Secretory Expression, Molecular Modification, And Physiological Function Of Bacillus Subtilis Aminopeptidase

Aminopeptidases (APs, EC3.4.11) are a group of exopeptidases that can selectivelycatalyze the cleavage of the N-terminal amino acid residues from peptides and proteins, whichhave agreat application in food, pharmaceutical, and chemical industry. As a commodityproduct, APs are mostly produced by wild type strains, and the price of APs is expensive dueto the low production. The application effect of APs is mostly determined by the substratespecificity and stability of the enzyme. Therefore, it is important to construct a strain withhigh yield of AP, alter the substrate specificity, and enhance the stability, which can improvethe product competitiveness of APs.A strain Bacillus subtilis Zj016was screened from fermented bean curd, which producedan extracellular AP (BSAP) and had a good potential in industrial application. In this study,the gene of BSAP was identified, and expressed in B. subtilis with the signal peptide of itself.Researches on characterization, molecular modification, and the physiological funcation ofBSAP were carried out. The results were summerized as follows:(1) Cloning and secretory expression of BSAP. The target gene was composed of1368bp encoding a protein containing455aa, and the first31aa was indicated to be a signalpeptide. The BSAP exhibited significant sequence similarity to AP encoded by the ywaD genebut with four different amino acids. It was indicated that BSAP can not be secreted into themedium with the signal peptide of itself in recombinant E.coli (pET-BSAP) but wassuccessfully secreted with the signal peptide of itself in recombiant B. subtilis (PMA-BSAP),and the yield reached52.4±2.04U mL-1which was about18times higher than that of wildtype and9times higher than that of recombiant E. coli. It was indicated that the optimaltemperature and pH of purified enzyme were50℃and8.5, and BSAP was stable at thetemperature below60℃and pH7.5-9.0. Except for Co2+, BSAP was inhibited more or lessby other divalent metal ions, especially Zn2+and Ni2+. In the case of substrate specificity,BSAP was most active toward hydrophobic amino acids with a large aliphatic chain (Leu) andbasic amino acids (Arg and Lys) and can not hydrolyze the amino acids with large side chains(Phe and Pro) and acidic amino acids (Glu and Asp). After optimization of the cultureconditions of B. subtilis (PMA-BSAP), the yield of BSAP was2.6times that beforeoptimization. The recombinant B. subtilis was used to scaled up in a15L fermentor, and theyield was similar to that in flask.(2) Altering the substrate specificity of BSAP. Based on homology modeling anddocking study, the structure of BSAP with binded substrate Bestatin was obtained. Fourresidues (Leu370, Asn385, Ile387, and Val396) located in the substrate binding region wereconfirmed to have a directed contact with the substrate and selected for saturationmutagenesis. Five mutants with different substrate specificity were obtained after screeningand the mechanism of the alteration of substrate specificity was also investigated. Comparingthe effect of different mutants for protein hydrolysis application, it was revealed that acombination of BSAPs with different substrate specificities exhibited the highest hydrolysis efficiency, and the hydrolyzing degree of which was1.26times that of wild-type BSAP.(3) Enhancing the stability of BSAP. Based on the molecular dynamics simulation, it wasfound that a thermal sensitive domain (PA domain) located in the non-catalytic region ofBSAP. The results of thermal stabilty and tryptic digestion analysis indicated that BSAPwithout PA domain (BSAP-ΔPA) exhibited better thermal stability and BSAP was moresusceptible to trypsin than BSAP-ΔPA. The kinetic parameters of BSAP and BSAP-ΔPAtoward aminoacyl-pNAs and Peptide A (12-aa long) indicated that deletion of PA domain didnot affect the catalytic ability and substrate specificity toward small molecular substrate(aminoacyl-pNAs) but improved the catalytic ability toward macromolecular substrate. Thehydrolysis of soybean protein indicated that BSAP-ΔPA exhibited better hydrolytic abilitythan BSAP.(4) Research on the physiological function of BSAP. Two mutant strains (BS-AP-K andBS-AP-R) were constructed on the basis of Xer/dif recombinant system. BS-AP-K was thestrain which the ywaD gene was deleted, and BS-AP-R was the strain in which the ywaD genewas replaced by ywaD-ΔPA gene. Comparing the growth among wild-type, BS-AP-K, andBS-AP-R in different media and at different temperatures, it was found that deletion of BSAPhad a negative effect on the growth, which became severer at higher temperatures. Theimpaired growth rate of BS-AP-K can be overcome by use of amino acid supplements.