The RSID-saliva test and the SALIgAE-saliva test are two recently developed forensic saliva detection kits. In this study, we compared the sensitivity and the specificity of the two test kits with the Phadebas amylase test by analyzing amylases from various sources including human, animals, plants, and micro-organism. The data demonstrate that the RSID-saliva test and the SALIgAE-saliva test offer higher sensitivity and specificity for the detection of saliva than the Phadebas amylase test. The detection limits of the RSID-saliva test, the SALIgAE-saliva test, and the Phadebas amylase test equate to 10, 4, and 1000 nL, respectively for human saliva. The RSID-saliva test and the SALIgAE-saliva test were further evaluated by analyzing semen, vaginal secretion, breast milk, blood, urine, sweat, and feces. The results of the two tests are in good agreement. The two tests reacted with urine, breast milk, and feces, but not with semen, vaginal secretion, blood, and sweat.

Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.Dehalogenases are enzymes that remove halogen from the carbon moiety (6). Several dehalogenases have been purified and characterized since their first detection in bacteria. These dehalogenases were categorized according to their substrate specificities (28) and recently according to their phylogenetic relationships (8). Much attention has been drawn to the 2-haloacid dehalogenases or halidohydrolases. These are hydrolytic enzymes that cleave the halogen-carbon bond(s) in halogenated aliphatic acids, yielding hydroxy-or oxoalkanoic acids from a substrate with a mono-or disubstitution, respectively (7, 28). At least 11 2-haloacid dehalogenase genes have been isolated and sequenced. Comparative study on the amino acid sequences of these enzymes has exhibited 37 to 67% homology (17, 32). Among these enzymes three conserved motifs have been identified. These include residues 4 to 18 in motif 1, residues 105 to 123 in motif 2, and residues 139 to 194 in motif 3 (1). Motif 1 contains a highly conserved aspartate and a threonine, motif 2 contains a highly conserved hydroxy residue (serine or threonine), and motif 3 contains a highly conserved lysine and a pair of aspartates. These conserved motifs were expected to convey functions essential for catalysis. Site-directed mutagenesis had confirmed the role of these motifs in the activity of dehalogenase L-DEX-YL and dehalogenase IVa (DehIVa) (18; B. C. M. Pang and J. S. H. Tsang, unpublished data).Most dehalogenases were identified from microorganisms isolated from enrichment cultures using specific halogenated substrates (3, 27). These microorganisms are capable of utilizing the substrates as sole carbon and energy sources. Burkholderia cepacia MBA4, isola...

Amino and carboxyl terminal deletion derivatives of dehalogenase IVa (DehIVa) of Burkholderia cepacia MBA4 were constructed and analyzed for enzyme activity and for protein integrity. The results suggested that the majority of the protein is indispensable. Point mutations on 29 conserved charged and/or polar residues were generated and characterized. Derivatives D11E, D11N, D11S and D181N were totally inactive while mutant N178D was defective in catalysis. Mutations of other conserved residues displayed varying effects. Mutation that enhances DehIVa activity has been shown to be inhibitory in other dehalogenase and essential conserved residues in DehIVa have been shown to be dispensable in others. This suggests there is no general rule for the importance of these conserved residues.