Evaluation of processing options for transgenic sugarcane tissue expressing bovine lysozyme
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Bovine lysozyme, an antimicrobial enzyme, was expressed in sugarcane using constitutive promoters. As a result, both stalks and leaves express this enzyme, albeit at different levels. The goal of this work was to assess the various options available for processing transgenic sugarcane tissue expressing bovine lysozyme and ultimately develop a process for efficient recovery of this enzyme from sugarcane tissue. Since sugarcane stalk is traditionally pressed for sucrose release, we wanted to assess whether the enzyme could also be released through conventional stalk processing. Single pressing of shredded / chopped sugarcane stalks was partially (50%) effective in releasing the enzyme from stalk tissue. This finding was weighed versus extraction of stalk and leaf tissue. In general, lysozyme extraction from leaves and stalks required particle size reduction, high-shear mixing and in some cases pH and ionic strength adjustment. We ultimately selected stalk extraction conditions that gave efficient release of active enzyme with a minimal processing time. Extraction of stalk tissue released a variety of stalk protein in addition to bovine lysozyme. We next examined potential ways to enrich the enzyme relative to host protein using either membrane filtration alone or in combination with ion-exchange chromatography. Preliminary results suggest that purification yield is affected by the method selected for enriching the protein.
