Studies On Bacillus Subtilis Aminopeptidase

Aminopeptidases are debittering exopeptidases that selectively release N-terminalhydrophobic amino acid residues from bitter polypeptides. A strain ,Bacillus subtis Zj016 ,withextracellular aminopeptidase activity was isolated from preserved beancurd. It was identifiedaccording to 16S rDNA genetics character, morphological characters, culture characters, andmain physiological characters. Optimizations of medium and enzyme-producing condition werecarried out on the basis of single factor experiment and Orthogonal design. Theenzyme-producing conditions were: temperature 30oC, rotate speed 200r/min, pH 7.2,inoculum size 5%, operational volume 25mL/250mL, germ culture time 6h. The optimizedmedium was :soluble starch 15.0g/L, peptone 10.0 g/L, beef extract 2.0g/L, casein5.0 g/L,KH₂PO₄·12H₂O 0.16g/L, K₂HPO₄ 2.68g/L, NaCl 1.0 g/L, MgSO₄·7H₂O 0.1 g/L. Theaminopeptidase activity was increased from 167.2U/mL to 766.0U/mL after optimization. The purification procedure consisted of ethanol fractionation and two chromatographic steps,which included gel filtration and hydrophobic interaction chromatography. This procedureresulted in a recovery rate of 12.6% and a 100.7-fold increase in specific activity. Theaminopeptidase appeared to be a dimeric enzyme with a molecular mass of 75 kDa. The activitywas optimal at pH 8.5 and 60°C. The temperature stability was 20～70 ℃ and the pH stabilitywas 8.0～10.0. Glycerol and peptone protected the enzyme activity. The enzyme was notinhibited by PMSF and DFP but inhibited by several divalent cations (Zn²⁺,Ni²⁺) and EDTA.Co²⁺ activated the enzyme activity. The enzyme seemed to be Zn-metalloenzyme and had twoZn²⁺ combined on the activation region. Km of the aminopeptidase was 1.02μmol/L and Vmaxwas 5675μmol/L/min. Hydrolysate of soy protein and casein(1%) by Trypsin treatment (E/S:1/100) was bitter.Treatment of aminopeptidase (E/S:10000U/g) could remove the bitterness. Amount of free aminoacid including hydrophobic amino acid was increased about 26%.