Purification and Characterization of Four Extracellular Proteases Isolated from Raw Milk Psychrotrophs

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Abstract
Extracellular proteases from psychrotrophic strains of Bacillus coagulans (LY 9), Bacillus sp. (LY 10), B. subtilis (LY 11) and Pseudomonas fluorescens (LY 13) were purified and characterized. The MW of the purified protease from P. fluorescens LY 13 was 4.50 .times. 104, and from the 3 Bacillus spp. ranged from 3.35 .times. 104 to 3.90 .times. 104. The proteases from LY 10 and LY 13 were monomeric proteins; the protease from LY 9 was in a polymeric form that contained up to 14 subunits. Only the protease from P. fluorescens LY 13 showed trypsin-like activity. All 4 proteases were inhibited by EDTA and were classified as metalloproteases. Casein was the preferred substrate for these proteases. Susceptibility of casein fractions to attack by these proteases varied with the enzyme source. Maximum enzyme activity was between pH 6.5 and 7.5. The protease from P. fluorescens LY 13 retained more activity after heating at 63.degree. C for 30 min than the proteases from the 3 Bacillus spp. Ca ion showed a protective effect by decreasing heat denaturation of the proteases from LY 9 and LY 11. This protective effect tended to be greater in the presence of Tris-HCl buffer (0.05 M, pH 7.5) plus 10% skim milk than in buffer only.