Improvement Of Thermostability Of β-mannanase From Thermoanerobacterium Aotearoense By Ca²⁺ Binding Site Mutation

β-mannanase is widely used in industrial fields such as bioenergy,food processing,feed manufacturing,and petroleum exploitation because it can hydrolyze the mannan in hemicellulose into small molecular substances.In industrial production,the hydrolysis of mannans often requiresβ-mannanase with high activity and good thermostability.We screened a strain of Thermoanaerobacterium aotearoense SCUT27 in the early stage,and successfully cloned itsβ-mannanase（Man25）gene and expressed Man25 in E.coli BL21（DE3）.Man25shows many excellent characteristics,such as high specific enzyme activity and good stability between pH 3-8.However,the thermostability of Man25 is poor,so in order to make Man25more suitable for industrial applications,this study improved the thermostability of Man25 by modifying the Ca²⁺binding site.First,we identified the Ca²⁺binding sites as E32,E34,S53,G56 and D143 through protein sequence alignment and homology modeling.Mannotriose and mannopentaose as the ligand was molecularly docked with Man25,and the results showed that the Ca²⁺binding site is far away from the catalytic active site.Therefore,we speculate that the modification of the Ca²⁺binding site has little effect on the catalytic activity of the enzyme.In order to screen more mutants,we have developed a method for the detection of intracellularβ-mannanase enzyme activity based on the leaky expression of the T7 promoter in E.coli BL21（DE3）and the cell cleavage by high temperature.The strain can be lysed when cultured at 50℃for 12 h,resulting in a clear hydrolysis circle,and its EI value is 3.78±0.07.The advantages of this method are convenience,rapidity,short cycle,high throughput,and no need to add inducers.Through saturation mutation of the Ca²⁺binding site and high-throughput screening of the mutation library,we finally obtained two mutants D143A and E32K/E34S with improvement of thermostability.Compared with Man25,D143A and E32K/E34S presented improvements in thermostability with 3.6-fold and 1.8-fold extended half-life respectively at 55℃,and specific activity increased by 20%and 7%respectively.In addition,the V_max of D143A and E32K/E34S were increased by 11%and 3.7%respectively,and their K_m values were not much different,indicating that the improvement of thermostability of Man25 did not cause the enzyme catalytic activity to be lost.Analysis of the two mutants D143A and E32K/E34S by molecular dynamics simulation showed that the radius of gyration and the accessible area of the solvent were reduced,indicating that the internal interaction in the two mutants was stronger and the protein folding was tighter.The reason for the improved thermostability may be related to the enhanced hydrophobic effect of the D143A mutation and the hydrogen bond produced by the E32K/E34S mutation.In this study,we successfully screened two mutants with higher thermostability in the Ca²⁺binding site mutation library with the help of the developed a method for the detection of intracellularβ-mannanase enzyme activity.The results show that the mutation of the metal ion binding site can improve the thermostability ofβ-mannanase.Our strategy provides a new idea for the modification of the thermostability ofβ-mannanase with metal ion binding sites.In addition,the simple preliminary screening method we developed can be applied to the screening of other thermophilic enzymes expressed in E.coli BL21（DE3）cells.