Stella H. Wilkes

[44] Aeromonas aminopeptidase

Publisher Summary Aeromonas aminopeptidase is a heat-stable extracellular enzyme that can be isolated in high yield from culture filtrates of the marine bacterial species, Aeromonas proteolytical. It is readily prepared in physically homogeneous form, free from traces of endopeptidase activity. The production and isolation of this aminopeptidase are described in this chapter. It details the procedure for enzyme production and enzyme isolation. The key properties of the enzymes, such as the purity, physical properties, stability, specificity and catalytic properties, inactivation and Inhibitions are described. The Leucyl-β -naphthylamide Assay, Leucyl-p-nitroanilide Assay, Gelatin-Digesting Test for Endopeptidase and the Oligopeptide Test for Endopeptidase Activity are also discussed in detail.

Aeromonas aminopeptidase: Purification and some general properties

Abstract An aminopeptidase was purified 87-fold from culture filtrates of the marine bacterium Aeromonas proteolytica . The stability of the aminopeptidase at 70 ° permitted the use of a heat treatment to inactivate a proteinase from which the aminopeptidase was otherwise difficult to separate. Purification of the aminopeptidase from heat-treated culture filtrate was accomplished by chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. Moving-boundary electrophoresis, ultracentrifugation, and substrate specificity studies indicated a high degree of homogeneity for the preparations. The enzyme hydrolyzed a number of peptides, amino acid amides, and amino acid β-naphthylamides. It was specific for substrates having a free α-amino group on a residue of the l -configuration; the nature of the amino acid in the N-terminus exerted a considerable effect on hydrolytic rates. The aminopeptidase, as isolated, did not require the addition of activating ions, but was strongly inhibited by ethylenediaminetetraacetate (EDTA). Addition of Zn ++ or Co ++ to the EDTA-inhibited enzyme restored activity to the original level; Mn ++ was partially effective, and Mg ++ , Ca ++ , and Ni ++ were ineffective. Analysis of several individual preparations revealed the presence of zinc in the aminopeptidase. A molecular weight of 29,500 was calculated from measurements of sedimentation velocity and diffusion.

[33] Aeromonas neutral protease

Publisher Summary Culture filtrates of Aeromonas proteolytica possess unusually high activity toward casein and hemoglobin substrates, surpassing the levels produced by several other microbial species known to yield large quantities of extracellular proteolytic enzymes. Fractionation of culture filtrates has revealed that this activity is due to two endopeptidases. This chapter discusses one of the endopeptidases. The chapter deals with assaying Aeromonas neutral protease both with hemoglobin and a doubly blocked dipeptide substrate. If the neutral protease is to be used in sequence or specificity studies, it is also necessary to assay for the presence of aminopeptidase in fractions obtained during the isolation of the protease and in the final product. Assays such as: Assay with Furylacryloyl Peptides, Hemoglobin Assay has been discussed. The chapter also discusses the enzyme production and isolation. It also discusses the physical, metal, catalytic properties, its purity and stability as well.

Partial Purification and Some Enzymatic Properties of a Proteinase from Aeromonas proteolytica

Summary A proteinase possessing the characteristics of an endopeptidase was partially purified from culture filtrates of Aeromonas proteolytica. The enzyme readily hydrolyzed hemoglobin, casein, insulin, CGPN, CGPEE, and CGP; with the latter substrate K m was 4.19 × 10−2 M. Chelating agents or dialysis at low pH inhibited the proteinase, which could be reactivated under favorable conditions by the addition of certain divalent metal ions.

Aeromonas neutral protease: Specificity toward extended substrates

Abstract Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P1′, 3 the residue furnishing the amino group to the scissile bond, produced significant changes in krmcat, whereas the identity of P1′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P1′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P1t , P2′, P3′) are involved while only two residues (P2, P1) on the amino terminal side of the sensitive bond are implicated.