Study On The Polysaccharide Degrading Enzymes From Seaweed By Epiphytic Bacteria From Indonesian Macroalgae

Indonesia,which is located in the tropes,is rich in macroalgae resources and has a high species richness.The surface of macroalgae incubates a large number and a wide variety of marine microbial communities,which is an important source for the development of new functional microorganisms and heat-stable marine biological enzymes.Seaweed polysaccharide-degrading enzymes are an important branch of marine biological enzymes and an important source of new bioactive compounds.However,the seaweed polysaccharide-degrading enzymes produced by Indonesian macroalgae epiphytes have not been effectively developed.In this study,Indonesian macroalgae was taken as the research object,the diversity of culturable polysaccharide degrading bacteria on its surface was analyzed.Metagenomic technology was used to screen out carrageenase gene,agarase gene and alginate lyase gene with novel sequences.After heterologous expression,the enzymatic properties were studied and its application in environmental treatment was explored.In this study,74 strains of polysaccharide degrading bacteria were isolated from the surface of 11 species of Indonesian macroalgae,including 20 carrageenan degrading bacteria,28 agar degrading bacteria and 26 algin degrading bacteria.Among them,Cobetia and Pseudoalteromonas were the dominant species.The phylogenetic analysis of24 representative strains with high degradation activity indicated that these strains belonged to the class?-Proteobacteria and Bacilli.It is mainly distributed in the genera of Cobetia,Pseudoalteromonas,Microbulbifer and Bacillus.Metagenomic sequencing and analysis were performed on the macroalgae samples Dictyota canaliculata,Liagorothamnion Mucoides and Sargassum turbinarioides with high production of polysaccharide degrading enzymes.The results showed that the bacteria species on the macroalgae surface were Vibrio,Klebsiella,Photobacterium,Acinetobacter,Cobetia,Escherichia,Leptolyngbya,Serratia,Enterobacter,Oceanicola,Lewinella,Robiginitalea and Cyanothece.According to the results of Cazy database annotation,Dictyota canaliculata and Sargassum turbinarioides have high abundance of PL family gene sequence annotation,and Liagorothamnion mucoides have high abundance of GH family gene annotation.The novel sequences of seaweed polysaccharide degrading enzyme genes(carrageenase gene Car1293,agarase gene Aga-5 and alginase lyase gene Aly448)were obtained for further study.The seaweed polysaccharide degrading enzyme genes Car1293,Aga-5 and Aly448were cloned and expressed.Sequence analysis showed that the length of Car1293 is 2589bp,coding 862 amino acids,theoretical molecular weight of 90 k Da;the length of Aga-5is 348 bp and encodes 115 amino acids with a theoretical molecular weight of 13 k Da;the length of Aly448 sequence was 1914 bp,coding 637 amino acids,and the theoretical molecular weight is 68 k Da.The recombinant plasmids Car1293+p ET-30(a),Aga-5+p ET-30(a)and Aly448+p ET-30(a)were synthesized and transformed into E.coli BL21(DE3)to achieve heterologous expression of the three polysaccharide degrading enzyme genes.The recombinant enzymes were purified by Ni-NTA affinity chromatography column.After purification,SDS-PAGE electrophoresis was used to verify the results.The molecular weight of the three recombinant enzymes was consistent with the theoretical single target bands.The results showed that Car1293,Aga-5 and Aly448 were heterologously expressed correctly in E.coli BL21(DE3).The enzymatic properties of recombinant polysaccharide degrading enzymes Car1293,Aga-5 and Aly448 were studied,and their application in bioremediation of algae pollution was explored.The results of enzymatic properties showed that the optimal temperature of polysaccharide degrading enzymes Car1293,Aga-5 and Aly448 were 50?and had good thermal stability,the optimal pH was 6.0,7.0 and 8.0,respectively.Moreover,Car1293 and Aly448 had good alkaline resistance.Different metal ions had different effects on the activity of different enzymes,Na⁺,Mn²⁺and Fe²⁺promoted the activity of Car1293 enzyme;Mn²⁺,Na⁺,Ca²⁺,Fe²⁺,Ba²⁺,K⁺,Fe³⁺and Ni²⁺can promote the relative enzyme activity of Aga-5;and Na⁺can promote the enzyme activity of Aly448.Thin layer chromatography(TLC)showed that all the three recombinases could degrade their specific substrates to algal oligosaccharides with low degree of polymerization.The composition and types of the degradation products need to be further determined.Recombinant enzyme Car1293,Aga-5 and Aly448 were used to decompose the algae of Grateloupia filicina,asparagus and Sargassum horneri,respectively.The results showed that the three enzymes had good degradation effect with corresponding algae,and the optimal reaction time was 6 h,6 h and 12 h,respectively.This study deepened our understanding of the diversity of polysaccharide degrading enzymes produced by the epiphytic bacteria of macroalgae from Indonesia.The obtained polysaccharide degrading enzymes with novel sequences are expected to be developed into potential industrial enzymes and have practical application value in bioengineering and environmental governance.