Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola.
This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.
Bai
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Department of Chemical Engineering, Iowa State University, Ames, Iowa 50010, USA.
Nikolov
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ZL
D015153:Blotting, Western; D004795:Enzyme Stability; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D005966:Glucuronidase / Q000302:isolation & purification*; D006358:Hot Temperature; D007700:Kinetics; D010316:Particle Size; D030821:Plants, Genetically Modified / Q000201:enzymology; D000074262:Rapeseed Oil; D011994:Recombinant Proteins / Q000302:isolation & purification; D012997:Solvents
D005229:Fatty Acids, Monounsaturated; D000074262:Rapeseed Oil; D011994:Recombinant Proteins; D012997:Solvents; D005966:Glucuronidase