Analysis Of The Genome And Seaweed Polysaccharide-degradation Enzyme Gene Of Pseudoalteromonas Carrageenovora ASY5

In this study,a carrageenase-producing bacteria,Pseudoalteromonas.carrageenovora ASY5 was isolated in the previous research of our lab,and it had certain research and application value.The genome of P.carrageenovora ASY5 was analyzed to explore potential functional genes,and new seaweed polysaccharide degrading enzymes from this bacterial strain were developed to achieve high value utilization of seaweed.The main findings obtained are as follows:(1)In order to understand the metabolism characteristics and gene functions of P.carrageenovora ASY5,whole genome sequences were determined and protein coding genes were predicted.Sequencing results showed that the genome of P.carrageenovora ASY5 has one chromosome of a total size of 3,652,029 bp and a plasmid of a total size of 837,079 bp with a 39.48%GC content.4305 genes were predicted,of which 4144 genes were protein-coding genes.The analysis of KEGG,GO,COG and functional genes indicated that the strain has complete metabolic pathways,including carbohydrate,fatty acid,amino acid and so on;most genes were related to metabolism of energy and amino acid transport.It also encoded proteins of seaweed polysaccharide-degradation,such as alginate lyase and β-galactosidases.(2)In this study,a new alginate lyase,Alg823,was cloned from the marine bacterium P.carrageenovora ASY5.Comparative sequence analysis and neighbor-joining tree results showed that the predicted amino acid sequence encoded by alg823 belongs to the polysaccharide lyase 6(PL6)family and has a molecular mass of about 82.6 kDa.The recombinant alginate lyase was characterized followed by NTA-Ni Sepharose purification.When sodium alginate was used as the substrate,Alg823 showed relatively high activity at pH ranging from 5.5 to 10.0 and temperatures ranging from 40℃ to 65℃ with the highest activity at 55℃ and pH 8.0.Notably,the recombinant alginate lyase retained more than 75%of the maximum enzyme activity after 30 min incubation at 50℃,suggesting that Alg823 was a heat-stable alginate lyase.The recombinant alginate lyase retains more than 80%of the maximum enzyme activity after 24 h incubation at a broad range of pH 6.0-10.0 at 4℃,which exhibited excellent pH stability.Metal ions Mg2+,Ca2+,Na+,and K+increased the activity of the enzyme.With sodium alginate as the substrate,the Km and Vmax values of Alg823 were 0.15 mg/mL and 1.84 U/g,respectively.The alginate oligosaccharides obtained by degradation of alginate by recombinant alginate lyase displayed reducing power and the scavenging abilities towards ABTS radical,hydroxyl radical and DPPH with EC50 of 3.78 mg/mL,7.26 mg/mL,and 10.61 mg/mL,individually.Its pH-tolerance and heat-adaptation make Alg823 a novel and potential candidate in the application of alginate oligosaccharides production.(3)Furthermore,an agarolytic β-galactosidase was cloned from the marine bacterium P.carrageenovora ASY5 and characterized.The β-galactosidase gene contained a 3099 bp fulllength gene sequence and encoded 1032 as residues of the protein with a molecular weight of about 117 kDa in SDS-PAGE analysis.Based on domain structure analysis and neighbor-joining tree,the β-galactosidase belongs to GH2 family.Then,the characterization of the recombinant βgalactosidase was studied and the results showed that the optimal temperature and pH of the enzyme was 30℃ and pH 8.5,respectively.And it is demonstrated that the recombinant βgalactosidase Gal1265 retained more than 65%of the maximum enzyme activity after incubation for 7 h at 25℃ and retained more than 50%of the maximum enzyme activity after incubation for 1 h at 30℃.Gal1265 could retain more then 60%of the maximum enzyme activity after incubation for 24 h over a range of pH 5.0-9.0.Na+,K+,Mg2+ and Mn2+could promote the enzyme activity,while the enzyme activity was stimulated by CTAB,SDS,EDTA,Cd2+,Cu2+,Zn2+,Ba2+,Fe3+and Al3+.With ONPG as the substrate,the Km and Vmax values of Gal 1265 were 0.43 mM and 3.25 mM·min-1,respectively.Gal1265 could act on agaroseoligosaccharides with D-galactose as the non-reducing end,but couldn’t digest the neoagarooligosaccharides with 3,6-anhydro-L-galactose as the non-reducing end.Gal1265 has great potential in the comprehensive utilization of marine red algae.