Screening Of The Agarolytic Bacterium Alteromonas Macleodii QZ9-9 From Hainan Macroalgae And Its Enzyme-Producing Properties And Degradation Products

Due to the problems of low level of science and technology,extensive production,and low added value in the application of seaweed polysaccharide in Hainan,this work aimed to isolate seaweed polysaccharide-degrading enzyme-producing strains with the potential industrialized application by the microbial operation methods with low energy consumption,no pollution and high added value.Sequencely,the strain,Alteromonas macleodii QZ9-9,efficiently possessing the capacity of degrading agar which is one of the main polysaccharides of macroalgae,was characterized.The main results are as follows:（1）Screening of strains that can degrade different types of seaweed polysaccharides of macroalgae.3 strains with the capacity of degrading algin,5 strains with the capacity of degradingλ-carrageenan,13 strains with the capacity of degradingκ-carrageenan,and27 strains with the capacity of degrading agar were isolated from 9 species of algae,35sediments from Xisha stations in Hainan Island,and 50 isolations from Xisha stations in the South China Sea.Among these strains,all the strains with the capacity of degrading algin belonged to Vibrio alginolyticu with the highest enzyme activity of 0.37 U/mL.Halomonas sp.λ4 displayed the highest λ-carrageenan-degrading enzyme activity of 6.36U/mL;6 Bacillus strains with the capacity of degradingκ-carrageenan were obtained,of which Bacillus inaquosorumκ11 exhibited the highest enzyme activity of 3.16 U/mL.11strains with the capacity of degrading agar belonged to Alteromonas,among which A.macleodii QZ9-9 isolated from Gracilaria had the strongest degradation ability with enzyme activity of 12.17 U/mL.Additionally,agar-degrading strains belonging to Paracoccus were reported in this study.（2）Biological characteristics of agar degrading strain A.macleodii QZ9-9.A.macleodii QZ9-9 and its extracellular agarase（12.17 U/mL）displayed the highest degradation circle diameter of 41.46 mm and 22.89 mm,and the first order degradation rate constants were 0.02 h^-1 and 0.77 U^-1,respectively.The optimal medium for producing agarase was:bran 1 g/L,agar 1.2 g/L,soybean meal 5 g/L,Na Cl 70 g/L,（NH₂）₂SO₄5 g/L,K₂HPO₄·3H₂O 6 g/L,MgSO₄·7H₂O 1 g/L,FeSO₄·7H₂O 0.1 g/L.The optimal fermentation condition for producing agarase was:p H 7.5,inoculum volume of6%,liquid volume of 20%,culture temperature of 35℃,and a rotating speed of 180 rpm.The optimum catalytic temperature of A.macleodii QZ9-9 agarase was 55℃,and the optimum catalytic pH of that was 6.0.The crude agarase had good thermal stability,and the crude agarase activity can remain above 93%after incubation at 30-50℃for 5 h.The agarase also showed good p H stability between 6.0 and 8.0.The crude agarase can tolerate Cu²⁺,Mn²⁺,Ni²⁺,and Fe³⁺,whereas Na⁺,K⁺,and Mg²⁺can promote the enzyme activity.A.macleodii QZ9-9 agarase was confirmed by Native-PAGE with a molecular weight of about 75 k Da.（3）Complete genome sequence of strain A macleodii QZ9-9 strain and its agarase genes.The total length of A.macleodii QZ9-9 genome was 4,974,508 bp including one chromosome and one plasmid.Four agarase（Qa,Qb,Qc,and Qd）genes were identified.The gene lengths of these four agarase were 2,388 bp（Qa）,876 bp（Qb）,2,340 bp（Qc）and 2,334 bp（Qd）,respectively.And their GC content ratio was 41.67%（Qa）,31.73%（Qb）,41.62%（Qc）and 43.74%（Qd）,respectively.The Qa,Qc and Qd belonged to the GH50 family,while Qb belonged to the GH16 family.Ex Pasy analysis showed that Qa(Met₁-Gln₇₉₅),Qb(Met₁-Glu₂₉₁),Qc(Met₁-Lys₇₇₉),and Qd(Met₁-Glu₇₇₇)were composed of 795,291,779 and 777 amino acids,respectively,the molecular weight of the full-length protein was 91.30 k Da,34.03 k Da,87.99 k Da,88.66 k Da,and the theoretical isoelectric point（p I）was 5.09,6.13,5.43,5.24,respectively.（4）The high value-added research of A.macleodii QZ9-9.This work found that the degradation products of strain A.macleodii QZ9-9 had certain antioxidant activity,and the DPPH scavenging activity of the products fermented for 50 h was the highest of 50.79%after reaction for 1 h.The low molecular weight metabolites of≤3 k Da（10 mg/mL）showed the highest DPPH scavenging activity,which was up to 85.85%.A total of 766metabolites were detected in the low molecule metabolites by metabolomics.The peptide-like metabolites,such as prolyl-histidine（2.68%）,isoleucyl-histidine（0.3%）,isoleucyl-proline（0.29%）,and arginyl-proline（0.27%）,and the maculosin（0.95%）were found in the top 20 metabolites with relatively high abundance.It was proved that the DPPH scavenging activity of 600μg/mL maculosin was 58.44%,which was lower than that of vitamin C of 85.33%with the same concentration.This study not only lays an important theoretical significance and practical value for the development of high value-added resources of regional characteristic macroalgae in Hainan,but also enriches the indigenous marine microbial resources for producing seaweed polysaccharides-degrading enzymes in Hainan.