Purification, characterization and production of bacterial ice nucleators from recombinant Escherichia coli and Saccharomyces cerevisiae

Bacterial ice nucleators primarily produced by ice nucleation-active (Ina+) bacteria possess an ability to trigger ice formation of supercooled water at temperatures as warm as --2 to 10°C. Ina protein, a unique membrane-associated protein encoded by a single ice nucleation gene (ina) has been demonstrated as the major component of bacterial ice nucleators. The objectives of this study were to clone the ina gene from Erwinia herbicola by the PCR and express it in E. coli and S. cerevisiae; to isolate and purify ice nucleators from Ina+ E. coli; to characterize the purified ice nucleators; and to investigate the effect of Ina+ yeast cells on food freezing processes.; A 3.6 kb DNA fragment containing the ina gene of Er. herbicola was isolated by PCR cloning and fused with six adjacent histidine residues in plasmid pQTH-21. E. coli transformants carrying pQTH-21 produce three types of ice nucleators (type 1, type 2 and type 3) with a threshold temperature of --3.6°C. Extracellular ice nucleators (ECINs) were found to be released from the cell surface of Ina+ E. coli.; The hexahistidine-tagged recombinant Ina protein was rapidly purified by metal chelation chromatography and the purified Ina protein showed a threshold temperature of --10°C. The results from delipidation of ECINs by phospholipases, detergents and organic acids indicate that phospholipids are required components for the production of type 1 and type 2 ice nucleators.; Growth temperatures significantly affect the production of type 1 and type 2 ice nucleators. Optimal production of type 1 and 2 ice nucleators was achieved when Ina+ E. coli cells were grown at temperatures below 22°C. Type 1 and type 2 ice nucleators can be induced by exposing the Ina+ E. coli cells grown at 37°C to temperatures of 18 to 22°C for several hours.; The ina gene released from pQTH-21 was placed under the control of a galactoseinducible promoter (GAL1) and introduced into Saccharomyces cerevisiae. Yeast transformants carrying the ina produced type 2 and type 3 but no type 1 ice nucleators with a threshold temperature at --5.6°C. Lower temperature growth (15 to 20°C) of Ina+ yeast was required for the production of type 2 ice nucleators. Type 2 ice nucleators can also be induced by moving Ina+ yeast cells from 25°C to 5°C and the maximum activity was achieved after induction at 5°C for about 12 hr.; The addition of Ina+ yeast to liquid foods significantly elevated ice nucleation to higher subzero temperatures, which provides application potentials for the freezing of concentrated food. This freezing of solid or semi-solid products in the presence of Ina+ yeast resulted in unique fiber-like textures.