New insights into the isopenicillin N transport in Penicillium chrysogenum.

In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRed protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane.

Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods.

Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.