Substrate specificity and properties of heat-stable proteases from Pseudomonas fluorescens

Extracellular proteases and lipases from strains P26, 32A, Pl, and P27 of Pseudomonas fluorescens were produced using a dialysis membrane method. The proteases were partially purified by molecular exclusion chromatography (MWC0=60,000) and selected biochemical characteristics were determined. Proteases of all strains were heat-stable (62.5 [degrees] C, 30 min), neutral ([greater than] 84Z of total activity between ph 6 and 8), metalloenzymes (inhibited by 10 mM EDTA). Optimal temperature for activity was approximately 37 [degrees] C, but all proteases were active at 5 [degrees] C and 25 [degrees] C. Activation energies on azocasein ranged from 4.5 (Pl) to -1 14.0 (32A) KcaLmole. All extracellular proteases had strong endopeptidase activity, but no exopeptidase (aminopeptidase) activity was detected. Proteolytic activity on purified milk proteins was measured using an o-phthaldialdehyde assay. Specific activities of the pseudomonal proteases on casein proteins were significantly higher (p [less than] 0.01) than on whey proteins. Hydrolysis of whole casein by proteases of strains P26, 32A and P27 was significantly greater than on a-, 6- or k- casein whereas protease of strain Pl hydrolyzed whole casein and a-casein at equal rates. The proteases varied in their abilities to hydrolyze a-, 8- and k-casein. Bovine serum albumin (BSA), B-lactoglobulin and a- lactalbumin were weakly hydrolyzed by the proteases. Among the whey proteins, proteases of strains P26 and 32A hydrolyzed BSA at a higher rate than B-lactoglobulin or a- lactalbumin. Peptides generated from the hydrolysis of oxidized ribonuclease A by proteases of known specificity (trypsin, chymotrypsin, Staphylococcus aureus protease) and the pseudomonal proteases were separated by RP-HPLC. The proteases of known specificity generated a low number of peptides in relatively high concentrations. Proteases of strains P26, 32A, Pl, and P27 produced higher numbers of peptides and the majority of these peptides were in low concentrations. Thus, the pseudomonal proteases exhibited a wider substrate specificity. Among the heat-stable proteases, those of strains P26 and 32A hydrolyzed a wider range of peptide bonds than proteases of strains Pl and P27.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Food science (MU)

Grantor dc:publisher

University of Missouri--Columbia

Year dc:date.issued

1986

Author dc:creator

• Mitchell, Steven Lynn

Advisor dc:contributor.advisor

• Marshall, Robert T.

Statement dc:rights

• OpenAccess.

Language dc:language.iso

eng, English

DOI

https://doi.org/10.32469/10355/98909

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/98909