The Research Of ?-Glutamyl Peptides Synthesized By Glutaminase From Bacillus Amyloliquefaciens And Their Antioxidant Activity

?-glutamyl peptides are derived from dehydration and condensation of?-carboxyl group of glutamine?or glutamic acid?with?-amino group of amino acid?or peptide molecule?,which are naturally distributed in animals,plants and microorganisms.?-glutamyl peptides were first discovered in 1956.Since?-glutamyl peptides were found to be physiologically active and in possession of unique taste characteristics,its researches have never been interrupted.?-glutamyl peptides can be obtained through extraction in natural resources or chemical synthesis.However,enzymatic synthesis is a method much more widely used in?-glutamyl peptides production.?-glutamyl transpeptidase is commonly applied in synthesis of?-glutamyl peptides.Researches have proved that some microbial-derived glutaminase can also catalyze?-glutamyl transfer reaction.Therefore,glutaminase is possibly applied in?-glutamyl peptides synthesis and researches on that can help expand its application area.In this thesis,?-glutamyl peptides synthesis was catalyzed by glutaminase from Bacillus amyloliquefaciens,in which cysteine and tryptophan are two amino acids used as?-glutamyl acceptor,glutamine used as?-glutamyl donor.Synthesis condition was optimized and the potential antioxidant activity of?-glutamyl peptides was researched.In the first place,the characteristics of glutaminase from Bacillus amyloliquefaciens were studied,two commercial glutaminase?named A and B?taken as subjects.Results revealed that glutaminase A and B can catalyze both glutamine hydrolysis and?-glutamyl transfer.Under the condition of pH 10.0,37?,the hydrolysis activity of glutaminase A and B were 66.21 U/g and5.27 U/g,respectively.The transpeptidation activity of glutaminase A was 101.28 U/g and glutaminase B had no transpeptidation activity in this condition.In conclusion,glutaminase A possessed better hydrolysis activity as well as transpeptidation activity.Therefore,glutaminase A was chosen to synthesize?-glutamyl peptides next step.Besides,LX-1000EP epoxy resin was applied to immobilize glutaminase A.The influences of factors including immobilized pH,adsorption time and relative enzyme addition on immobilized ratio were studied in response surface method.The optimized immobilized condition was worked out as followed:immobilized pH 10.5,adsorption time 1 h,relative enzyme addition 3.92%.On this condition,immobilized ration reached 76.77%.UPLC-MS/MS was applied to identify the items in both the Gln-Cys reaction system and Gln-Trp reaction system.Four?-glutamyl peptides were detected in the former system,including?-EC,?-EEC,?-EEEC,?-EEEEC.Also,?-EW,?-EEW,?-EEEW and?-EEEEW were identified in the latter one.After that,HPLC was used to quantify the products in two reaction systems.Based on the yields of the products,reactive conditions were optimized as follows,pH,temperature,enzyme volume and substrate concentration being 10.0,37?,0.1%and 0.1mol/L,respectively.Under this condition,the yields of?-EC,?-EEC,?-EEEC,?-EEEEC were40.92%,22.79%,1.75%and 0.64%,respectively.While,the yields of?-EW,?-EEW,?-EEEW were 51.02%,26.12%and 1.91%,respectively.The conversion rate of cysteine and typtophan were 66.10%and 79.05%,respectively.In the last chapter,the antioxidant activity of?-glutamyl peptides were estimated with indexes of DPPH radical scavenging rate,reducing power,Fe²⁺chelating rate,ABTS radical scavenging rate,O₂?^-scavenging rate.Results showed?-EC possese a stronger antioxidant ability than GSH does.The EC₅₀ of?-EC to scavenge DPPH radicals,ABTS radical and O₂?^-were 0.0525 mg/mL,3.1858?g/mL and 0.0196 mg/mL,respectively,which was followed by S-1,?-EEC,?-EW,S-2 and?-EEW.Different conclusion was obtained in terms of the ability to chelate Fe²⁺.?-EEC was most capable of chelating Fe²⁺,followed by?-EEW,S-1,?-EC,S-2,?-EW.