Computer-aided Design To Improve The Stability Of Agarase Aga50D

The continued growth of the world’s population has led to an increasing demand for resources that cannot be met by land supply alone.People are therefore looking to the oceans,and marine algae are attracting attention for their use as a source of food,materials and renewable energy.Agar,the main component of the cell wall of red algae,is usually used in the food industry as a thickening and gelling agent with low added value.In contrast,the oligosaccharides extracted from agar have a variety of beneficial biological activities,including anti-inflammatory,antioxidant,probiotic,antibacterial and skin whitening,making agar oligosaccharides have a greater potential for application in food,medicine and agriculture,which greatly improves the economic benefits of agar.Biological methods are considered as the main method for sustainable commercial production of agar oligosaccharides due to the advantage of being able to specifically hydrolyze agar and friendly to the environment.However,most wild-type agarases have an optimum temperature of 30°C-40°C,while agar forms into gel below 40°C,which greatly limits the efficiency of enzyme hydrolysis.Therefore,it is of great importance to improve the thermal stability of agarases to make them suitable for industrial production.With the rapid development of structural biology,bioinformatics and computer technology,computational design of proteins has become a reliable means to modify protein properties.The agarase Aga50D,derived from Saccharophagus degradans 2-40,is capable of degrading agarose into neoagarobiose（NA2）as only products,but its low thermal stability limits its application potential.Therefore,this project combines various computational tools to identify flexible and thermosensitive regions by molecular dynamics simulations and PROSS computational strategies to improve the structural stability of the Aga50D molecular structure,and experimentally validate the performance of the mutant.The main findings are as follows.Modification of Aga50D flexible and thermosensitive sites which was indentified by molecular dynamics simulations to enhance structural stability.The flexible sites were identified with the help of B-factor and RMSF_298K,and thenΔRMSF andΔP-DSSP were extracted from high and low temperature molecular dynamics trajectories to identify the thermosensitive sites;the 92 sites obtained from the screening were further screened by PSSM and 1784ΔΔG calculations to finally obtain 22 mutant designs.The effectiveness of the calculated method to improve the catalytic activity was experimentally verified up to81.82%,among which three mutants improved the enzyme activity by>200%,namely A348S（255.2%）,R66Q（243.4%）and V166M（202.7%）.Further enzymatic experiments on the superior mutants showed that the mutants had the same optimal reaction temperature and p H as the wild type and better catalytic efficiency than the wild type;the mutants were characterized to be farther away from the active center,which is believed to be a remote metastable effect to improve the catalytic efficiency of the enzyme.Based on the combinatorial mutant design provided by the PROSS strategy,the epistatic effects were excluded by disassembling and characterizing the combinatorial design.We disassembled combinatorial mutant sites provided by PROSS and performed 20 single point mutation construction and property characterization,and found that the thermostability of S747Q and A86D was significantly enhanced,among which the A86D mutant had the most significant thermostability enhancement withΔT_mincreased by 23.2℃;the relative enzyme activity of A86D under optimal conditions was also better than that of wild type with 133.6%.The results of enzymatic experiments showed that A86D has the same optimum reaction temperature and p H as the wild type,has a wider temperature tolerance range,and the catalytic efficiency is better than that of the wild type.Further verification of the thermostability of A86D revealed that it retained 82.46%of the original enzyme activity after 60 min incubation at 60℃,72.18%after 180 min incubation,and still retained 30.83%of the original enzyme activity after 6h incubation,while the wild enzyme Aga50D was poorly thermostability and had been completely inactivated after 30 min incubation at 60℃.The obtained mutant A86D was used for the preparation of new agarose（NA2）products,and it was found that the yield of NA2 obtained by enzymatic hydrolysis of agarose by A86D mutant at 50°C for 3 h was 5152.6μM,which was 6 times higher than that of the wild type;the yield of NA2 obtained by enzymatic hydrolysis of agarose by A86D mutant at 60°C for 3 h was 5513.6μM,while the wild type was completely inactivated and failed to play a hydrolytic role.Therefore,A86D is fully capable of working above the agarose condensation temperature of 40°C and has excellent potential for industrial applications.Besides,the presence of Aga50D dimer was confirmed by Native electrophoresis,Size Exclusion Chromatography,Small-angle X-ray Scattering and Differential Scanning Fluorescence,and the thermostability of the enzyme was positively correlated with the percentage of dimer.Further molecular dynamics simulations initially verified the mechanism of A86D to improve thermal stability.A86D was able to promote the formation of Aga50D dimer interfacial electrostatic interactions,improve the probability of dimer formation,enhance the compactness of conformation and have a more stable structure.With the help of computer-aided design,the mutant that can meet the stability possessed by industrial production was screened,which improved the application value of agarase Aga50D and provided a basis for the modification of other agarases to improve their performance.