Exploring And Characterization Of Polysaccharide Degrading Enzymes From Macroalgae-associated Bacteria Collected From Antarctica

A large number of unknown microbial populations can be discovered in Antarctic region to obtain bioactive substances with unique functions,in addition,the Antarctic coastal beach also distributed abundant macroalgae resources,especially the high proportion of endemic species.Of the 117 species of macroalgae reported from Antarctica,57 are endemic to the Antarctic region.The surface of macroalgae harbors a large number of microorganisms and their produced enzymes,such as GH?glycoside hydrolase?and PL?polysaccharide lyase?which not only involved in the degradation of macroalgae but also plays an important role in the carbon cycle of the ocean.However,there still lack sufficient understanding of the seaweed polysaccharide degrading enzyme produced by Antarctic marine bacteria,especially associated bacteria collected from Antarctic macroalgae.In this study,the Antarcitc macroalgae-associated bacteria and their polysaccharides-degrading enzymes were analyzed and recognized by metagenomic method.In order to exploit the industrial application prospect of seaweed polysaccharide degrading enzyme,the novel carragenase genes,agarase genes and alginase lyase genes were screened,expressed and characterized.Firstly,the genomic DNA of macroalgae-associated bacteria from six macroalgae samples?Melanthalia abscissa,Callithamnion tetragonum,Plocamium cartilagineum,Phaeurus antarcticus,Pachymenia orbicularis,Desmarestia Antarctica?was extracted and sequenced.The results showed that a total of 395273,808276,499224,534251,296596,309247 gene sequences were obtained from six samples.The dominant species on the macroalgae surface were Leucothrix,Psychromonas,Chondrus,Bacteroides,Burkholderia,Cellulophaga,Granulosicoccus,Palmaria,Lewinella,Chryseobacterium,Flavobacterium,Absidia,Acanthamoeba,Achromobacter,Acinetobacter,Agrobacterium.The carbohydrate-active enzymes?CAZymes?highest abundance genes of the macroalgae-associated bacteria was GT?Glycosyl Transferase?family,followed by GH?glycoside hydrolase?and CBM?carbohydrate binding module?family.Analysis the diversity of GH and PL membersshowed,the abundance of GH and PL members from p.antarcticus and p.cartilagineum was higher than the other four macroalgae samples.In particular,we found a large number of GH96 and GH117 family members from Antarctic macroalgae-associated bacteria,and the novel sequences of seaweed polysaccharide degrading enzyme genes?carragenase genes car1383,agarase genes aga1904 and alginase lyase genes aly644?were obtained for further study.Then,the novel gene sequences of carragenase,agarase and alginase lyase were heterologous expressed.Sequence analysis showed that the length of car1383 is 1041 bp,coding 346 amino acids,theoretical molecular weight of 40 k Da;The length of aga1904 is 2011 bp,coding 640 amino acids,theoretical molecular weight of 72 k Da;The length of aly644 is 2238 bp,coding 745 amino acids,theoretical molecular weight of 82 k Da.The above sequences were successfully connected to the p ET-30?a?vector and heterogenously expressed into the E.coli BL21?DE3?cell.The recombinant protein was isolated and purified by Ni-NTA His Tag Kit,and the content and purity of the target protein were detected by SDS-PAGE.The results showed that the purified target proteins all presented single target bands and their molecular weights were consistent with the theoretical ones.This result confirmed that the target gene car1383,aga1904 aly644 was correctly expressed in E.coli BL21?DE3?and purified recombinant proteins Car1383,Aga1904 and Aly644 were obtained.Finally,the biochemical characteristics of recombinant carragenase Car1383,agarase Aga1904 and alginase lyase Aly644 were investigated.The results showed that all the optimum temperature of Car1383,Aga1904 and Aly644 were 50? and the optimum p H was 6.0,and Car1383 and Aga1904 could still maintained the initial activity of 30% and over 40% at p H 11.0.Metal ions had different influnces on the enzyme activity of carragenase Car1383,agarase Aga1904 and alginase lyase Aly644.Most of them,such as Mg²⁺,Zn²⁺,Fe³⁺,Cu²⁺,Mn²⁺,EDTA,Ni²⁺ and Ba²⁺ could inhibit the activity of carragenase Car1383,agarase Aga1904 and alginase lyase Aly644.The Km values of Car1383,Aga1904 and Aly644 were 6.51 mg/m L?15.36mg/m L and 16.75 mg/m L respectively,it showed that the three enzymes had high affinity for their substrate.The results of thin layer chromatography?TLC?showed that the three recombinant polysaccharide degrading enzymes could degrade the polysaccharide substrates into oligosaccharides and monosaccharides,but the detailed structure and composition of the enzymatic hydrolysis products need to be further analyzed and determined.This study has effectively expanded our understanding of the bacterial diversityon the surface of Antarctic macroalgae and their polysaccharides degrading enzymes.And our results had also deepened our understanding of how polysaccharide degrading bacteria dealed with the hundreds of millions of tons of polysaccharides produced by algae each year and their role in the carbon cycling system of the ocean.At the same time,the novel polysaccharide degrading enzymes obtained in this study are expected to be exploit to potential industrial enzymes.