Molecular design of lysozyme for the improvement of protein function.

为了利用蛋白质工程技术改善鸡溶菌酶的功能，进行了以下实验。结论：1.鸡溶菌酶的N端含有额外的蛋氨酸残基，在大肠杆菌中得到表达，并折叠成活性构象。建立了一种处理溶菌酶N-端序列的方法。建立了在酵母中表达和分泌溶菌酶的方法，并用该方法制备了多种突变型溶菌酶。从突变的溶菌酶的性质中，获得了以下信息。(1)当催化羧基Glu35和Asp52发生突变时，溶菌酶活性几乎完全丧失。(2)Asp52位突变的溶菌酶与底物结合能力较弱。(3)位于35位的游离残基的pK异常，从而抑制了该位置电荷的出现。(4)用谷氨酰胺取代Trp108使酶失活和不稳定。(5)用天冬氨酸取代β-折叠中的天冬氨酸46，活性降低到25%以下。(6)用Gly替代Asn37或Asp101可提高裂解活性。(7)Arg14-His15缺失可提高几丁质酶活性。(8)与α-乳清蛋白类似的钙结合部位的还原溶菌酶的折叠受钙浓度的控制。为了探索提高天然溶菌酶功能的途径，我们分离了多种鸡型溶菌酶，并测定了它们的氨基酸序列和性质。

In order to improve the function of chicken lysozyme by protein engineering, the following experiments were carried out.1. Various mutant chicken lysozymes which always contain an extra methionine residue at the N-terminal were expressed in E. coli, and folded to the active conformations.2. A method to process the N-terminal sequence of lysozymes thus obtained was developed.3. A method for the expression and secretion of lysozyme in yeast was established and various mutant lysozymes were prepared by the method.4. From the properties of the mutant lysozymes, the following informations were obtained. (1) When catalytic carboxyls, Glu35 and Asp52, were mutated, lysozyme activity was almost completely lost. (2) Lysozyme with the mutation at Asp52 has the weak substrate binding ability. (3) The pK of dissociable residue located at position 35 was abnormal to depress the appearance of charges at this position. (4) Replacement of Trp108 with Gln make the enzyme inactive and unstable. (5) Substitution of Asp for Asn46 which is in the beta-sheet lowered the activity to less than 25%. (6) Substitution of Gly for Asn37 or Asp101 increased the lytic activity. (7) Deletion of Arg14-His15 enhanced the chitinase activity. (8) The folding of the reduced lysozyme where the calcium binding site similar to alpha-lactalbumin is constructed was controlled by the concentration of calcium.5. In order to learn the way to improve lysozyme function for nature, various chicken type-lysozymes were isolated, and their amino acid sequences and properties were determined.