14A-8 |
Comparison of protease produced by Pseudomonas fluorescens M3/6 at two growth conditions |
C. D. MEJIA1, S. S. Nielsen2, and K. D. Hayes1. (1) Dept. of Food Science, Purdue Univ., 745 Agriculture Mall Dr., West Lafayette, IN 47907- 2009, (2) Dept. of Food Science, Purdue University, 745 Agricultural Mall Dr., West Lafayette, IN 47907
Studies suggest that the plasmin zymogen, plasminogen (PG), associates with casein in milk and is activated during cheese ripening. Previous research proved that a metalloproteinase produced by Pseudomonas fluorescens M3/6 at 4°C increases the activity of plasminogen activators (PAs), which benefits cheese ripening. If the growth of this culture at a higher temperature accelerates production of the protease, a protease isolate could be added to increase cheese ripening rate. The aim of this study was the comparison of isolates of P. fluorescens M3/6 proteases produced at 4°C and 23°C. Bacterial cells were incubated at 4°C and 23°C in reconstituted nonfat dry milk for 45 and 14 days, respectively. Growth was measured by standard psycrotrophic plate count, and protease production was monitored by an azocasein assay. The protease was purified using ammonium sulfate fractionation and ion exchange chromatography. The protease effect on PG activation was evaluated by a coupled assay in which the protease influences PA to convert PG to plasmin, which reacts with the chromogenic substrate Spectrozyme PL®. The molecular weights were visualized using SDS-PAGE and caseinolitic profiles using casein SDS-PAGE. The action on purified caseins (α, β, κ) was determined along with the N-terminal sequence of the proteases. Results showed that the proteases produced at 23°C and at 4°C had the same enhancing effect (250% increase) on urokinase-type PA (P < 0.05). The same molecular weight and the same caseinolitic effect were observed. Nine of the ten N-terminal amino acids residues of both samples were identical, indicating that their primary structure is highly similar if not identical. The protease production and its specific activity at 23°C were significantly greater (P < 0.05) than the protease produced at 4°C, suggesting that the same protease was produced at a higher metabolic rate of the bacterial cells at 23°C.
Session 14A, Dairy Foods: General developments in dairy technology I
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