Studies Of Bacillus Licheniformis E7 Aminopeptidase Expression And Its Efficient Proteolysis

Aminopeptidase（AP,EC 3.4.11）is a class of exopeptidases,that selectively degrades N-terminal hydrophobic amino acid residues of polypeptide chains and proteins to release the free amino acids.Aminopeptidase can effectively reduce the bitterness of protein hydrolysates,improve food flavor and nutritional value.It acts synergistically with endopeptidases such as alkaline protease to promote the deep hydrolysis of proteins,which has been applied in the preparation of various active peptides.In this study,a Bacillus licheniformis strain with high aminopeptidase production was screened.The aminopeptidase gene was cloned and overexpressed in Escherichia coli BL21（DE3）and Bacillus subtilis 168,respectively.The enzymatic properties of recombinant enzymes were studied.The combination of recombinant enzymes with alkaline protease efficiently improved the degree of hydrolysis of soybean protein and casein to release multitudinous peptides and amino acids.The specific results are as follows:（1）Aminopeptidase producing strain was screened and identified,and its gene fragment was cloned.An aminopeptidase-producing strain was screened from the soil by combining the casein plate hydrolysis transparent circle method and the enzyme assay method.The strain was identified as Bacillus licheniformis and named B.licheniformis E7 by analyzing the morphology,physiological and biochemical characteristics and comparison of its 16S r DNA sequence.The complete aminopeptidase gene pep N was cloned from B.licheniformis E7 genomic DNA by genome walking technology,with a total length of 1,446 bp encoding 481 amino acids.（2）The recombinant E.coli BL21/p ET28-pep N and B.subtilis 168/p MA0911-pep N were constructed and the recombinant proteins were expressed.Using p ET-28a（+）and p MA0911 as expression vectors,and E.coli BL21（DE3）and B.subtilis 168 as hosts,recombinant expressing aminopeptidase strains E.coli BL21/p ET28-pep N and B.subtilis 168/p MA0911-pep N were constructed.SDS-PAGE analysis showed that the recombinant aminopeptidases could be solubly expressed in E.coli as intracellular protein（named as Ec Pep N）,and in the B.subtilis as extracellular and intracellular forms(named as Bs Pep N^ex and Bs Pep Nⁱⁿ).The molecular weight of recombinant proteins was about 52 k Da,which was consistent with the theoretical value.Ec Pep N was expressed with the highest level under the conditions of 0.1 m M IPTG and 20℃,while Bs Pep N^exand Bs Pep Nⁱⁿ showed the best expression at induction temperature of 30℃.（3）The recombinant aminopeptidases were purified and characterized.The recombinases were purified by Ni²⁺-affinity chromatography,and the target proteins were eluted with 100 m M imidazole.SDS-PAGE analysis showed that the purified enzymes presented a single band around52 k Da.The enzymatic properties study indicated that the optimal reaction conditions for Ec Pep N was p H 9.0 and 50℃,Bs Pep N^ex was p H 9.0 and 45℃,and Bs Pep Nⁱⁿ was p H 7.0 and 45℃.And their highest specific activities were 365.04,401.33 and 113.77 U·mg^-1,respectively.The recombinant Ec Pep N,Bs Pep N^ex and Bs Pep Nⁱⁿ were stable at p H 6.0-9.0,with half-lives of 36 h,6h and 12 h at 45℃.The Ec Pep N showed the best salt tolerance.Its residual enzyme activity remained above 55%after incubation in 3 M Na Cl for 22 days.Among the seven substrates tested,the recombinant aminopeptidase indicated preference for the substrate Ala-p NA.Therefore,they are defined as alanine aminopeptidase.The recombinases Ec Pep N,Bs Pep N^ex and Bs Pep Nⁱⁿ showed K_m values of 1.20,1.01 and 1.09 m M,towards Ala-p NA,and the k_cat/K_m values of Ec Pep N and Bs Pep N^ex were about 4 times higher than that of Bs Pep Nⁱⁿ.（4）Product spectrum of soybean protein and casein hydrolyzed by recombinant aminopeptidases and alkaline protease was analysed.With commercial aminopeptidase as a control,the recombinant aminopeptidases and alkaline protease synergistically hydrolyzed soybean protein.In the hydrolysates,more than 75%were polypeptides with molecular weight less than 500 Da,and more than 2,000 mg·L^-1 of abundant free amino acids,which was 50 times more than that catalyzed by alkaline protease alone.In the casein hydrolysates catalyzed by double enzymes,the polypeptides less than 500 Da accounted for about 78%,and the content of small peptides less than150 Da was 6 times that of alkaline protease hydrolysis.The proteolytic effects of recombinant aminopeptidases at different expression forms had little difference,and the hydrolytic efficiency was equivalent to that of commercial leucine aminopeptidase.This study provides important research ways for improving the deep protein hydrolysis and enhancing the values of protein-rich bioresources.