Modification Of The Thermostability Of Bacillus Subtilis Keratinase

Keratinases are a class of proteases that specifically catalyze keratin substrates and have a wide potential for applications in feed additives,green tanning,cosmetics and pharmaceuticals.However,the temperature tolerance of the application environment has been the key limiting its catalytic efficiency.Therefore,the development of mutants with good thermal stability has become the focus of keratinase research.In this study,we used the recombinant keratinase KerZ1 as the research target and obtained several keratinase mutants with significantly improved thermal stability based on the fixed-point mutation technique by calculating the free energy of protein folding,the B-factor value of amino acid residues and the flexibility of the Loop region.The main findings are as follows.（1）Enhancement of keratinase KerZ1 thermal stability based on protein folding free energy calculationBased on the calculation of protein folding free energy（ΔΔG）,eight potential sites related to the thermal stability of KerZ1 were obtained.The mutant N122F with improved thermal stability was obtained by targeted mutation,and the t_1/2 of N122F was 25.4 min at 60℃,which was 1.4 times higher than that of KerZ1（18.1 min）.Meanwhile,the T₅₀ of N122F was increased by 3.1℃ compared with that of KerZ1.The catalytic efficiency(k_cat/K_m)of N122F was not affected.（2）Saturation mutations improve the thermal stability of keratinase KerZ1Six sites potentially altering the thermal stability of KerZ1 were obtained based on the calculation of B-factor values of amino acid residues.The mutants T77C and T77E with improved thermal stability were obtained by targeted saturation mutation combined with high-throughput screening.t_1/2 of T77C and T77E were 40.3 min and 41.2 min,respectively,at 60℃,which were 2.2 and 2.3 times higher than those of KerZ1（18.1 min）.Meanwhile,the T₅₀ of T77C and T77E increased by 9.0℃ and 7.3℃,respectively,compared with KerZ1.The catalytic efficiency(k_cat/K_m)of T77E increased by 8.1%compared with KerZ1,while that of T77C decreased.（3）Rational design of the Loop region improves the thermal stability of keratinase KerZ1The stability of the Loop region in KerZ1 was predicted by B-factor analysis and molecular dynamics simulation,and three unstable Loop regions were obtained.Based on the statistical analysis of theβ-turn angle,amino acid mutations were introduced in theβ-turn angle of the Loop region,and mutants A128D and L240N with significantly improved thermal stability were obtained.The results showed that the t_1/2 of A128D and L240N at 60℃were 30.4 min and 34.3 min,respectively,which were 1.7 times and 1.9 times of the half-life of KerZ1.The T₅₀ of A128D and L240N were 5.8℃and 6.6℃higher than that of KerZ1,respectively.Meanwhile,the catalytic efficiency(k_cat/K_m)of A128D and L240N was not significantly affected.（4）Complex mutations to improve the thermal stability of keratinase KerZ1To further enhance the thermal stability of keratinase KerZ1,seven compound mutants were obtained by compound mutation of the excellent mutants obtained by fixed-point saturation mutation and Loop region modification.Among them,the t_1/2 of A128D/L240,T77E/L240N and T77C/A128D were 42.3 min,60.2 min and 90.7 min,respectively,which were 2.3,3.3 and 5.0 times longer than the half-life of KerZ1.The catalytic efficiency(k_cat/K_m)of T77E/L240N was improved by 10.3%compared with KerZ1,while the catalytic efficiencies of A128D/L240 and T77C/A128D were reduced.