino lipid.The morphological and physiological and biochemical characteristics of the bacteria also support that the strain B2Z047 should be Agarivorans albus.2.The whole genome sequencing of the strain B2Z047 was completed,and the alginate lyase was annotated and bioinformatics analysis was performed.The whole genome of strain B2Z047 was sequenced,and 13 potential alginate lyase genes were identified by annotation.The analysis found that Algl belongs to the PL6 family,Alg9 and Alg10 belong to the PL17 family,Alg2,Alg4,Alg5,Alg6,Alg7,Alg8,Algl2₈21 and Alg12₂857 belong to the PL7 family,of which Alg12₈21 and Alg12₂857 have only 5 amino acid differences.Alg3 and Algl1 cannot yet determine the family classification.Among the 13 alginate lyases,only Alg9 and Alg10 did not have annotated signal peptides.It is likely that the products of these two genes are intracellular or periplasmic alginate lyase.Domain analysis showed that in these enzyme molecules,Alg3,Alg4,Alg7,Alg8,Algll,Alg12₈21 and Algl2₂857 are single.domain enzyme components,Algl,Alg2,Alg5 and Alg6,Alg9 and Alg10 are multi-domain components.Among them,Algl,Alg2,Alg5 and Alg6 have carbohydrate binding modules.According to genetic analysis,it was found that the strain B2Z047 has a variety of alginate oligosaccharide penetrating transport proteins and enzymes such as DEHU reductase,KDG kinase,KDPGQ aldolase,which are involved in the further degradation of oligosaccharide cleavage products,indicating that the bacteria has complete degradation of alginate metabolic pathway.3.The separation,purification and characterization of the alginate lyase produced by the strain B2Z047 have been completed,and the corresponding genes of the enzyme are determined by mass spectrometry as algl2₈21 and alg12₂857.The Native-PAGE of the fermentation broth of strain B2Z047 only showed two active bands,one of which showed strong algin degradation activity,and the other was weaker.An electrophoretic-pure alginate lyase is purified from the fermentation broth by ultrafiltration and concentration,ion exchange chromatography and gel filtration chromatography.Through SDS-PAGE and renaturation,the molecular weight of the alginate lyase was measured to be approximately 35 kDa.The mass spectrometry identification of the purified alginate lyase was consistent with the result of the active electrophoresis band in PAGE.It was determined that the alginate lyase secreted by the bacteria into the fermentation broth was a mixture of Alg12₈21 and Alg12₂857.The purified alginate lyase was characterized by its enzymatic properties.The study found that the alginate lyase AlgY has extremely strong temperature stability and is completely inactivated after incubation at 100℃ for 70 min.The optimal reaction temperature of the AlgY is 55℃,the optimum reaction pH is 7.5.The substrate preference shows that the enzyme prefers to degrade polyG and hardly degrade polyM.The main degradation products of this enzyme are 1-4 sugars.Except for Cu2+and Mn2+,which can significantly inhibit enzyme activity,other metal ions have no obvious effect on enzyme activity.The enzyme also has certain resistance to low concentrations of SDS.4.The fermentation process of Agarivorans albus B2Z047 was analyzed.RT-qPCR and fermentation broth mass spectrometry analysis showed that the expression of 13 alginate lyase genes in B2Z047 varies,most of which are expressed in background,and three genes,alg9,alg10 and alg12,are specifically induced.The fermentation process of alginate lyase production in strain B2Z047 was analyzed.The study found that in the shake flask medium with alginate as the carbon source,the bacteria grew rapidly,and the bacterial density reached the maximum at 18 h,while it took 28 h in the 2216E medium without alginate as carbon source.After 12 hours of culture,the activity of alginate lyase in the fermentation broth reached the maximum.The total sugar content in the fermentation broth continued to decrease during the growth of the bacteria,and the total sugar content in the 12 h was reduced to the minimum.The reducing sugar content in the fermentation broth reached the highest at 8 h,then decreased rapidly,and reached the lowest at 12 h.The enzymatic activity of the culture medium with alginate is up to 0.6 U,and the enzyme activity of the culture medium without alginate is basically maintained at 0.1 U,so alginate has a certain inducing effect on the expression of alginate lyase gene.RT-qPCR was used to determine the expression levels of 13 alginate lyase genes when the fermentation was up to 12 h.It was found that in the medium containing alginate,the expression levels of alg9 and alg12 genes were 6 times higher than in 2216E medium without alginate,alg10 was 7 times higher,and the expression levels of other alginate lyase genes were not no significant change.In 2216E medium,the expressions of alg3,alg4 and alg11 were up-regulated twice,and alg6 and alg7 remained basically unchanged.Since algl2₈21 and alg12₂857 differ by only 8 bases,it is not certain which gene is the up-regulated expression of alg12 gene.Proteins were analyzed in the fermentation broth of the two media.The results showed that 13 alginate lyases were detected in the culture medium supplemented with alginate as carbon source,which matched with the alginate lyases analyzed by genome sequencing.Nine alginate lyases were detected in 2216E medium,but Alg7,Alg9,Alg 10 and Algl2₂857 were not detected.Although the specific peptide of Alg12₂857 was not detected in the 2216E medium,it may still contain the enzyme due to its 98.5%similarity with Algl2₈21.Protein mass spectrometry analysis in the fermentation broth also showed that there were agarase,xylanase,amylase and cellulase in the fermentation broth.5.The heterologous expression of 13 alginate lyase genes in E.coli was completed,9 of which have enzymatic activity,and the enzymatic properties of these recombinant alginate lyase genes were characterized.Expression vectors were constructed using pET28a and pET29b plasmids for 13 alginate lyase genes,and these expression vectors were transferred into Escherichia coli（BL21）for heterologous expression.All genes are expressed,except for Alg 1,Alg6,Alg8 and Alg11,the other 9 expressed components have enzymatic activity.Experiments show that Alg 12₈21,Algl2₂857 prefer to degrade polyG,Alg2,Alg3,Alg4,Alg5,Alg6,Alg7 and Alg8 can degrade both polyM and polyG.Alg9 and Alg10 belong to the oligosaccharide lyase.Alg9 specifically degrades dimer of mannuronic acid and guluronic acid.The degradation band shows that the activity on mannuronic acid is greater than guluronic acid.Alg10 can degrade timer and tetramer of mannuronic acid and guluronic acid,but cannot act on dimer.The pH effect of the recombinase shows that the optimal reaction pH of most enzymes is neutral or slightly alkaline.The pH reaction range of Alg7,Algl2₈21 and Alg12₂857 is relatively wide.Alg3 has a very narrow pH suitable for the reaction,and only has high enzyme activity when the pH is 9.5,and the enzyme activity is significantly reduced when the left and right deviations are 0.5 pH units.The optimal reaction temperature of recombinant alginate lyase is between 35-55℃.Although Alg12₈21 and Algl2₂857 have only 5 amino acid differences,their optimal reaction temperature is 45℃ and 50℃,and when the reaction temperature is increased to 60℃,the enzyme activity of Algl2₂857 drops below 20%of the highest enzyme activity,while Algl2₈21 can maintain above 90%enzyme activity.The other enzymatic properties between Algl2₈21 and Alg12₂857 are basically the same.The difference of 5 amino acids has a certain influence on the reaction temperature range of the enzyme,which can be further explored.Chapter 3 purified alginate lyase also had the highest enzyme activity of 90%at 60℃,so the majority of purified enzymes were Alg12₈21.By measuring the temperature stability of the recombinant alginate lyase Alg12₈21 and Alg12₂857,the thermal stability of Alg12₈21 is higher than that of Alg12₂857,and it retains more than 80%of its activity at 100℃ for 20 min,while Algl2₂857 retains 70%of its activity.