Microbial diagnostics using enzymes in catalytically inactive, binding competent assays


Roger Laine1, Betty C. R. Zhu2, and W. C. Jennifer Lo1. (1) Department of Biological Sciences, Anomeric, Inc, Department of Chemistry, Louisiana State University, LSU Agricultural Center, Baton Rouge, LA 70803, (2) Department of Biological Science, Louisiana State University, Baton Rouge, LA 70803
Vibrio parahemoliticus secretory chitinase 1, 94kD, cloned in E. coli, and utilized as fluorescein labeled in catalytically inactive assays (Fungalase®) has been shown to accurately diagnose “eosinophilic fungal rhinosinusitis (EFRS)” by Taylor, Ponikau, et al. in "Otolaryngology -Head and Neck Surgery" (2002, In press). Silver stain could not be effectively used in mucinous, eosinophilic-rich samples, however, the specificity of the binding site for chitin in this enzyme is sufficient to distinguish chitin cell walls among contaminating mucin, eosinophils and bacteria. Fungalase® is a general fungal stain for chitinous fungi in pathological samples. Catalytically inactive hen eggwhite lysozyme E35Q mutation (Kirsch, 1989), was fluoresceinated to provide a general bacterial stain “Bacterase™” analogous to Fungalase®. This peptidoglycan binding protein recognizes both peptidoglycan and chitin but maintains a tight 6-sugar binding site with no catalytic activity. Thus, both Fungalase and Bacterase act as monovalent antibodies or monovalent lectins by recognizing specific structures.