Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water.

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of <10 CFU mL-1E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of <10 CFU per 100 mL.

Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.

The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 µg protein/cm(2) and 5.74 µg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

Covalent attachment of lactase to low-density polyethylene films.

Polymer films to which bioactive compounds such as enzymes are covalently attached offer potential for in-package processing of food. Beta-galactosidase (lactase) was covalently attached to surface-functionalized low-density polyethylene films. A two-step wet chemical functionalization introduced 15.7 nmol/cm2 primary amines to the film surface. Contact angle, dye assays, X-ray photoelectron spectroscopy, and appropriate protein assays were used to characterize changes in film surface chemistry after each step in the process of attachment. Glutaraldehyde was used to covalently attach lactase to the surface at a density of 6.0 microg protein per cm2 via reductive amination. The bond between the covalently attached lactase and the functionalized polyethylene withstood heat treatment in the presence of an ionic denaturant with 74% enzyme retention, suggesting that migration of the enzyme into the food product would be unlikely. The resulting polyethylene had an enzyme activity of 0.020 lactase units (LU)/cm2 (approximately 4500 LU/g). These data suggest that enzymes that may have applications in foods can be covalently attached to inert polymer surfaces, retain significant activity, and thus have potential as a nonmigratory active packaging materials.