In the case of production by an enzymatic method, enzymes are used according to the chemical nature of the cell wall. In plant cells, cellulases and pectinases are the most common. Different enzymes are required for bacterial and fungal cells .
In plant cells, cellulases digest the cell walls and pectinases digest the central lamellae. If you want to digest the cell walls and middle lamellae in one step (sometimes it is better to pre-digest the plant material), you dissolve both enzymes together with an osmotic (mostly mannitol ) in distilled water, fill this mixture into a Petri dish and add the finely chopped plant material It must be ensured that the pH of the enzymes is reached. After a few hours, the incubation time varies from plant to plant, isolated protoplasts are obtained, which can now be cultivated further, for example to regenerate new plants. However, protoplasts also open up possibilities for hybridizations by protoplast fusion in an electric field or for genetic engineering changes.
In the early days of protoplast isolation ( 1920s ), protoplasts were obtained by prior plasmolysis and subsequent extraction of the protoplast with the aid of a needle, which had the disadvantage that only a low yield of protoplasts was obtained. The enzymatic method described was then established in the 1960s .
- Paul Präve: Handbook of Biotechnology. Oldenbourg Industrieverlag, 1994, ISBN 978-3-835-66223-0 , p. 244.