Isolation, Identification Of An Agarase-producing Bacterium NTa,the Optimal Cultivation Of Agarase Production And Characterization Of The Agarase

Agarase is part of glycoside hydrolases that degrade agar into agaro-oligosaccharides,based on the mode of action on the the agarose,agarase are classified into two groups:?-agarases and?-agarases.Agaro-oligosaccharides,which degraded by agarase,have remarkable properties,so its can extensively apply to the food,pharmaceutical and cosmetic industries.Besides,agarase is used as the important enzyme for biological studies.In this study,a new agarase-producing strain,NTa is isolated from the coastal sediments in Xiamen,is identified via 16S rRNA gene sequence and biochemical reactions,and the production of agarase is enhanced via a series of statistical methods,moreover characterization of the agarase is studied.The major conclusions are showed as follows.?1?16S rRNA phylogenetic,phenotypic and biochemical analyses showed that:the agar-degrading bacterium NTa belonged to the genus Stenotrophomonas sp..?2?The optimal carbon,nitrogen,NaCl and temperature were comfirmed,the results showed that:the optimum carbon was agar,nitrogen sources,NaCl concentration and temperature of the agarase-producing bacterium NTa were agar,yeast extract,5%and 28°C,respectively.The agarse appeared to be subject to catabolite repression,since its synthesis was repressed when glucose was added to the medium in culture.The existence of NH₄⁺affected negatively the growth of strain and agarase production.During the fermetation,agarase were synthesized in the logarithmic grown phase late rapidly,and its kinetic model showed that the syntheses of agarase were strictly associated with the growth.?3?Among the seven tested variables,yeast extract and initial culture pH were identified as the most significant factors for agarase production via a Plackett-Burman design?PBD?.Then,the levels of the two significant variables were further optimized through a single steepest ascend experiment and central composite design?CCD?with response surface analysis.The ANOVA results demonstrated that the model was highly significant,so it can be used to navigate the design space.The highest agarase production was obtained in the medium consisting of 1.03%yeast extract at initial culture pH 8.0.Under the optimal conditions,the experimental value of agarase activity reached 2.628 U/mL.Optimization process resulted in an increased yield of agarase activity by 62.5%over the basal medium.?4?Stenotrophomonas sp.NTa could produce extracellular agarases.The optimum temperature and pH of strain NTa agarase were 40°C and 7.0,respectively.The enzymatic activity was stable below the temperature of 30°C.It also showed stability over a pH range of7.0-9.0.Ca²⁺could activate agarase activity,and Na⁺,K⁺and Mg²⁺had no significant influence.However,Ag⁺,Ba²⁺,Fe²⁺,Mn²⁺,Cu²⁺,Zn²⁺,Fe³⁺and EDTA inhibited the enzyme activity.The agarase had good resistance to some inhibitors,detergents and denaturant.The results of substrate specificity detection of agarase towards different substrates indicated that it was highly agar specific.?5?The mainly components of hydrolytic product analyzed by chromogenic substrate and TLC,HPLC and LC-MS showed that hydrolysis of agar by NTa produced neoagarotetraose and few neoagarobiose,and the category of agarase by NTa was?-agarase.Stenotrophomonas sp.NTa was a new type of agarase-producing strain,and it had provided a foundation for agarase production as well as its application in the production of agaro-oligosaccharide.