Molecular Designs for Functional Food Proteins by Genetic Modification

(1)对溶菌酶、α-乳清蛋白和卵泡抑制剂的功能特性进行了基因改造。溶菌酶的构象稳定性通过用苏氨酸取代91位丝氨酸来增强分子内疏水填充而大大提高。富含丝氨酸的肽段附着在巨噬细胞的c端，显著增强了溶菌酶的杀菌作用(2)发现糖基化α-乳清蛋白（α-LAs）可以激活巨噬细胞（J774.1）。来说明函数。我们在酵母毕赤酵母中获得了糖基化和非糖基化的α-LAs突变体。毕赤酵母分泌的糖基化α-LA能显著刺激巨噬细胞的活化，而非糖基化α-LA则不能。为了进一步确认碳水化合物链的作用，我们构建了多种α-LAs突变体。突变体N45D在45号位置缺失了n-链糖基化位点，产生了非糖基化型和糖基化型。前者显著降低了Mφ的激活，后者则表现出对Mφ的强烈激活。突变体D46N在46号位置添加糖基化信号，只产生糖基化的α-LA。D46N强烈激活Mφ。这些结果表明，糖基化的a-LA激活了Mφ，而非糖基化的a-LA则没有。此外，通过Mφ验证了糖基化α-LA的FMA细胞抗肿瘤活性。(3)将卵泡抑制剂cDNA整合到酵母毕赤酵母基因组中。用10 mM Tris-Hcl缓冲液（pH-8.0）在DEAF sepharose柱上用离子交换层析纯化重组ovoinhibitor。重组ovoinhibitor分子量为46 kDa，通过SDS-PAGE计算。重组卵泡抑制剂对胰蛋白酶、凝乳胰蛋白酶和弹性酶具有天然卵泡抑制剂的抑制活性。另一次尝试在毕赤酵母中表达卵泡抑制剂弹性酶抑制域的小片段。由卵泡抑制剂第6和第7结构域组成的弹性酶抑制片段成功整合到毕赤酵母基因组中，并在培养基中分泌。SDS-PAGE计算分子量为15 kDa。重组碎片化弹性酶抑制域对弹性酶和凝乳胰蛋白酶均有抑制活性。卵泡抑制剂主要以糖基化形式分泌。对糖基化对蛋白酶抑制活性的影响也进行了研究

(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