Studying The Function Of The CBM Domains In A GH16 ?-agarase

In this study,a gene encoding a GH16 agarase has been cloned from a strain Microbulbifer sp.And evolutionary tree has been constructed to identify the GH16?-agarase in this strain by blasting the sequence in NCBI.Agarases containing different CBM domains were recombinanted and characterized.The main results were as follows:1?Through the contrast analysis of sequence,it shows that the full sequence of GH16 agarase contained an active site and two CBM domains.Aga-ms-R contained the CD and two CBM domains,while Aga-ms-Ri contained the CD without CBM domain;Aga-ms-R2 was containing the CD and only one CBM domain.2?After gene clone and expression,recombinant agarases were purified and characterized.The optimum reaction temperature and pH of the recombinant enzyme Aga-ms-R and Aga-ms-R2 were 50 ? and 7,respectively,while they were stable in the pH range of 5.0?9.0.After preservation at 70 ? for 30 mins,still some of their enzyme activity remained.The optimum reaction temperature and pH and stability at pH range of 5.0-9.0 of the recombinant enzyme Aga-ms-Ri were just like the above two enzymes.However,almost no enzyme activity of Aga-ms-Ri exsited after preservation at 70 ? for 30 mins.These three recombinant enzymes were purified and the molecular weights were:Aga-ms-R of 63.62 KDa,Aga-ms-Ri of 31.18 KDa,and Aga-ms-R2 of 47.59 KDa.The specific activity of Aga-ms-R was 42.89 U/mg,the kinetic constant Km was 7.463 mg/mL,and Vmax was 2.804?mol/(mL·min),and the specific activity of Aga-ms-Ri was 30.67 U/mg,Km was 8.943 mg/mL,and Vmax was 0.358 ?ol/mL min.The specific activity of Aga-ms-R2 was 48.57 U/mg,the Km value was 1.571 mg/mL,and Vmax was 0.864 ?mol/(mL·min).,The data from this experiment revealed that CBM domains in P-agarase from GH16 family played an important role to maintain better thermal stability and higher enzyme activity.Therefore,the cut-off recombinant enzymes would be more suitable for different industry applications.3?By analysis with thin layer chromatography,high performance liquid chromatography and mass spectrometry,the enzymatic hydrolysates of the three recombinant enzymes were accurately identified to be only DP4.The substrate of three recombinases was highly specific.The GH16 agarase hydrolyze the agar specially,while Alginate or carrageenan not.In conclusion,the deletion of CBM domains in ?-agarase could not lead to different enzymatic products,and CBM domains showed strong specificity for their substrates.In this study,that the comparison of enzyme properties of GH16 agarases with different substrate-binding domain would provide theoretical guidance for the application of CBM domains in agarase.