Molecular design of lysozyme for the improvement of protein function.

为了利用蛋白质工程技术提高鸡溶菌酶的功能，进行了以下实验.在大肠杆菌中表达了N端多一个蛋氨酸残基的各种突变型鸡溶菌酶。coli，并折叠成活性构象.建立了一种对所获得的溶菌酶N端序列进行处理的方法.建立了溶菌酶在酵母中表达和分泌的方法，并利用该方法制备了多种突变溶菌酶.从突变溶菌酶的性质中，获得了以下信息。(1)当催化羧基Glu 35和Asp 52突变时，溶菌酶活性几乎完全丧失。(2)Asp 52突变的溶菌酶与底物结合能力较弱。(3)位于35位的可解离残基的pK异常，从而抑制了该位置的电荷出现。(4)用Gln取代Trp 108使酶失活和不稳定。(5)用β折叠中的Asn 46取代Asp使活性降低至小于25%。(6)Gly取代Asn 37或Asp 101增加了裂解活性。(7)Arg 14-His 15缺失后几丁质酶活性增强。(8)还原型溶菌酶的折叠受钙离子浓度的控制，其中的钙离子结合位点与α-乳清蛋白相似.为了探索提高天然溶菌酶功能的途径，本研究分离了多种鸡型溶菌酶，并测定了其氨基酸序列和性质。

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.