Characterization Of The Agarases From The Marine Bacteria Stenotrophomonas Sp.NTa And Vibrio Sp.JMUAZ5

In this thesis,two agarase-producing strains NTa and JMUAZ5 were isolated from the coastal sediments of mangrove forest in Xiamen.Agarase from strain NTa was purified using chromatography,and then the enzyme and the antioxidant activity of its enzymatic hydrolysate were characterized.Strain JMUAZ5 was preliminarily identified at molecular level.Characterization of its crude agarase enzyme and the antioxidant activity of its enzymatic hydrolysate were also conducted.The major conclusions are showed as follows.?1?Seven agarase-producing bacteria were isolated from the coastal sediments of mangrove forest in Xiamen.16S rRNA gene sequence analysis showed that six strains were preliminarily identified as Vibrio sp.,one strain was identified as Stenotrophomonas sp..16S rRNA phylogenetic analyses showed that strain JMUAZ5 and Vibrio diabolicus P4-1?GenBank accession no.KC261281?were most closely related.This strain was named Vibrio sp.JMUAZ5.The agar-degrading bacterium NTa had been authenticated and belonged to Stenotrophomonas sp..?2?Stenotrophomonas sp.NTa agarase was purified to homogeneity with a specific activity of 8.2 U/mg,a purity fold of 9.1 and a yield of 2.0%.The molecular mass of the purified enzyme was estimated to be 179.7 kDa and 89 kDa by gel filtration chromatography and SDS-PAGE,respectively,which showed that the agarase is a dimmer.?3?The optimal pH and temperature of the purified agarase were 10.0 and 40°C,respectively.It was stable at temperature below 40°C and in the pH range from 5.0 to 11.0.Na⁺,K⁺,Ca²⁺,Mg²⁺and Ba²⁺could promote the agarase activity.Whereas,Zn²⁺,Mn²⁺,Cu²⁺,Cd²⁺,Co²⁺,Fe²⁺,Al³⁺and Fe³⁺inhibited the enzyme activity,Ag⁺had no influence on its activity.Stain NTa agarase had a good resistance against the detected inhibitors,detergents and denaturant.The K_m,V_max,k_cat and k_cat/K_m values of the agarase towards agarose were 11.3 mg/ml?25.4 U/mg?760.1 s^-11 and 67.0 s^-1 mg^-1 ml,respectively.By the thin layer chromatography and MALDI-TOF-MS analysis,the predominant products of Stenotrophomonas sp.NTa agarase degrading agar were biose,tetraose,hexaose.In addition,the enzymatic hydrolysis products of agar exhibited the antioxidant activities in reducing power,scavenging hydroxyl radical,scavenging2,2-diphenyl-1-picrylhydrazyl?DPPH?radicalandscavenging2,2?-azinobis-?3-ethylbenzothiazoline-6-sulfonic acid??ABTS?radical.?4?The substrate specificity analysis showed that the crude agarase from strain Vibrio sp.JMUAZ5 had significant substrate specificity towards agar.The optimum temperature and pH of the crude agarase were 40°C and 8.0,respectively.Its enzymatic activity was stable at 20°C and25°C.It also showed stability across a pH range of 6.0-10.0.Li⁺,Co²⁺,Ca²⁺and Al³⁺stimulated the agarase activity.Whereas,Ag⁺,Zn²⁺,Mg²⁺,Ba²⁺,Mn²⁺,Cu²⁺,Fe²⁺and Fe³⁺impeded the enzyme activity.Na⁺,K⁺,and Ni²⁺had no effect on its activity.The crude enzyme had good resistance to detected inhibitors,detergents and denaturant.In addition,the enzymatic hydrolysate of the crude agarase,agaro-oligosaccharides,had reducing ability and scavenging power on ABTS radicals.