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Figure 4 | BMC Biotechnology

Figure 4

From: Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

Figure 4

IPNS produced by H. polymorpha strain IPNS 4.2 cultivated at 25°C is enzymatically active. H. polymorpha IPNS 4.2 cells were cultivated on methanol-containing media to the mid-exponential growth phase at 37°C (well 4), 30°C (well 2) and 25°C (well 3). As controls, H. polymorpha WT cells, cultivated on methanol at 25°C (well 5), and P. chrysogenum cells, cultivated for 40 h on penicillin production media supplemented with phenylacetic acid (well 1) were used. Cell free extracts (40 μg of protein each) were incubated for 20 min at 25°C in a buffer containing ACV to allow production of IPN. Panel A: Reactions were terminated by methanol addition. After protein precipitation, an equal volume of reaction product was added to each well of a bioassay plate on which M. luteus had been plated as indicator strain. The results indicate that extracts prepared from IPNS 4.2 cells cultivated at 25°C and 30°C contain active IPNS enzyme, resulting in the formation of a halo. In contrast, extracts from IPNS 4.2 cells grown at 37°C show no activity. Panel B: As control, the reaction products were further incubated in the presence of penicillinase for an additional 30 min at 25°C. After that, the reaction mixtures were terminated by methanol addition and the reaction products analyzed on a bioassay plate as described in A. The absence of halos on the plate demonstrates that the IPNS 4.2 extracts indeed allow formation of the β-lactam ring.

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