Study On Improving The Thermal Stability Of Β-agarase Aga50D Based On Oriental Immobilization

Enzymatic hydrolysis is considered to be the predominant commercial scale method for the sustainable production of agar oligosaccharides due to its advantages such as its ability to specifically break down agar polysaccharides without the production of harmful compounds.However,free agarases suffer from several disadvantages（low thermal stability,narrow p H range,effective activity in aqueous environments and loss of catalytic activity after cycling）,leading to limited applications in industry.Therefore,the use of immobilization techniques to improve the thermal stability and operational stability of the enzyme as well as to achieve reusability can reduce the cost of the reaction and has good prospects for industrial applications.Directional covalent immobilization allows the enzyme to be immobilized in a certain direction（away from the active center）on a suitable carrier,thus enabling better binding of the enzyme to the substrate.With the flourishing development of structural biology,bioinformatics and computer-aided methods,mutation design of proteins has become an effective method to modify protein properties.In this study,six candidate mutation sites were finally obtained by screening the flexible,rigid and thermosensitive regions and energy calculations;and six mutants ofβ-agarase Aga50D were obtained;finally,the functional magnetic nanoparticles were used as carriers to covalently immobilize theβ-agarase Aga50D mutant by site-specific targeting to obtain immobilizedβ-agarase with significantly enhanced stability.The main findings were as follows:（1）Molecular dynamics simulations were used to identify the flexible and rigid as well as the thermosensitive regions of the enzyme molecule and combined with energy calculations for mutation design ofβ-agarase Aga50D.Flexible and rigid regions were identified by B-factor and RMSF_298K,and heat-sensitive regions were identified by high and low temperature molecular dynamics simulation trajectories(ΔRMSF_323K-298K);Then,by means of Rosetta Cartesian DDG,we calculated the conformational free energy change（ΔDDG）from cysteine-induced mutation of selected amino acids to determine the favorable forward mutation,and PYMOL was used to check whether the amino acids were present on the molecular surface.Finally,two mutation sites in the flexible region were obtained:R66C and K588C;The four mutation sites in the rigid region were K211C,E308C,N452C and S558C.（2）The R66C,K211C,E308C,N452C,S558C and K588C genes were obtained by PCR amplification,constructed into the expression vector p ET28a and successfully expressed in E.coli genetically engineered bacteria（BL21）;after affinity chromatography using nickel columns,high purity mutants were obtained and the enzymatic properties of the wild-type and mutant enzymes were determined separately.The results showed that among the six mutant designs,the enzyme activities of five mutants were increased compared with the wild enzyme,among which the relative enzyme activities of E308C and N452C were increased by 137.0%and 130.2%,respectively,while the relative enzyme activity of K211C was slightly lower（98.1%）.The optimum reaction p H was 7.0 and the optimum reaction temperature was 30°C for both the wild enzyme and all mutant enzymes.The overall effect of the mutation design on the enzymatic properties ofβ-agarase Aga50D was not significant and was consistent with the expected mutation design results,and the resulting mutant enzymes could be further used for oriented immobilization.（3）The maleimide-modified magnetic nanoparticles（MAL-NH₂-MNPS）were obtained by sulfo-SMCC cross-linking of aminated magnetic nanoparticles,which could spontaneously bind to the cysteine residues ofβ-agarase Aga50D mutant.The optimal reaction conditions for immobilization were:reaction time of 6 h,enzyme loading（enzyme:carrier）of 1:50,cross-linking reagent ratio of（cross-linker:carrier）1:50.The protein loading efficiency of different mutant enzymes R66C,K211C,E308C,N452C,S558C and K588C were 51.9%,42.3%,41.4%,60.0%,44.7%and 45.4%,respectively.45.4%,and the activity retentions were 96.7%,87.9%,60.5%,94.9%,91.2%and 96.9%,respectively.After incubation for 30 min and 60 min at 40°C,the wild enzyme retained 79.8%and 37.8%of the original enzyme activity respectively,while after 6 h of incubation,almost all enzyme activity was lost;the immobilized enzyme S558C retained 99.5%and 45.4%of the original enzyme activity after incubation for 30 min and 60min at 40°C,respectively.The immobilized enzyme S558C retained 99.5%and 97.3%of the original enzyme activity after incubation for 30 min and 60 min at 40°C,and still retained 86.7%of the original enzyme activity after 6 h of incubation.The immobilized enzyme retained around20%to 50%of the relative enzyme activity after 5 repeated uses.In the presence of organic reagents,the free enzyme lost all its activity,while most of the immobilized enzymes retained more than 90%of their activity.（4）Sulfhydryl-modified magnetic nanoparticles（SH-NH₂-MNPS）,obtained by cross-linking aminated magnetic nanoparticles with traut reagent,can spontaneously form intermolecular disulfide bonds with cysteine residues onβ-agarase Aga50D mutant,thereby targeting the immobilization ofβ-agarase.The optimal reaction conditions for immobilization were:reaction time of 12 h,enzyme loading（enzyme:carrier）of 1:50,cross-linking reagent ratio of（cross-linker:carrier）10:50.The protein loading efficiencyof R66C,K211C,E308C,N452C,S558C and K588C mutant were 63.5%,41.7%,42.3%,71.6%,61.9%and 55.1%,respectively,and the activity retenions were 92.2%,94.4%,68.3%,88.0%,81.3%,96.8%,respectively,most of the mutant enzymes retaining more than 80%of their enzyme activities.For example,at 45°C,the wild enzyme retained 60.8%and 22.2%of the original enzyme activity after incubation for 30 min and 60 min,respectively,while the wild enzyme lost all enzyme activity after incubation for 6 h;mutant E308C retained 88.8%of the original enzyme activity after incubation for 30 min and 60 min at 45°C,respectively.After incubation for 30min and 60 min at 45°C,the mutant E308C retained 88.8%and 82.7%of the original enzyme activity respectively,and after incubation for 6 h,the immobilized enzyme E308C still retained56.4%of the original enzyme activity.The immobilized enzyme retained more than 30%of the relative enzyme activity after 4 repeated uses.In the presence of organic reagents,the free enzyme lost all of its activity,while most of the immobilized enzymes retained more than 70%of their activity.（5）The product of immobilizedβ-agarase Aga50D was characterized as a single neoagarobiose using thin-layer chromatography and liquid-phase mass spectrometry.The enzymatichydrolysis product,neoagarobiose,has certain antioxidant capacity and a strong scavenging ability for ABTS radicals,with a maximum scavenging rate of 91.4%for ABTS radicals and an average scavenging rate of 59.6%for DPPH radicals,as well as a weak scavenging ability for OH radicals,with a scavenging rate of only 13.3%when neoagarobiose reaches a higher concentration.