The Screening Of Thermo-stable Aminopeptidase-Producing Strain, The Enzymatic Properties And Gene Cloning

Aminopeptidases catalyze the cleavage of amino acid residues at the N-terminal positionof peptides. Because aminopeptidases can mostly hydrolyze hydrophobic amino terminus ofthe peptide chain, they can reduce bitterness and increase free amino acids which improve thenutritional value of food. They have broad application prospects in the processing resourcesof the various proteins. In this study, the strain isolated from soil, producing anaminopeptidase, was identified and named Pseudomonas aeruginosa NJ-814. This researchincluded its fermentation, purification, enzymatic properties, cloning and expression of gene.The optimized fermentation medium was as follows: mannitol20.0g·L-1, soybean cakepowder10.0g·L-1, K2HPO4·3H2O2.5g·L-1, NaH2PO4·2H2O0.5g·L-1and CoCl2·6H2O1.2g·L-1. The optimal initial pH, liquid volume, inoculation volume and the fermentation time foraminopeptidase production were6.0,50mL/250mL,5%and34h. The enzyme activityreached3.54U·mL-1which was5.80times than the initial activity.The aminopeptidase was purified to homogeneity using (30-50%) ammonium sulfateprecipitation and DEAE-Sepharose ion exchange chromatography. Peptide map fingerprintingshowed that the sequence of the enzyme was highly similar to aminopeptidases from P.aeruginosa18A, P. aeruginosa PAO1, P. aeruginosa PAb1and keratinase from P. aeruginosa.The purified enzyme showed maximum activity at pH9.0and80oC. Half of the activity canremain after incubation at80oC for119min, and it exhibited better heat stability. It was stablewithin pH range of7.5-10.5. Overall, it was activated by Co2+and inhibited byphenylmethanesulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA), Mn2+,Zn2+, Ba2+, Cu2+, Ni2+, Fe2+and Mg2+. It had a better tolerance for the majority of hydrophilicand hydrophobic organic solvent. In particular, the tolerance of aminopeptidase againsthydrophilic organic solvent was stronger than the hydrophobic. Within severalaminoacyl-p-nitroanilines (AA-pNA), Lys-pNA was proven to be the optimal substrate,followed by Leu-pNA and Arg-pNA. The Michaelis-Menten constant (Km) of the enzyme forLys-pNA and Leu-pNA were2.32mmol·L-1and9.41mmol·L-1, respectively. The maximumreaction velocity (Vmax) were5.36mmol·L-1min-1and12.11mmol·L-1min-1, respectively.When soybean or pea protein gluten meal served as substrate, their hydrolysis was measuredin free amino acid content and molecular weight distribution, the results showed that whenaminopeptidase was applicated in the hydrolysis cooperatively with alkaline protease, theycan strongly hydrolyze the hydrophobic residue of leucine amino-terminal. There is a greatapplication prospect in debittering proteinsThe lysine aminopeptidase gene (kap) was cloned from the genomic DNA of P.aeruginosa NJ-814by PCR. The expression vector pET-28a-kap was constructed to partlyexpress fusion protein in E. coli BL21(DE3). It can be speculated that this enzyme maybecontains two zinc ions in its active site by amino acids sequence analysis.