The Research On Immobilization Of Agarase With Magnetic Nanoparticles And Bioactivity Of Its Products

Red algae are a kind of marine algae with a wide distribution.Agarose extracted from red algae can be used as a gelling and thickening agent,and is widely used in food industry,but its added value is not fully reflected.Agarose oligosaccharides and oligosaccharide monomer 3,6-anhydro-α-L-galactose（L-AHG）with various biological activities can be obtained by enzymatic hydrolysis or acid hydrolysis of agarose.The main biological activities of agarose oligosaccharides and L-AHG including anti-inflammatory,antimicrobial,and whitening activities with potential applications in food,pharmaceutical,and cosmetic industries.Biological enzymatic methods are suitable for continuous production of agarose oligosaccharides and L-AHG due to the advantages of mild reaction conditions,high specificity and high catalytic efficiency.However,the optimum temperature of agarase from natural sources is about 30℃,while the agarose solution changes from sol state to gel state when cooled to 30℃～40℃,which limits the continuous application of agarase.Therefore,it is great significance to improve the catalytic efficiency and thermal stability of agarase for the efficient preparation of agarose oligosaccharides and L-AHG.With the development of immobilization technology,immobilized enzyme is considered to be an effective method to improve the stability of enzyme.Therefore,in this research,we use magnetic nanoparticles as the carrier of immobilized agarase,optimizes the application of biotin/streptavidin affinity system in immobilized agarase,and achieves efficient production of agarose oligosaccharides and L-AHG.And the biological activity of the products was preliminarily explored.The main conclusions are as follows.Firstly,β-agarase was biotinylated with different activated groups,and the enzyme was immobilized on magnetic nanoparticles connected with streptavidin by biotin/streptavidin affinity.The immobilized enzymes were characterized by TEM,XRD,TGA,and FT-IR.The results show that the average particle size of the immobilized enzymes was 195 nm,with typical Fe₃O₄ spinel structure.Both amino activated and carboxyl activated immobilized enzyme can realize the reuse of enzyme,while amino activated immobilized enzyme had better stability.The residual activity of amino activated immobilized enzyme after incubation at 50℃for 6 h was 24.74%,which was 1.84 times that of the carboxyl activated immobilized enzyme.Moreover,amino activated immobilized enzymes could improve the affinity of the enzyme to the substrate,and carboxyl activated immobilized enzymes had a higher inhibitory effect on the catalytic efficiency of the enzyme.Secondly,the effect of biotin side chain length on the immobilization effect and stability was studied.The results showed that the enzyme activity retention and stability of the immobilized enzyme increased with the increase of biotin side chain length.When the side chain was polyethylene glycol 24（PEG24）,the enzyme activity retention rate of the immobilized enzyme reached 135.67%,and after incubation at 40℃for 6 h,it still retained84.18%of the residual activity,while the free enzyme only had 30.50%of the residual activity,and its half-life at 50℃was 7.24 times that of the free enzyme;it also showed good reusability,retaining 53.64%of the initial enzyme activity after 7 repetitions;the yield of neoagarose（NA2）at 45℃was 1.89 times that of the free enzyme.Then,based on the above amino activation and PEG24 side chain affinity method,β-agarase andα-agarase were co-immobilized to prepare antioxidant and antimicrobial L-AHG,and the effects of the ratio of two enzymes,initial protein content,p H value and temperature on the co-immobilized bienzyme were studied.The results showed that the optimum conditions of co-immobilized bienzyme were p H of 8.0,temperature of 25℃,initial protein concentration of 1.25 mg/m L,and the ratio ofβ-agarase toα-agarase was 1:1.5.The enzyme activity of co-immobilized bienzyme was 2.5 times higher than that of single immobilized bienzyme,and the co-immobilized bienzyme had better thermal stability than free enzymes.After incubation at40℃for 3 h,79.45%of the initial enzyme activity was retained,while the free enzymes only had 21.40%residual enzyme activity;The co-immobilized bienzyme had good reusability and storage stability,and the maximum reaction rate of the enzyme was increased from 1.64μmol/min to 1.70μmol/min.Finally,the biological activity of the enzymatic hydrolyzate was explored.The antioxidant results indicate that L-AHG has better free radical scavenging ability than NA2 and D-galactose.The scavenging rate of hydroxyl radical by L-AHG was 47.16%,while the highest scavenging rate of NA2 and D-galactose was less than 6%.L-AHG had broad-spectrum antimicrobial activity,while NA2 and D-galactose did not show obvious antimicrobial effect.The minimum inhibitory concentrations of L-AHG on Bacillus subtilis,Staphylococcus aureus,Salmonella typhimurium,and Escherichia coli were 1.25 mg/m L,1.25 mg/m L,0.63 mg/m L,and 0.63mg/m L,respectively.It was preliminarily confirmed that L-AHG destroyed the integrity of bacterial cell membranes,resulting in the death of the bacteria.