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Fed-batch cultivation of Bacillus licheniformis with an increased level of mineral salts in the media to increase yield

IP.com Disclosure Number: IPCOM000216080D
Publication Date: 2012-Mar-23
Document File: 7 page(s) / 113K

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Fed-batch cultivation of Bacillus licheniformis with an increased level of mineral salts in the media to increase yield

The solubility of proteins can be changed by changing the composition of the buffer the protein is solubilised in. Addition of different an-ions and cat-ions has different influence on the solubility of different proteins: Some proteins show increased solubility with increased ion-strength, while other show less solubility.

In the case of enzymes produced by microbial cultures the solubility can be changed by adding different salts. Some enzyme class show in general increased solubility at higher ion-strength (e.g. some alpha-amylases), while other show lower solubility at higher ion-strength (e.g. some subtilisin proteases).

In the case where the protein solubility has to be manipulated in a cultivation broth in which the protein is produced by a microorganism, some restrictions apply to the ranges and types of salts that can be used to increase the ion-strength. Some ions might be toxic to the cells, or might in another way harm the productivity of the cells, while other ions might be taken up by the cell, and thereby have less effect.

The increased solubility found for some alpha-amylases by increasing the ion- strength in the culture broth can be a process advantage, e.g., by keeping the enzyme in solution makes the primary separation during recovery much easier, as the cells then easily can be separated from the soluble enzyme.

Production of proteases has always been a challenge, as the proteases tend to degrade themselves by the phenomenon called auto-proteolysis. This phenomenon is in general concentration dependent, meaning that higher concentration increases the speed of auto-proteolysis.

There are different approaches that can be used to reduce the effect of auto- proteolysis. Reducing the temperature and/or changing the pH to values that are sup- optimal for the protease activity is one way to reduce/circumvent this problem. Another way is to reduce the concentration of free active protease in the media, either by decreasing the solubility or inhibit the protease activity.

Decrease of the protease solubility can either be achieved by changing the protease protein (e.g. changing the amino acid sequence), or by changing the cultivation broth composition, pH or chemical/physical factors to make the protease less soluble. Increase of the concentration of different ions in the media is one way to decrease the solubility of the proteases. In the case where the strain used for production of the protease is very productive, this may lead to fast precipitation of the protease in the broth, and in this way result in very significant yields of the protease of interest.

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Salt tolerance of Bacillus licheniformis

In order to test the salt tolerance of a B. licheniformis strain producing a subtilisin of interest we performed the following experiments:
Different salts ((NH4)2HPO4, KCl; NaCl; NaNO3; Na2SO...