Function-structure of protein-polysaccharide cpmplex constructed by protein engineering.

蛋白质-多糖偶联物具有良好的热稳定性和乳化性等功能性质。为了阐明功能性质改善的分子机制，我们利用基因修饰的方法构建了糖基化溶菌酶。对编码蛋清溶菌酶的互补DNA进行定点突变，将Arg-21和Gly-49分别替换为Arg-21和Gly-49，在19和49位引入两个N-糖基化位点(Asn^&lt；19&gt；-Try^&lt；20&gt；-Tr^&lt；21&gt；和Asn^&lt；49&gt；-Ser^&lt；50&gt；-Thr^&lt；51&gt；将单糖化和双糖化的溶菌酶(G49N、R21T、R21T/G49N)分别在酵母表达载体上插入单双突变体hew1 cDNA，在酿酒酵母中表达。突变的溶菌酶主要以多甘露糖基形式表达，少量表达两种寡甘露糖基形式。多甘露糖基溶菌酶G49N和R21T分别在第49和19位糖基化，约300个甘露糖残基，而R21T/G49N上的多甘露糖链长度约为272和18个甘露糖残基。R21T/G49N具有比R21T和G49N两种单一多甘露糖溶菌酶更好的乳化性能。在单一多甘露糖溶菌酶中，G49N的乳化性能略好于R21T。这表明多糖的结合对其功能性质是必不可少的。

Protein-polysaccharide conjugates showed the excellent functional properties such as heat stability and emulsifying properties. In order to elucidate the molecular mechanism of the improvements of functional properties, we constructed the glycosylated lysozyme using genetic modification. Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site-directed mutagenesis to introduce two N-linked glycosylation sites (Asn^<19>-Try^<20>-Tr^<21> and Asn^<49>-Ser^<50>-Thr^<51>) into both positions 19 and 49 by substituting Arg-21 with Thr and Gly-49 with Asn, respectively. The single and double glycosylated lysozymes (G49N,R21T,R21T/G49N) were expressed in Saccharomyces cerevisiae carrying the yeast expression plasmid inserted the single and double mutant HEWL cDNAs. The mutant lysozymes were predominantly expressed a polymannosyl form with a small amout of two oligomannosyl forms. The polymannosyl lysozymes G49N and R21T were glycosylated at positions 49 and 19 with approximately 300 mannose residues, respectively, while the length of the polymannosyl chains attached to R21T/G49N was approximately 272 and 18 mannose residues. The R21T/G49N showed better emulsifying properties than two types of single polymannosyl lysozymes R21T and G49N.In a case of single polymannosyl lysozyme, G49N showed somewhat better emulsifying properties than R21T.The removal of polymannosyl chain from lysoyme with Endo- beta -N-acetylglucosaminidase (Endo-H) resulted in a dramatic decrease in the emulsifying properties of polymannosyl lysozymes. This suggests that the plysaccharide attachment is essential for the functional property.