Molecular Modification And Immobilization Of Laccase-like From Bacillus Licheniformis

Laccase is a green catalyst with widespread applications in various fields such as food,paper,wastewater treatment,pharmaceuticals,sensors,and fuel cells.However,the enzyme has disadvantages in practical applications such as poor activity,poor stability,and difficulty in reuse.Therefore,it is important to improve the catalytic activity and thermal stability of laccase and to effectively recover free laccase.The BILac-like studied here is derived from Bacillus licheniformis and possesses both laccase and purine nucleoside phosphorylase activities.In this study,the catalytic activity to the substrate 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid（ABTS）and thermal stability of the enzyme were enhanced by site-directed mutagenesis.Then the metal inorganic salt immobilization technique was used to immobilize the enzyme,which improved the reusability and storage stability of the enzyme,and laid the foundation for the application of laccase in environmental and other fields.（1）The recombinant plasmid p MA5-BILac-like was constructed and BILac-like was expressed in Bacillus subtilis.The enzyme was molecularly modified based on homology modeling and molecular docking analysis using ABTS as substrate:the Mutant Library I was based on mutation of amino acids by substrate binding energy to enhance enzyme activity,and the catalytic efficiency(k_cat/K_m)of the screened mutants K226S,C189A,K217F,V245Y,E227Y,and S247F reached 22,10.9,5.9,4.3,3.1and1.3 times of the wild type.In order to improve the thermal stability,the Mutant Library II was constructed by predicting the disulfide bond according to the homologous structure model of Bl Lac-like.Mutants S251C,G151C,G187C,A156C/K200C（V1）were screened,and their half-lives at 40°C were 3.2,2.4,2 and 3.2 times that of the wild type,respectively.（2）The high-quality mutants screened by the two mutant libraries were subjected to combined mutation,and finally the mutant C189A/S251C with improved catalytic efficiency and thermal stability was obtained.The catalytic efficiency for the substrate ABTS of the mutant C189A/S251C can reach 8453 L·mmol^-1·s^-1,which is 6.3 times higher than that of the wild type,and the specific enzyme activity also increased from 4.68 U·mg^-1 to 28.49 U·mg^-1;The T_m value increased from 54℃to 57℃and the half-life at 40℃increased from 2.5 h to 5.5 h in the wild type;In the study of p H stability and optimum temperature,it was found that the mutant had higher tolerance to acidic environment than the wild type,and the optimum temperature was also increased from 37°C to 50°C.（3）The metal inorganic salts immobilization on mutant C189A/S251C was studied.and the immobilized enzyme Ni-C189A/S251C was constructed under the optimal preparation conditions.The enzymatic properties showed that the catalytic efficiency of Ni-C189A/S251C reached 29844.24 L·mmol^-1·s^-1,which was 3.5 times higher than that of the free enzyme,and the specific enzyme activity increased from 28.49 U·mg^-1 to 83.86 U·mg^-1.Moreover,the immobilization method effectively enhanced the storage stability and reusability of BILac-like:after 8 days of storage at room temperature,the enzyme activity of the free enzyme was only43%of the initial value,while the immobilized enzyme retained more than 60%of the enzyme activity;Ni-C189A/S251C can maintain 50%activity after repeated use for 5 times.