Molecular Designs for Functional Food Proteins by Genetic Modification

(1)通过基因改造提高了溶菌酶、α-乳清蛋白和卵抑制剂的功能特性。在91位丝氨酸被苏氨酸取代后，溶菌酶的构象稳定性大大提高，从而增强了分子内的疏水堆积。溶菌酶C-末端含有富含丝氨酸的多肽，可显著增强溶菌酶的杀菌作用。（2）糖基化α-乳白蛋白（α-LAs）可激活巨噬细胞（J774.1）。以阐明其功能。我们在毕赤酵母中生产了糖基化和非糖基化的突变体α-LA。酵母分泌的糖基化α-LA对巨噬细胞的活化有显著的促进作用，而非糖基化α-LA则无此作用。为了进一步证实糖链的作用，构建了各种突变的α-LA。缺失45位N-糖基化位点的突变体N45D同时产生非糖基化和糖基化两种类型，在45位的位置， 关于我们 76号，α-LA。前者使M φ活化明显减弱，后者则表现出较强的M φ活化。突变体D46N在46位添加糖基化信号，仅产生糖基化的α-LA。D46N对M φ有强烈的激活作用。这些结果表明，糖基化的α-LA激活M φ，而非糖基化的α-LA不激活M φ。进一步证实了糖基化α-LA经M φ途径在FMA细胞中的抗肿瘤活性。（3）将Ovoinhibitor cDNA整合到毕赤酵母基因组中。分泌的重组ovoinhibitor通过离子交换层析在DEAF琼脂糖柱上用10 mM Tris-Hcl缓冲液（pH-8.0）纯化。SDS-PAGE分析表明，重组蛋白的分子量为46 kDa。重组蛋白对胰蛋白酶、糜蛋白酶和弹性蛋白酶具有天然蛋白酶抑制剂的抑制活性。另一个尝试是在毕赤酵母中表达ovoinhibitor中弹性蛋白酶抑制结构域的小片段。结果表明，由ovoinhibitor中的第6和第7结构域组成的弹性蛋白酶抑制片段已成功整合到毕赤酵母基因组中，并分泌到培养基中。SDS-PAGE测得其分子量为15 kDa。重组片段化弹性蛋白酶抑制结构域对弹性蛋白酶和胰凝乳蛋白酶均具有抑制活性。卵抑制剂主要以糖基化形式分泌。还研究了糖基化对蛋白酶抑制活性的影响。

(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