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A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

The heartwood of bald cypress,Taxodium distichum (L.) Rich., resisted feeding attack by the Formosan subterranean termite,Coptotermes formosanus Shiraki. Hexane-extracted heartwood, however, was consumed at > 12 times the amount of sound heartwood eaten. A bioassay usingT. distichum sapwood as a feeding substrate was employed to assess the antitermitic activity of successive hexane, acetone, and methanol extracts of heartwood shavings and isolates derived from the active hexane extract. Two fractions, eluted from the crude hexane extract by liquid chromatography, significantly reduced termite feeding compared to the parent extract, while a third fraction was less active than the original hexane extract. Each fraction contained one major component. All three components were structurally related diterpenes. The two most active heartwood constituents were identified by GC-MS and NMR as ferruginol and manool, while the third and least active, but most prevalent, compound in heartwood was identified as nezukol. Results of bioassays suggest that these allelochemicals act principally as feeding deterrents with accompanying termite mortality due to starvation.