Purification and Properties of an Extracellular Proteinase of Psychrophilic Escherichia freundii

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Abstract
A proteinase was purified from the culture filtrate of psychrophilic Escherichia freundii, by a combination of procedures, such as precipitation with ammonium sulfate, batchwise treatment with CM‐cellulose, chromatography on DEAE‐Sephadex and gel filtration on Sephadex G‐100. The final enzyme preparation was homogeneous with respect to sedimentation in the ultracentrifuge, acrylamide‐gel electrophoresis and immunodiffusion. The enzyme was an alkaline proteinase with the optimum pH at 10.0. The optimum temperature was 25°C at pH 10.0. The proteinase of psychrophilic E. freundii was fairly thermolabile. The purified enzyme was inhibited by metal‐chelating agents but not by diisopropylphosphofluoridate and p‐chloromercuribenzoate.The molecular weight of the proteinase was estimated to be 45800 by sedimentation‐equilibrium studies, 45000 by gel‐filtration method and 51000 by polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulfate. The calculation of minimum molecular weight on the basis of zinc content yielded a value near 45400 which indicated the presence of 1 g‐atom zinc per mol enzyme. The proteinase had a sedimentation coefficient of 4.12 S, a diffusion coefficient of 77.4 μm2/s, a partial specific volume of 0.718 ml/g, an isoelectric point of pH 5.2, A1%1cm at 280 nm of 12.45, and an intrinsic viscosity of 0.048 dl/g. The amino‐acid composition indicated 429 residues per molecule and the absence of sulfhydryl groups and disulfide bridges. The NH2‐ terminal amino acid of the enzyme was shown to be glycine.The extracellular Escherichia proteinase has been found to be a zinc‐containing alkaline metallo‐proteinase.

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