Biochemical Analysis And Application Of An Agarase From The Microbulbifer Sp

Agarase can degrade agar polysaccharides into agarose oligosaccharides with a 2 to 10 degree of polymerization.Agar oligosaccharides have a variety of biological activities.In this article,the recombinant agarase MS-GH42 was isolated and purified and its physicochemical properties were studied.The agarase degradation products were isolated,purified,identified and studied.The specific findings were as follows:1.The crude enzyme solution was purified by ammonium sulfate practionation,dialysis,DEAE-anion exchange chromatography.Then the purified enzyme solution was addressed to obtain a unique protein with a single peak through the gel chromatography column G3000PWXL liquid phase analysis and SDS-page gel electrophoresis,regarded as pure enzyme weight.The weight of protein molecular was 72 kDa.The agarose purification rate was 42.3 times,the enzyme activity recovery was 31.4%,and the specific activity was 112.5 U/mg.2.The enzymatic properties of the collected pure enzyme were studied.The optimum reaction pH of the recombinant enzyme was 7.0,which was neutral agarase;the optimal reaction temperature was 40?;and the stability was between pH 7.0-9.0.Better,after incubation at 40 ? for 1 h,the enzyme activity is maintained at more than 85%,with good temperature stability.3.Through calculating the activation energy(Ea),enthalpy change(?H?),free energy((?G?),and entropy change(?S?),we found that the reaction was a non-spontaneous endothermic reaction with a high degree of chaos,whose Ea was 213.79 KJ/mol.The Km was 2.181 mg/mL,the Vma.was 3.635 U/(ml min),and the kcat was 5.483 s-1 of this agarase calculated from the double reciprocal method,which indicated that it had a good affinity for the substrate.The result of metal ions activities showed that Fe3+,Co2+,Mn2+,Cu2+,Ni2+,and Pb2+ had strong inhibitory effects on recombinant agarose.Na+ and K+ had significantly promoting effects on enzyme reaction at concentrations of 10 mmol/L than 1 mmol/L.The result of NaCl activity showed that at the concentration from 0 to 1.5 mol/L,the NaCl could promote enzymatic reaction,and improve the salt tolerance.4.The enzymatic hydrolysates were analyzed by TLC,HPLC,LC-MS,and NMR to confirm the presence of Neoagarobiose(NA2),Neoagarotetraose(NA4),Neoagarohexaose(NA6),and Neoagarooctaose(NA8).With the increase of enzymatic reaction time,the quantities of NA2 and NA4 gradually increased,which were the final products.A large amount of agar substrate was digested and concentrated to obtain 25 mg/mL oligosaccharide mixture.Then mixture was separated by Sephadex LH-20.The apparently distinct peaks of product in previous step were regarded as highly purified NA2 and NA4.5.Four different components of agarose oligosaccharides were used to study anti-oxidation activity,and Vc was used as a control.The result showed that different components of oligosaccharides all had different degrees of antioxidative activity,but their efficiency was not as good as Vc.In the hydroxyl radical scavenging experiment,NA6 and above had the best clearance rate of 48.12%.In the ABTS radical scavenging experiment,the highest NA2 scavenging rate was 76.21%(oligosaccharide concentration of 10 mg/mL).At oligosaccharide concentration of 10 mg/mL,NA2 had the best DPPH free radical scavenging rate of 29.14%.In the reducing force experiment,the reducing power of NA4 at concentration of 10 mg/mL was 0.366,which was 2.17 times than other enzymatic hydrolysis mixture at the same concentration.