Analysis Of The Interaction Between Alkaline Protease 2709 And Eglin C

Proteases are hydrolytic enzymes that break down proteins into short peptides,polypeptides,and amino acids.Protease has been widely used in various industrial processes such as washing powder,meat tenderization,leather softening treatment,feather processing,etc.Microbial-derived proteases are mainly used in industry.How to improve enzyme activity and simplify the purification of proteases has always been the focus of research in this field.Eglin C is a small heat-stable protein from leeches,belonging to the potato chymotrypsin inhibitor family,which can inhibit elastase,subtilisin,cathepsin,?-lytic protease,and chymotrypsin.First,p QE30-eglin C and p QE30-?(1-8)eglin C recombinant vectors were constructed,and transformed into Escherichia coli BL21(DE3)for recombinant expression,the purified eglin C and the deletion of 8 amino acid residues at the N-terminal?(1-8)eglin C were obtained through Ni²⁺-Sepharose4B Fast Flow affinity column and Superdex-75 gel column.15%SDS-PAGE revealed that the molecular weight of the target protein was about 8 k Da.The inhibitory activity of eglin C on different proteases was analyzed.The IC₅₀values of eglin C inhibitory activities for chymotrypsin,Bacillus licheniformis 2709 alkaline protease,Bacillus subtilis PB92alkaline protease,and Bacillus subtilis neutral protease are:0.20±0.15,0.24±0.19,3.33±0.47,52.46±0.38?mol/L,respectively.Eglin C and?(1-8)eglin C had no inhibitory activity on trypsin.The IC₅₀value of?(1-8)eglin C to 2709 alkaline protease is 0.30±0.43?mol/L.Compared with eglin C,its inhibitory ability to 2709 alkaline protease is basically stable.It shows that the deletion of the N-terminus of eglin C may not be related to the inhibitory properties of proteases,but from the stability analysis,it is found that the protease will hydrolyze the N-terminus,which is also the result of the instability of the N-terminus.The ZDOCK server is used for molecular docking between eglin C and 2709 alkaline protease,it was found that the 4amino acid residues(Asn219,Ser100,Gly62,Ser99)of 2709 alkaline protease and the 3 amino acid residues(Asp46,Arg48,Asn50)of the reactive site binding loop of eglin C formed an interface interaction.In order to test the results of molecular docking simulation,the key residues Arg48 and Asn50 are both mutated to Ala by using site-directed mutagenesis technology,the analysis of the inhibitory activity of the 2709 alkaline protease revealed that the IC₅₀values of eglin C R48A and eglin C N50A mutants inhibitory activities for 2709 alkaline protease are:8.97±0.33?mol/L,1.96±0.42?mol/L,respectively.It was analyzed by fluorescence spectroscopy,the interaction of eglin C and its mutants R48A and N50A with 2709 protease was studied.The dissociation constants of eglin C,eglin C R48A,eglin C N50A and 2709 alkaline protease are 2.60×10^-7mol/L,2.70×10^-7mol/L,5.98×10^-6mol/L,respectively.We compared the semi-inhibitory activity and dissociation constant of eglin C wild-type and mutant on 2709alkaline protease and found that Arg48 is only responsible for the inhibition of protease activity,while Asn50 not only binds to 2709 protease,but also participates in the inhibition of protease activity.But the effect on protein binding is stronger than inhibition of activity.Finally,the prepared Ni²⁺-sepharose4B-His₆-Tag-eglin C and?(1-8)eglin C affinity column was used to purify the 2709 alkaline protease from Bacillus licheniformis,it was found that the Ni²⁺-sepharose4B-His₆-Tag-?(1-8)eglin C affinity column was more stable,and the purification effect of protease was better.which greatly simplifies the operation steps compared to conventional protease purification.In short,we recombinantly expressed eglin C protein,the binding relationship between eglin C and several alkaline proteases was analyzed,and it was found that it has a strong binding ability with alkaline protease 2709.Asn50 site has a significant effect on the binding of eglin C to alkaline protease 2709,the constructed alkaline protease purification system can realize the separation and purification of alkaline protease 2709 in one step.