Molecular Designs for Functional Food Proteins by Genetic Modification

(1)通过基因修饰改善了溶菌酶、α-乳清蛋白和卵子抑制因子的功能性质。通过91位丝氨酸取代苏氨酸加强分子内疏水堆积，大大提高了溶菌酶的构象稳定性。富含丝氨酸的多肽附着在C末端，大大增强了溶菌酶的杀菌作用。(2)糖基化的α-乳球蛋白(α-LAS)能激活巨噬细胞(J774.1)。以阐明其功能。我们在巴斯德毕赤酵母中产生了糖基化和非糖基化突变体α-LAS。在毕赤酵母中分泌的糖基化α-LA能显著刺激巨噬细胞的活化，而非糖基化的α-LA则不能。为了进一步证实糖链的作用，构建了各种突变体α-LAS。缺失45位N连接糖基化位点的突变体N45D在POSI…上产生了非糖化型和糖化型更多第76条，α-LA。前者显著降低M-φ的活性，后者则表现为对M-φ的强烈激活。突变体D46N在第46位添加糖基化信号，仅产生糖基化的α-LA。D46N能强烈激活M-φ。这些结果表明，糖基化的a-LA能激活M-φ，而非糖基化的a-LA则不能。进一步证实了糖基化α-LA通过M-φ表达的FMA细胞具有抗肿瘤活性。(3)卵泡抑制因子基因已整合到毕赤酵母基因组中。用10 mM Tris-HCl缓冲液(pH-8.0)作离子交换柱层析，纯化重组卵抑制因子。经SDS-PAGE分析，重组卵泡抑制物的相对分子质量为46 kDa。重组卵子抑制因子对胰酶、凝乳酶和弹性蛋白酶的抑制活性与天然卵子抑制因子相同。另一项尝试是在巴斯德毕赤酵母中表达卵子抑制因子中的弹性酶抑制区小片段。成功地将卵子抑制因子第六和第七结构域组成的弹性蛋白酶抑制片段整合到毕赤酵母基因组中，并在培养基中分泌。经SDS-PAGE分析，其相对分子质量为15 kDa。重组片段弹性蛋白酶抑制域对弹性蛋白酶和胰凝乳酶均有抑制活性。卵子抑制物主要以糖基化形式分泌。糖基化对蛋白水解酶抑制活性影响的研究也较少。

(1) The functional properties of lysozyme, α-lactalbumin and ovoinhibitor were improved by genetic modification. The conformational stability of lysozyme was greatly enhanced by substitution of serine with threonine at position 91 to strengthen the intramolecular hydrophobic packing. The bactericidal action of lysozyme was dramatically strengthened by the attachment of serine-rich peptides to the C-terminaus(2) Glycosylated a-lactalbumins (α-LAs) were found to activate macrophage cell (J774.1). To elucidate the function. we produced the glycosylated and non-glycosylated mutant α-LAs in yeast Pichia pastoris. The glycosylated α-LA secreted in Pichia pastoris remarkably could stimulate the activation of macrophage cell, wheroas the non-glycosylated a-LA could not. To further confirm the role of carbohydrate chain, various mutant α-LAs were constructed. The mutant N45D deleted the N-linked glycosylation site at position 45 produced both non-glycosylated type and glycosylated type, at posi … More tion 76, α-LA. The former greatly decreased the activation for Mφ, the later revealed strong activation for Mφ. The mutant D46N added the glyrcosylation signal at position 46 produced only glycosylated α-LA. The D46N strongly activated Mφ. These results indicate that glycosylated a-LA activates Mφ, while non-glycosylated a-LA does not. Furthermore, the anti-tumor activity using FMA cell of glycosylated α-LA through Mφ was confirmed(3) The ovoinhibitor cDNA was integrated into yeast Pichia pastoris genome. The secreted recombinant ovoinhibitor was purified by ion-exchange chromatography on a DEAF sepharose column with 10 mM Tris-Hcl buffer (pH-8.0). Recombinant ovoinhibitor has a molecular weight 46 kDa, calculating from SDS-PAGE. Recombinant ovoinhibitor showed the inhibitory activity against trypsin, chymotrypsin and elastase as native ovoinhibitor. Another attempt was carried out to express the small fragment of elastase inhibitory domain in ovoinhibitor in Pichia pastoris. The elastase inhibitory fragment consisting of sixth and seventh domain in ovoinhibitor was successfully integrated into Pichia genome and secreted in cultivation medium. The molecular weight was 15 kDa, calculating from SDS-PAGE. Recombinant fragmented elastase inhibitory domain showed both inhibitory activity against elastase and chymotrypsin. Ovoinhibitor predominantly secreted in the glycosylated form. The effect of glycosylation on the inhibitory activity against proteases was also investigated Less