Characterization Of A Recombinant ?-agarase From A Marine Bacterium Microbulbifer Sp.AG1 And Research On D136N Mutant With Improved Thermostability

In this study,a marine agarase-producing bacterium strain AG1 was isolated from mangrove soil samples in Xiamen,and an agarase gene was cloned from this strain.The agarase gene was analyzed by bioinformatics analysis.It was heterologously expressed in E.coli,and the recombinant agarase was obtained.Characterization of the recombinant agarase and antioxidant activity of the enzymatic hydrolysates were studied.Besides,thermal stability of the recombinant agarase was improved by site-directed mutagenesis.The major conclusions are showed as follows.On the basis of 16S rRNA gene sequence alignment,the strain was assigned to Microbulbifer sp.AG1.The genomic DNA of this starin was used as the template for amplification of agarase gene by using PCR.The amplified products were cloned into p MD18-T vector and then sequenced.The results showed that the cloned gene was 1302 bp,encoding a protein with 433 amino acid residues.Sequence alignment of this protein showed it was an agarase which belonged to GH16.The three-dimensional structure of Microbulbifer sp.AG1agarase was constructed by homology modeling and presented?-strand rich structure.The whole structure revealed a jelly-roll fold,which was composed of two oppositely paralleled?-strands.By site directed mutagenesis,Glu147 and Glu 152 might be the putative catalytic active sites of this agarase.The recombinant agarase without the signal peptide part was expressed and purified from E.coli BL21(DE3)with a molecular mass of 75.6 k Da.The recombinant agarase can specifically hydrolyze agar,and it could actively hydrolyze p-nitrophenyl-?-D-galactopyranoside but not p-nitrophenyl-?-D-galactopyranoside.These results indicated that this agarase was a?-agarase that could specifically hydrolyze the?-glycosidic bond.Using agarose as the substrate,the optimal temperature and p H for the enzyme were 60°C and 7.5,respectively.The recombinant agarase maintained 67%and 19%of residual activities after incubation at 50°C and60°C for 1 h,individually.Except SDS and guanidine hydrochloride,the recombinant agarase had a relatively good resistance against the detected inhibitors,detergents and urea denaturant.Ba²⁺,Mn²⁺,Ni²⁺,Co²⁺,Cu²⁺,Zn²⁺,Fe²⁺,Al³⁺and Fe³⁺suppressed the enzymatic activity,while K⁺and Ca²⁺had no influence on agarase activity.The K_m,V_max,k_catand k_cat/K_m values of the recombinant agarase towards agarose were 3.8 mg/ml,91.7 U/mg,3468.2635s^-1and 913.143s^-1mg^-1ml.By TLC and MALDI-TOF-MS analysis,the predominant product of the recombinant agarase was neoagarotetraose.The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.Using site-directed mutagenesis,the single point mutant of D136N for the recombinant agarase was constructed.D136N had the same optimum temperature and p H compared with the wild type agarase(WT),but it showed better thermostability:For mutant D136N,85%of residual activity was retained after incubation at 60°C for 1 h.The microstructure analysis showed that after mutation the hydrogen bond number increased one more between 136 amino acid residue and its surrounding residues.The additional hydrogen bond in mutant of D136N could account for the enhanced themostability.