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The genes encoding pro-carboxypeptidase Ys (pro-CPYs) from Saccharomyces cerevisiae and Hansenula polymorpha were cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was present in the form of inclusion bodies, accounting for about 35% of the total cellular protein. The inclusion bodies solubilized in 6 M guanidinum chloride were renatured by dilution 1:60 into renaturation buffer. Further refolding and activation were followed by the addition of 60 μg ml−1proteinase K into the diluted solution. The specific activities of in vitro activated S. cerevisiae and H. polymorpha CPYs were found to be similar, representing about 25% of that of native S. cerevisiae CPY.

The effect of permeabilization of G. suboxydanscells with toluene on the oxidation of D-sorbitol to L-sorbose was investigated. Treatment of the cells with 10% toluene resulted in a three fold increase in the specific sorbitol dehydrogenase activity and a two fold increase in the efficiency of D-sorbitol conversion to L-sorbose of the free cell suspension. When the permeabilized cells were immobilized in calcium alginate, the operational stability during air-lift reactor operation was also found to increase with up to three times longer half-life(44 days) of catalytic activity compared with immobilized intact cells.