Improved Thermostability Of CALB By Rational Design Combined With Immobilization

Candida antarctica lipase B（CALB）belongs to serine hydrolases with anα/βhydrolase fold.CALB has been widely applied in industries due to its excellent catalytic performance.However,the relatively low thermostability of the wild-type CALB cannot meet the needs of hash industrial conditions such as the elevated temperatures.In order to improve the thermostability of CALB and expand the application of CALB in industry,rational design combined with immobilization strategies were designed,and the mutants with improved thermostability were screened out.In addition,we tried to increase the expression of CALB in Pichia pastoris by fed-batch fermentation.The results were summarized as follows:（1）Potential thermostable mutations were selected based on the change of free energy calculated by the software PoPMuSiC and FoldX,as well as the spatial positions of amino acid residues.The mutation A146G,A151P,and L278M have been verified that they can effectively improve the thermostability of CALB.After combining the three mutations,the thermal stability of the combined mutants was further improved.The half-life of A146G-L278M was6.28 h at 50°C,which was 1.35-fold higher than that of the wild type,and the T_m values was increased by 3.3°C.Besides,the optimal reaction temperature of A146G-L278M was 50°C,which was 5°C higher than the wild type,and the specific activity was 2-fold higher than that of the wild type.The kinetic parameters in an ester synthesis reaction indicated that the catalytic efficiency k_cat/K_m of A146G-L278M for hexanoic acid was 4.1-fold higher than that of the wild type.（2）The mutant CALB-4X was obtained by the introduction of the disulfide bond T180C-A240C predicted by software MODIP and BridgeD in A146G-L278M.The half-life of CALB-4X was 121 h at 50°C,which was 26-fold higher than that of the wild type,and the T_m values was increased by 4.0°C.The optimal reaction temperature of CALB-4X was 5°C higher than the wild type,and the kinetic parameters in an ester synthesis reaction showed that the catalytic efficiency k_cat/K_m for hexanoic acid was 3.8-fold higher than that of the wild type.In addition,the inhibition constant K_i indicated that ethanol has a stronger inhibitory effect on the wild type.（3）The mechanism of the thermostable mutants was analyzed.According to molecular dynamics simulation analysis,the overall RMSD shift of the mutants A146G-L278M and CALB-4X are lower than that of the wild type,indicating that the offset of the C_αatom of the protein main chain has decreased after mutation,and the protein structure has better stability.The RMSF of the mutants and the wild-type were generally similar,indicating that the mutation did not affect the structure of CALB too much.（4）CALB-4X was immobilized by siliceous mesocellular foams.The protein loading was98.8 mg·g^-1,the immobilization yield was 47%,and the enzyme activity recovery rate reached80%.The half-life of the immobilized enzyme MCFs-C8-CALB-4X at 50°C was 481 h,which was 4-and 103-fold higher than that of the free enzyme CALB-4X and the wild type,respectively.The immobilized enzyme had higher enzyme activity at 60°C than the free enzyme.Furthermore,the immobilized enzyme has good reusable stability,it can still retain 77%of the activity after using 6 times repeatedly.（5）Improvement of the expression of the thermotolerant mutants in Pichia pastoris by fed-batch fermentation.The enzyme activities of the mutant strains GS115-calb-A146G-L278M and CALB-4X in 3 L fermentor could reach 582 and 542 U·mL^-1,respectively,which were 2.5 and 2.4-fold higher than the wild-type,and the amount of extracellular protein respectively reached 2.4 and 2.5 mg·mL^-1.