Study On The Improvement Of Thermal Stability And Expression Of Proteases And The Application To Improve The Nutritional Value Of Soybean Meal

Protease is a kind of hydrolase that can cut off the peptide bond of protein to produce free amino acids and polypeptides,which is one of the most studied enzymes in enzyme engineering.Due to its advantages of high efficiency,mildness and no chemical pollution,protease is widely used in feed,detergent,food industry,medicine,cosmetics,textiles and environmental governance,and its related development and application are also highly concerned in the global enzyme market.However,most of the wild protease enzymes found at present have low activity and poor thermal stability,which cannot meet the needs of industrial applications.Therefore,the improvement of thermal stability and expression of protease has always been a hot spot in this research field.On the other hand,the utilization of protease for the protein source of agricultural and industrial waste meal is a green and sustainable biological method,which is of great significance for the reuse of agricultural and industrial waste meal and the prevention of environmental pollution.So far,the biological method to improve the nutritional value of soybean meal feeding mostly focuses on the use of different types of microorganisms for fermentation,and the related research on enzymolysis of soybean meal is less,and the process is not comprehensive and in-depth enough.The process that can meet the industrial production and application has not been reported.In this study,the protease PEPA and NPI from Aspergillus were heterologously expressed in Pichia pastoris,and the molecular modification for improving their thermal stability was carried out based on B-factor and molecular dynamics simulation（MD）;Increase the expression of protease from signal peptide,chaperone protein related to folding secretory pathway and transcription activator;Finally,the research on enzymolysis of soybean meal with protease as the main enzyme reagent aims to reduce the antigen protein in soybean meal and increase the content of small molecular nutrients,so as to improve the feeding nutritional value of soybean meal and optimize the production process of related enzymolysis soybean meal.The main research results are as follows:1.Heterologous Expression and Enzymatic Properties of PEPA and NPI Proteinase Genes in Pichia pastorisBased on the NCBI database information,the acid protease gene PEPA from Aspergillus niger CBS513.88 and the neutral protease gene NPI from Aspergillus oryzae were synthesized by codon optimization.Two protease genes with precursor peptide were cloned and recombined into p PIC9K vector,and then fermented after screening of antibiotic concentration.After 48 h enrichment culture in BMGY medium and 72 h induction culture in BMMY medium with methanol,the specific activities of PEPA and NPI reached 382.825 U/m L and 812.09 U/m L respectively.The optimum temperature of PEPA and NPI is 50℃and 55℃respectively,and the optimum p H values are p H 3.0 and p H 7.0 respectively.The thermal stability of both PEPA and NPI was found to be poor by the determination of their thermal stability,and the enzyme activity was greatly lost,which was less than 35%,after 30 min tolerance at the optimal temperature.The p H stability of PEPA and NPI was measured,and it was found that the p H stability of the two was good near the optimal p H,and they were tolerant for 1 h respectively near the optimal p H,the former retained at least 50%of the relative residual enzyme activity,while the latter retained more than80%of the relative residual enzyme activity.In the experiment of determining the influence of metal ions on the activity of two protease enzymes,the metal ions Cu²⁺,Mn²⁺and organic reagentsβ-Mercaptoethanol significantly activated the activity of protease NPI,while SDS strongly inhibited it;Fe³⁺and organic reagents SDS and EDTA strongly inhibit the activity of protease PEPA,while Zn²⁺and Ca²⁺have certain activation effects.2.Rational design based on B-factor and molecular dynamics simulation to improve the thermal stability of proteases PEPA and NPIIn view of the poor thermal stability of the wild type protease,the acid protease PEPA and neutral protease NPI were normalized by the B-factor value of the x-ray crystal structure,combined with structural analysis and molecular dynamics simulation,to screen the residues with high flexibility.Partial overlap PCR was used to carry out single point mutation and compound mutation of the two mutants,and finally the dominant compound mutants N11R/N12S-D76R and D632G/A633V-S57W were obtained.The optimal temperature of both mutants was3℃higher than that of the wild type,and the half-life was 1.77 times and 2 times of that of the wild type,respectively.There was no significant loss of enzyme activity before and after the mutation.3.Screening and modification of high expression signal peptide of protease in Pichia pastorisIn order to improve the expression of protease in Pichia pastoris,this study attempts to screen the dominant fusion signal peptide expressed by protease,and mutates the N terminal and H terminal that have a significant impact on the secretion and expression of exogenous proteins,Results A high copy of acid protease PEPA（N2R/T7A-PEPA/p PIC9K）with an specific activity of 802.54 U/m L and a high copy of neutral protease NPI（S7A/E9A-NPI/p PIC9K）with an specific activity of 1801.49U/m L were obtained.4.Coexpression of chaperone protein of folding secretory pathway enhances the expression of protease in Pichia pastorisIn this study,strains N2R/T7A-PEPA/pp IC9K and S7A/E9A-NPI/p PIC9K of（3）were used as receptor bacteria,and the expression of protease was increased by co-expressing chaperones HAC1,PDI,Cne1,Bip of the folding secretion pathway.Results After single factor screening and combination screening of chaperone protein,and then screening of high concentration G418 resistance,the dominant recombinant bacteria PEPA-HAC1-PDI and NPI-HAC1-PDI were finally obtained,and their specific activities were increased by 16.6%and 21.16%respectively compared with the supernatant of receptor bacteria.5.Study on Enzymatic Hydrolysis of Soybean Meal with Low Antigen and Establishment of Related Industrial Production LineIn this study,protease was used as the main enzyme reagent to study the enzymatic hydrolysis of soybean meal,focusing on the elimination of the main anti nutritional factor antigen protein in soybean meal and the improvement of the content of small molecular nutrients,improving the nutritional value of soybean meal and optimizing the relevant production process.Finally,when the ratio of water to soybean meal was 40:100（V/M）,the quality of enzyme addition reached 4‰（the ratio of protease addition was not less than 4×10⁵U/g（U/m））,when the enzymolysis temperature is between 35℃and 45℃,the soybean meal is enzymatically hydrolyzed by compound soybean meal hydrolytic enzyme No.2,including protease NPI,for 32hours,and finally the reducing sugar content of enzymolysis soybean meal is increased by at least 4 times,the polypeptide content is increased by at least 2 times,and the free amino acid content is increased by at least 2 times,achieving the expected enzymolysis effect.Finally,the industrial production line of low antigen soybean meal feed was successfully established by combining this enzymatic soybean meal process with local feed production companies.