Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.

Dectin-1, a specific pattern recognition receptor for β-1,3/β-1,6-glucans, is expressed mainly on phagocytes. Human dectin-1 (hDectin-1) and mouse dectin-1 (mDectin-1) were separately expressed on HEK293 cell surfaces for examination of the binding abilities of a synthetic particulate β-glucan (pβG), a product extracted from Saccharomyces cerevisiae, in this study. The binding of zymosan-FITC to hDectin-1 and mDectin-1 was inhibited by pβG at similar concentrations for 50% inhibition of binding (IC₅₀). However, the kinetics of the time course and dose response to zymosan stimulation observed for U937 and J774A.1 differed. Superoxide anion production was increased in U937 but reduced in J774A.1 when cells were treated with pβG, zymosan, or laminarin, whereas ovalbumin endocytosis was enhanced in U937 and J774A.1 treated either with pβG, zymosan, laminarin, or barley-glucan. These results indicate that the binding affinity of pβG to hDectin-1 is similar to the binding affinity to mDectin-1, and that stimulation by pβG as well as various forms of β-1,3-glucans on U937 and J774A.1 resulted in upregulation of cell activity and ovalbumin endocytosis. Additionally, other coreceptors on U937 and J774A.1 may be involved in directing different responses to superoxide anion production in these two types of cells. These results will likely contribute to further investigations on identifying the biological forms of β-glucans capable of binding its specific receptor as the effective immunomodulators.