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Purification and Characterization of Four Extracellular Proteases Isolated from Raw Milk Psychrotrophs¹

Department of Animal Sciences, Food Science Section, University of Kentucky, Lexington 40546-0215

ABSTRACT

Extracellular proteases from psychrotrophic strains of Bacillus coagulans (LY 9), Bacillus sp. (LY 10), Bacillus subtilis (LY 11), and Pseudomonas fluorescens (LY 13) were purified and characterized. The molecular weight of the purified protease from Pseudomonas fluorescens LY 13 was 4.50 x 10⁴, and from the three Bacillus species ranged from 3.35 x 10⁴ to 3.90 x 10⁴. The proteases from LY 10 and LY 13 were monomeric proteins, whereas the protease from LY 9 was in a polymeric form that contained up to 14 subunits. Only the protease from Pseudomonas fluorescens LY 13 showed trypsin-like activity. All four proteases were inhibited by ethylenediaminetetraacetate and would be classified as metallo proteases. 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 Pseudomonas fluorescens LY 13 retained more activity after heating at 63°C for 30 min than the proteases from the three Bacillus species. Calcium 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-HC1 buffer (.05 M, pH 7.5) plus 10% skim milk than in buffer only.

¹ Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article No. 84-5-3.

² Department of Food Science, Tunghai University, Taichung Taiwan, Republic of China.

³ To whom correspondence should be addressed.