| ?-Glutamyl peptides are widely distributed in nature and involved in various physiological activities of organisms.Glutaminase?EC 3.5.1.2?is a hydrolase with transpeptide effect.In this paper,a glutaminase for catalyzing?-glutamyl transpeptide reaction was screened out,and an unreported?-glutamyl peptide,?-[Glu]n?1-5?-Gln,was synthesized enzymatically.Furthermore,the effects of external conditions on its synthesis were also studied.Finally,the applications of?-[Glu]n?1-5?-Gln in calcium chelating ability and freezing resistance were explored.The experimental results were shown as follows:Firstly,the enzymatic characteristics of two glutaminases?E1 and E2?from Bacillus amylobacillus were studied.The results showed that both E1 and E2 could catalyze hydrolysis and transpeptide reactions.Under the conditions of p H 10.0 and 37.0?,the hydrolase activity of E1 was better than that of E2,however there was no significant difference in transpeptidase activity among E1 and E2?P<0.05?.Under the conditions of p H 4.0-10.0 and4.0-45.0?,E1 was more tolerable than E2.Try,Pro,Arg,Thr,and Gln all acted as the optimal?-glutamyl receptors for catalyzing the?-glutamyl transpeptide reaction by E1.Therefore,E1 was selected as the enzyme for subsequent experiments.Secondly,E1 was used to synthesize?-[Glu]n?1-5?-Gln,and the effects of solid concentration and initial p H value on its synthesis were investigated.The results showed that E1 had the ability to synthesize?-[Glu]n?1-5?-Gln.UPLC-MS/MS identified that the enzymatic reaction products contained?-Glu-Gln,?-Glu-Glu-Gln,?-Glu-Glu-Glu-Gln,?-Glu-Glu-Glu-Glu-Gln,and?-Glu-Glu-Glu-Glu-Glu-Gln.The increase of solid concentration or initial p H value was beneficial to the synthesis of?-[Glu]n?1-5?-Gln.Thirdly,calcium was chelated with?-[Glu]n?1-5?-Gln using aqueous system synthesis,and the synthesis process was optimized by single factor assay and orthogonal experiment.The structure characterization and cytotoxicity experiment of the product were also carried out.The results showed that?-[Glu]n?1-5?-Gln had calcium chelating ability.The optimal conditions were shown as follows:reaction time of 30 min,reaction temperature of 70.0?,peptide concentration of 0.10 g/m L,and peptide calcium ratio of 20:1?w:w?.Under these conditions,the chelation rate was 91.84%,and the calcium content of chelate was 4.39%.Both the amino group and the carboxyl group participated in chelation reaction.?-[Glu]n?1-5?-Gln and?-[Glu]n?1-5?-Gln chelated calcium did not show obvious toxicity to LO2 cells at concentration range of 0.3125 to 10 mg/m L.Finally,the antifreeze activity of?-[Glu]n?1-5?-Gln was explored by cooling curve and DSC experiments,and the hypothermia protection effect on yeast and the influence on the content of sulfhydryl groups in frozen dough were further investigated.The results showed that?-[Glu]n?1-5?-Gln had freezing resistance.The antifreeze activity of?-[Glu]n?1-5?-Gln prepared in an initial p H of 12.0 and reaction time of 6.0 h,was 2.38.?-[Glu]n?1-5?-Gln had hypothermia protection effect on yeast,and its protective effect on the disulfide bonds of frozen dough was also obvious. |
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