Fractionation of transgenic corn for recovery of recombinant enzymes

Two germ-separation methods, dry-milling and density separation by flotation, were evaluated for recovering recombinant β-glucuronidase (rGUS) that accumulated primarily in the germ of transgenic corn. The dry-milling process consisted of (i) seed tempering, (ii) degerming with a horizontal-drum degermer/dehuller, (iii) particle size fractionation with standard sieves, (iv) germ and endosperm separation by roller milling and sifting, and (v) removal of hulls by aspiration. Sieves nos. 5, 6, and 7 retained the majority of germ, and subfractions from these sieves were pooled as a germ-rich fraction. Mass balances showed that the germ-rich fraction, which constituted 17% of the total dry-milled corn weight, contained 49% of rGUS activity and 64% of the total recoverable oil. Germ fractionation by flotation was tested as a proof-of-concept method aimed at separating corn fractions based on their difference in specific gravity (sp gr). The process consisted of impact-grinding of corn kernels followed by density separation using 1.15 or 1.3 specific gravity sodium nitrate solution. The oil-containing germ fraction floated, whereas the heavier endosperm fraction sedimented. The flotation method was simpler and resulted in higher enzyme recovery, that is, the germ-rich fraction was 20% (w/w) of the initial corn weight, and accounted for 80% of rGUS activity and 77% of total oil. The sodium nitrate solution did not have an adverse effect on the enzyme activity.