Site Directed Mutagenesis Of L-asparaginase Tli10209 And Its Inhibitory Effect On Breast Cancer Cell Line MCF-7

L-Asparaginase（L-Asparaginase）can degrade L-asparagine（L-Asn）into ammonia and aspartic acid,which is widely distributed in many species such as bacteria,fungi,algae and mammals.Currently,L-asparaginase from bacteria is widely used.Due to the lack of asparagine synthetase in tumor cells,proteins that depend on exogenous L-Asn synthesis are needed.The application of L-asparaginase can reduce the content of L-Asn in cells,thus inhibiting the growth of tumor cells while normal cells are affected less because they can synthesize L-Asn endogenously.Therefore,L-asparaginase has been used as a first-line drug for treating acute lymphocytic leukemia,and L-asparaginase also has the potential to treat other hematological malignancies or solid tumors.However,due to the glutaminase activity of known L-asparaginase,the reduction of glutamine content in the blood during treatment can cause side effects such as pancreatitis,neuropathy,and cerebral hemorrhage.Its immunogenicity and the above-mentioned side effects limit its clinical application and often lead to treatment interruption.Therefore,how to modify the existing L-asparaginase molecules to reduce glutaminase activity and increase L-asparaginase activity in order to reduce the dosage of enzyme therapy is the main direction of current L-asparaginase transformation research.L-Asparaginase also has good therapeutic potential in some solid tumors,including breast cancer.As the most common tumor in the world,breast cancer has a long treatment period or more side effects with non-targeted therapy.Therefore,the development of new high-efficacy and low-side-effect treatments or drugs can greatly reduce the burden of patients.Thermophilic L-asparaginase has good stability and low activity at body temperature,so it can be used to treat tumors through appropriate strategies.In order to fully utilize the advantages of thermostable enzyme,this paper fixed the mutated enzyme with improved activity after molecular modification with synthesized gold nanorods,so that the immobilized enzyme can be activated by near-infrared（NIR）light.The enhanced permeability and retention effect（EPR）effect of gold nanorods itself can realize passive targeting,and its excellent photothermal conversion efficiency can construct a therapeutic system with photothermal therapy and amino acid deprivation therapy.Subsequently,MCF-7 breast cancer cells were used to evaluate the therapeutic effect of the system.The main research contents and results of this paper are as follows:（1）The L-asparaginase Tli10209 cloned and expressed from thermophilic bacteria Thermococcus litoralis DSM 5473 in the laboratory was homologous and docked with the substrate molecule.By sequence alignment with high enzyme activity of thermophilic L-asparaginase sequence,8 amino acid residues were selected for site-directed mutagenesis to construct 12 single-point mutants,and the expression and purification of mutants were successfully completed.（2）Characterization of the constructed mutants.Among the six beneficial mutations in single-point mutations,three overlapped mutants were further constructed,including 15mutants in total.Nine mutants with higher enzyme activity than wild type were found,and the ones with significantly higher enzyme activity at high temperature were T70A/D50G,K48L,T70A.At 37℃,the enzyme activities of T70A and T70A/D50G were 8.12 and 5.97 times higher than that of the wild type,respectively.The results of the optimal temperature of mutants showed that only 5 mutants had the same optimal temperature as the wild type,which was 90℃,and the other 10 mutants had their optimal temperatures reduced by 10-20℃;the results of the optimal p H showed that most of the mutants had their optimal p H increased by1-2;the kinetic parameters results showed that only a few mutants（E134N,T70A/D50G）mainly increased the affinity between enzyme and substrate to improve the enzyme catalytic efficiency and enzyme activity,and most of the mutants increased the enzyme activity by changing the k_cat and k_cat/K_m values.（3）The mutants with increased enzyme activity under physiological conditions were used on MCF-7 breast cancer cells.When the same concentration of mutants was used to treat cells,T70A showed the strongest inhibitory effect on cell proliferation,and the cell survival rate was66.9%,which was 24.6%lower than the wild type（91.5%）.The proliferation inhibition effect of other mutants was also stronger than that of the wild type.The five mutants with the highest enzyme activity were immobilized on the prepared gold nanorod surface through gold-sulfur bonds,and the inhibition effect of the immobilized enzyme on cell proliferation before and after NIR activation was determined.It was found that under the same concentration condition,the cell survival rate of T70A group after NIR activation was only 23%,which was 36%lower than that of the wild type.In summary,9 advantageous mutants of thermophilic L-asparaginase Tli10209 were screened and characterized in this study.Beneficial mutants showed better results in inhibiting the proliferation of MCF-7 breast cancer cells,whether using free enzymes or immobilized enzymes.