Heat-stable extracellular proteases of psychrotrophic pseudomonads : purification and physicochemical properties

Bartlett, Francis M. (1987) Heat-stable extracellular proteases of psychrotrophic pseudomonads : purification and physicochemical properties. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

There is a trend in the dairy industry towards the storage of bulk raw milk at refrigerated temperatures for attended periods of time before processing. This practice has the disadvantage of providing conditions favourable for the growth of psychrotrophic (cold-tolerant) microorganisms. Most raw milk psychrotrophs are heat sensitive Gram-negative bacteria, many of which produce heat-resistant extracellular proteaes. The heat-stability of these enzymes enables them to retain some activity in milk following conventional pasteurization, as well as ultra high temperature (UHT) pasteurization. The heat-resistant proteases have been implicated in such defects as the development of off-flavours, gelation and clearing of milk, and reductions in cheese yields. -- The heat-stable extracellular proteases of six psychrotrophic pseudomonads isolated from raw milk were purified to homogeneity by affinity chromatography using CBZ-DL-phenylalanine TETA Sepharose-4B. The yields of purified enzymes were 38 to 59% before concentration by ultrafiltration. The molecular weights, which were determined by gel-filtration, were 38 to 40kD, and the proteins had isoelectric points (pI) from 5.7 to 6.2. The optimum pH for proteolytic activity was between 7 and 8, while the optimum temperature ranged from 30 to 40°C. A dramatic loss of activity was noted for each protease at 45°C. -- Each of the proteases were metalloproteases as indicated by their sensitivity to the metal chelating agent EDTA. Restoration of activity to EDTA-treatcd proteases was achieved by the addition of Mg, Mn or Ca ions. The predominant metal ion present in the three proteases examined was Ca (5-8 g-atoms/mole) followed by Mg and Zn, with trace amouts of Mn. The amino acid content of each of the proteases were similar, with high numbers of aspartic acid, serine, glycine, and alanine residues. The N-terminal amino acid determined for three of the proteases was found to be threonine. The T16 protease contained two aminosugars, glucosamine and galactosamine, and was classified as a glycoprotein. -- Five of the six proteases exhibited a preference for α-casein as a protein substrate, while T16 showed greatest activity towards κ-casein. None of the proteases was able to break down the whey proteins, α-lactalbumin, or β-lactoglobulin. Collagenase and elastase activities were noted for each protease. The T16 protease was quite stable, with a D-value at 150°C of 2.2 min. The presence of Ca or Mg ions stabilized the T16 protease against inactivation at 90°C for 10 min while lactose had no effect. The secondary structure of the T16 protease consisted of "random coil" (67%) and β-structure(33%) with no α-helix. Upon heating from 45 to 55°C the secondary structure-tended to become more random. -- All of the proteases cross-reacted with the anti-serum to the T16 protease, including the protease of psychrotrophic pseudomonads from Ontario, British Columbia, Ireland, U.S.A., Australia and the Neterlands. An enzyme-linked immunosorbent assay (ELISA) was developed using anti-Tl6 IgG, which was sensitive to a minimum concentration of 720 μg/ml of purified T16 protease. The mole % G+C content of the DNA psychrotrophic pseudomonads from different geographical regions were similar (58 to 62%) with the exception of 015 (44%).

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