Growth medium for
The present invention provides improved media for the cultivation of Clostridium histolyticum and culture supernatants for the biotechnological production of collagenase enzymes. The nutrient media according to the invention comprise one or more peptones from a non-mammalian source, preferably plant-derived peptones. The media can additionally comprise fish gelatin. The invention provides media, culture supernatants comprising Clostridium histolyticum collagenase, and methods to produce said collagenase.
1. A liquid growth medium comprising water, a fish gelatin, a peptone from a non-mammalian source, whereby fish peptone is excluded from the non-mammalian source, wherein the fish gelatin is produced by partial hydrolysis of collagen extracted from skins, bones, cartilage, connective tissues, organs and intestines, wherein the concentration of fish gelatin in the growth medium is between about 2% and about 10%, whereby the percentage indicates weight by volume when the isolated fish gelatin is dry matter and volume by volume when the isolated fish gelatin is liquid matter, and additionally comprising an inoculum of bacteria of the genus Clostridium.
2. The growth medium of claim 1 wherein the fish gelatin is selected from the group consisting of high molecular weight fish gelatin, gelatin from a kosher species of fish, and liquid fish gelatin.
3. The growth medium of claim 1 wherein the peptone of non-mammalian source is a plant product.
4. The growth medium of claim 3 wherein the plant product is from selected from the group consisting of soy bean, broad bean, pea, and potato.
5. The growth medium of claim 4 wherein the growth medium comprises two or more different plant peptones, whereby the aggregate concentration of the plant peptones in the growth medium is between about 2% and about 10% weight by volume.
6. The growth medium of claim 1 wherein the peptone is selected from the group consisting of broad bean peptone (BP), peptone E1 from potato (E1P), soy bean peptone E110, vegetable peptone no. 1 from pea (VG100), and vegetable peptone phosphate broth from pea (VG200), and wherein the concentration of the peptone in the growth medium is about 5% weight by volume.
7. The growth medium of claim 1 wherein the peptone is VG100 and the concentration of the peptone in the growth medium is about 2% weight by volume.
8. The growth medium of claim 1 wherein the pH is between 7 and 8.
9. The growth medium of claim 1 wherein the growth medium is sterilized prior to adding the inoculum.
10. The growth medium of claim 9 wherein the inoculum is of Clostridium histolyticum bacteria.
11. A method for producing a supernatant of a Clostridium histolyticum liquid culture, the supernatant containing one or more proteases with collagenase activity, the method comprising the steps of
providing the growth medium according to claim 10 ,
growing and cultivating the inoculum of Clostridium histolyticum bacteria, whereby the bacteria secrete the one or more proteases with collagenase activity into the liquid culture, and
separating cellular and other particulate matter from the liquid culture,
thereby obtaining a culture supernatant with one or more proteases with collagenase activity.
12. A method for producing one or more purified proteases with collagenase activity from Clostridium histolyticum , comprising the steps of
providing the growth medium according to claim 10 ,
growing and cultivating the inoculum of Clostridium histolyticum bacteria, whereby the bacteria secrete the one or more proteases with collagenase activity into the liquid culture,
separating cellular and other particulate matter from the liquid culture, thereby obtaining a culture supernatant, and
isolating the one or more proteases with collagenase activity from the culture supernatant, thereby producing the one or more purified proteases with collagenase activity from Clostridium histolyticum.