Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin.

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.

Growth temperature controls the production of a single extracellular protease by Pseudomonas fluorescens MF0, in the presence of various inducers.

The psychotrophic strain Pseudomonas fluorescens MFO is known to express several enzymatic activities in milk, including extracellular proteolytic activity, optimally when cells are grown at 17.5 degrees C. In order to study the nature of the mechanisms controlling the production of the extracellular protease, we devised a defined medium in which this enzymatic activity was induced by an amino acid and small peptides. Regardless of the inducer, optimal proteolytic activity appeared at 17.5 degrees C. SDS-PAGE and isoelectrofocussing revealed a single protease produced by P. fluorescens MFO with all the inducers used and at all temperatures examined. The level of proteolytic activity correlated with the amount of enzyme in the supernatants.

Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens.

In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MF0 is maximal at a growth temperature of 17.5 degrees C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MF0, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5 degrees C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.