Effect of Processing on the Recovery of Recombinant β-Glucuronidase (rGUS) from Transgenic Canola

• Published in: Biotechnology progress 2001, Vol.17 (1), p.168-174
• Main Authors: Bai, Y., Nikolov, Z. L.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Blotting, Western; Brassica napus; Canola Oil; Enzyme Stability; Fatty Acids, Monounsaturated - chemistry; Fundamental and applied biological sciences. Psychology; Glucuronidase - isolation & purification; Hot Temperature; Kinetics; Methods. Procedures. Technologies; Others; Particle Size; Plants, Genetically Modified - enzymology; Recombinant Proteins - isolation & purification; sodium phosphate; Solvents; Various methods and equipments
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp000143p

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using β‐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 °C and for more than 5 months at 10 °C. At 70 °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 °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 μm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.