Purification and properties of two intracellular aminopeptidases produced by Brevibacterium linens SR3

Two aminopeptidases, designated as aminopeptidase I (API) and aminopeptidas e II (APII), were isolated from the disrupted cells of Brevibacterium linen s SR3 and purified to homogeneity by a combination of Phenyl-Sepharose, DEA E-Sepharose, Q-Sepharose and Sepharose CL-6B chromatography. Optimal temper ature for enzymatic activity was 45 degrees C for API and 37 degrees C for APII, and the pH optimum was found to be 8.5 for API and 8.0 for APII. Diva lent metals ions like Co2+ and Ni2+ increased enzymatic activity while Ca2, Cu2+ and Fe2+ had an inhibitory effect. The enzymes were strongly inhibit ed by EDTA, 1,10-Phenantroline and cysteine, indicating that both aminopept idases were metalloenzymes. The K-m values of API and APII for L-leucine-p- nitroanilide were 1.0 and 0.25 mM, respectively. Native molecular masses of aminopeptidases I and II were 80 and 220 kDa after gel filtration chromato graphy while molecular masses of 14 and 18 kDa, were seen, after SDS-polyac rylamide gel electrophoresis. (C) 2000 Elsevier Science Ltd. All rights res erved.