Identification Of Aminopeptidase Originated From Lysinibacillus Sphaericus And Heterologous Expression

Proteolysates often have a bitter taste,which hinders their application in the food processing industry.Aminopeptidase is a kind of exopeptidase,which can destroy the bitter peptide in the proteolysates,and is widely used in food processing industry.The goal of this research is to identify a novel aminopeptidase from Lysinibacillus sphaericus to provide a candidate of debittering enzymes for the food and feed industry.In this study,aminopeptidase-coding genes were predicted in L.sphaericus C3-41 genome,their Escherichia coli expression strains were constructed,and the aminopeptidases were purified.With the artificial substrates L-Leucine-p-nitroaniline（Leu-pNA）and L-Proline-p-nitroanilide（Pro-pNA）,their aminopeptidase activities were tested to select a good one for further study.By this way,Amp0279 was identified,and then its enzymological properties were determined,including the optimal reaction temperature,the optimal reaction pH,thermal stability,pH stability,the effect of metal ions and inhibitors.The activities of Amp0279 and the commercial Flavourzyme（?）were compared in the optimal conditions.In addition,a secretory expression system of Amp0279 was constructed in the food-safe strain Bacillus subtilis WB800N,and the production and activity of Amp0279 were tested,which laid a foundation of the use of Amp0279 in the food and feed industry in the future.Experiments were carried out according to the above route.The main results are as follows:（1）15 genes encoded aminopeptidases were predicted in L.sphaericus C3-41,and E.coli expression strains of them were constructed.And then 10 soluble aminopeptidases were purified,including Amp0116,Amp0279,Amp0293,Amp0832,Amp2281,Amp2720,Amp3285,Amp3546,Amp4139 and Amp4212.Artificial substrates Leu-pNA and Pro-pNA were used to detect their aminopeptidase activity,and Amp0279 was found to have good hydrolytic activity on Leu-pNA.（2）Bioinformatic analysis of Amp0279 amino acid sequence showed that Amp0279 belonged to the M29 protein family and was a metalloenzyme with Co²⁺as its active center.It was found that the optimal temperature of Amp0279 was 50℃,the optimal pH was 8.0.Amp0279 could hydrolyzed Leu-pNA at the temperature range of30-55℃ and the pH range of 3.0-10.0.Mn²⁺and Co²⁺could improve Amp0279activity significantly,and the optimal concertration of Co²⁺was 100μM.The metallase inhibitor ethylene diamine tetraacetic acid（EDTA）could completely inhibit the activity of Amp0279,while aspartic acid protease inhibitor Pepstain A and serine protease inhibitor phenylmethylsulfonyl fluoride（PMSF）displayed no effect.In the optimal conditions,the specific enzyme activity of Amp0279 was 3.54×10⁴U/mg,which was slightly over that of the commercial Flavourzyme（?）（3.37×10⁴U/mg）.（3）The constitutive intracellular expression of Amp0279 was achieved in B.substilis WB800N by a shuttle plasmid pHT315-8E21b,and the secretory expression was achieved by a shuttle plasmid pHT43.Enzymatic activity could be detected from the crude culture supernatant of recombinant strain B.substilis WB800N（pHT43-amp0279）without concentration and purification.In conclusion,a novel Co²⁺-dependent leucyl aminopeptidase Amp0279 from L.sphaericus C3-41 was screened and secreted expressed in B.substilis WB800N.This work provided a reference for subsequent study of this enzyme in industrial application.