Improving The Low-temperature Activity And Expression Element Optimization Of Keratinase KerZ1

Keratinase,a protease that can specifically degrades keratin,has shown advantages in improving tanning processes and in the cosmetic industry.Keratinase KerZ1,recombinantly expressed by Bacillus subtilis,can efficiently hydrolyse keratin substrates at the optimum reaction temperature（60℃）,but its catalytic activity is low at moderate and low temperatures below 60℃,making it difficult to adapt to the low temperature requirements of leather dehairing processes and cosmetic applications.Therefore,this study has improved the low-temperature catalytic activity of keratinase KerZ1 through directed evolution combined with simulation calculations and optimized the 5’-UTR expression element of its mRNA,laying the foundation for improving the utility of keratinase KerZ1in the tannery industry and cosmetics,as well as providing a valuable modification idea for other industrial enzymes.The contents and conclusions are as follows.（1）Through directed evolution,we screened for a keratinase mutant T3I/V45D/S100D that has the advantage of low-temperature activity.The specific activity of the mutant T3I/V45D/S100D was increased by 25.96%,29.20%and 112.91%compared to the KerZ1 at the optimum temperature of60℃,moderate temperature of 40℃and low temperature of 20℃,respectively.The half-life time(t_1/2)of the mutant T3I/V45D/S100D was almost the same as the original enzyme,and there was almost no loss of thermal stability.The mechanism for the enhanced low-temperature catalytic activity of this mutant may be due to the increased negative charge within the active pocket and the altered electrostatic potential on the surface of the enzyme molecule.The mechanism for the enhanced low-temperature catalytic activity of this mutant may be due to the increased negative charge within the active pocket and the altered surface electrostatic potential of the enzyme molecule.（2）Through homology comparison and simulated amino acid mutation,site directed mutation was introduced into the loop region of keratinase kerz1 to enhance its flexibility.Then,the three positive amino acid mutants introduced into the loop13 were compounded to construct the compound mutant T210S/N211S/T212G.The determination of enzymatic properties showed that the specific activity of the compound mutant was 45.68%and 85.74%higher than KerZ1 at 40℃and 20℃,respectively.The low temperature activity enhancement mechanism of the mutant T210S/N211S/T212G mainly comes from the reduction of hydrogen bonds between loop3 and loop13,but this also leads to the loss of thermal stability of the mutant at the optimum temperature.（3）The root mean square fluctuation（RMSF）values of residues in the Loop region of keratinase KerZ1 were analysed to obtain four potentially relevant sites for low-temperature activity,and a low-temperature-activity advantage mutant V26K was obtained by targeted saturation mutagenesis combined with high-throughput screening,showing an increase of 22.60%and 129.65%in specific activity over KerZ1 at 40℃and 20℃,respectively.The catalytic efficiency of the enzyme was also improved by 4.61%.（4）Four compound mutants were constructed by compounding the obtained mutants with advantageous low temperature catalytic activity of keratinase.Among them,complex mutant T3I/V45D/S100D/T210S/N211S/T212G showed a 6.80%increase in t_1/2 compared with KerZ1,while their specific activities at 20℃to 60℃were increased by 234.18%,70.56%and 31.87%compared with KerZ1.Meanwhile,its k_cat/K_m at 40℃reached 30155.30 g^-1·L·s^-1,the highest level for the low-temperature catalytic activity mutant of keratinase in this study.（5）Optimization of the 5’-UTR region of the mutant T3I/V45D/S100D/T210S/N211S/T212G expression element by Gibbs free energy calculation and sequence simplification strategies.The mutant KerZ1-LtAB-C4-Sim3C was screened for a 60℃enzyme activity of 204.44±1.19 k U·m L^-1after the shake-flask fermentation for 24 hours,which was 54.02%higher than T3I/V45D/S100D/T210S/N211S/T212G and 85.57%higher than KerZ1.After the fermentation in a5 L fermentor for 24 hours,the recombinant strain B.subtilis WB600 KerZ1-LtAB-C4-Sim3C showed enzyme activities of 797.05±20.86 k U·m L^-1 and 349.55±5.02 k U·m L^-1 at 60℃and 40℃,respectively,which were 42.98%and 80.79%higher compared to the original strain B.subtilis WB600 KerZ1.The advantages of its enzyme production capacity and low temperature catalytic activity of the enzyme were well-verified.