Summary: A new simple procedure for the production of protoplasts of with high regeneration rates and efficient transport activity is described, involving the use of a preparation of Novozym 234 with very low protease activity. The combination of a heat pretreatment at 55 °C for 15 min with the use of the protease inhibitor aprotinin resulted in a 97% reduction of Novozym 234 protease activity with respect to untreated controls. Polysaccharide-hydrolysing activity was inhibited much less, to 60% of the untreated Novozym 234 level. Protoplasts could be successfully produced with the new low-protease Novozym 234 preparation, showing a threefold increase in regeneration capacity compared to control protoplasts obtained with the original preparation. The rates of 3--methylglucose uptake and the capacity to accumulate this sugar analogue were also higher in protoplasts obtained by the new method.


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