Optimization And Structure Analysis Of A Protease With Highly Beta-Lactoglobulin Degradation Ability

With popularity of the dairy sensitization,desensitized dairy products have become a research hotspot.At present,the most promising preparation method is the protease hydrolysis method,which can effectively reduce and eliminate the allergen while retaining the nutritional parts of dairy.Therefore,a protease which can effectively degrade allergens own application value and could help us to understand the mechanism of protease desensitization better.Subtilisin from Bacillus subtilis is widely used in light industry because of its excellent industrial property.However,due to its poor thermal stability,there is a necessity to improve its stability through a structural modification to couple with the industrial process.In addition,because of its special catalytic pocket structure and wide substrate selectivity,subtilisin is very suitable for the rational design starting point to customize mutants with special selectivity.The crystal structure can provide a lot of structural information.These information can help us improve the success rate of rational design.Based on this,we obtained a protease?subtilisin E-S7?form high-throughput screening which could effectively degrade the milk allergen beta-lactoglobulin.The research about its enzymatic property,crystal structure and selectivity modification were conducted in this study.The main results are as follows:?1?Strain screening and protease identificationSkim milk plate hydrolysis ring method and allergen beta-globulin?BLG?ELISA kit were applied to screen the bacteria.Totally 16000 bacteria were tested and 42 bacteria with better BLG degradation ability were selected.Then 16s rDNA and functional gyrA gene sequencing were applied to these bacteria.The best result come from a Bacillus subtilis JN-S7and its preservation Numbers is CCTCC M2016532.Using peptide fingerprint analysis,the critical protease,subtilisin E-S7?SES7?,in the target bacteria was identified.Its gene sequence was extracted to construct recombinant expression plasmid pET-24a?+?/aprE.Recombinant protease was obtained then through preliminary separation and purification.Combined with sensory evaluation and high-performance liquid chromatography,we analyzed the taste and free amino acid content of hydrolyzed milk powder after hydrolyzed by SES7.?2?Protease enzymatic properties analysis and crystal structure analysisThe X-ray crystallographic structure of the mature form of subtilisin E-S7?SES7?at 1.90???resolution is reported here.The best crystallization condition is 0.15 M ammonium sulfate,15%?w/v?PEG 4,000 and 0.1 M Tris-HCl?pH 8.0?.Structural comparisons between the previously reported propeptide-subtilisin E complex?1SCJ?and our mature form subtilisin E-S7?6O44?,an S221C mutant,provide insight into active site adjustments involved in catalysis and specificity.To further investigate the protease substrate selectivity mechanism,we used SES7 to hydrolyze skim milk and analyzed the hydrolysates by LC-MS for peptide identification.The cleavage pattern suggests a high preference for proline at substrate P2position.The results based on the peptide analysis are consistent with our structural observations,which is instrumental in future protein engineering by rational design.Furthermore,the ACE-inhibitor and NLN-inhibitor activity of the hydrolysates were determined to assess the utility of SES7 for further industrial applications;IC₅₀-ACE=67±0.92?g/mL and IC₅₀-NLN=263±13?g/mL.?3?Thermal stability improvement by the b-fit strategyWe performed structure-based engineering to improve the thermostability of the subtilisin E-S7?SES7?peptidase.The B-value of each residue was redefined in a normalized B-factor calculation,which was implemented with a novel bioinformatics analysis strategy to identify the critical area?loop 158–162?related to flexibility and to screen for suitable thermostable motif sequences in the PDB database that can act as transplant loops.In total,445 structures were analyzed,and 29 thermostable motifs were identified as candidates.Iterative homologous modeling was performed using these motifs as a starting point for obtaining a desirable chimera loop.Five different mutations were introduced into this loop to construct new thermostable SES7 proteins.Differential scanning fluorimetry?DSF?revealed increases in the Tm of M5 of 7.3°Ccompared to the wild-type.The X-ray crystallographic structure of the best SES7 mutant?S221C and M5?was resolved at 1.96???resolution.The best crystallization condition is 0.2 M sodium bromide,20%?w/v?PEG 3,350The crystal structure shows that M5 forms more hydrogen bonds than the wild-type protein,which is consistent with our design and molecular dynamics simulations?MDS?results.This new B-FIT strategy is a powerful tool for protein engineering,as a mutant subtilisin with improved thermostability and promising industrial applications was obtained.?4?Rational design of protease substrate selectivityWe generated subtilisin E-S7?SES7?with altered substrate specificity.Protein engineering to modulate the electrostatic interactions of SES7 with amino acids at substrate S1 position led to a⁵-fold increase in selectivity toward positively charged substrates.The activity was restored by a double loop-graft mutation;the S1 cave was further adjusted by evaluating the bottleneck radius?BNR?.From kinetic and BNR analyses of wild-type and 23mutants of SES7,we determined that the protease loses activity beyond the BNR range of2.02–2.58???;k_cat/K_m reached a maximum when BNR was 2.53???.With the change in specificity,the best mutant L1-2&L2-5 eliminated the allergen?-lactoglobulin,retained other nutrient proteins in the milk powder,and generated angiotensin-converting enzyme-inhibitory activity hydrolysates.This demonstrates the feasibility of protease engineering by rational design of the S1 cave charge distribution,geometry,and bottleneck radius to generate a tailored protease for the milk powder industry.