一个耐热碱性磷酸酯酶突变型的研究  

A Study of a Mutant of Thermostable Alkaline Phosphatase

作　　者：袁有忠[1] 赵思汇[1] 周宗祥[1] 季朝能[1] 盛小禹[1] 毛裕民[1] 

机构地区：[1]复旦大学遗传学研究所遗传工程国家重点实验室,上海200433

出　　处：《Acta Genetica Sinica》2000年第4期361-368,共8页

基　　金：国家自然科学基金!39870402

摘　　要：为了进一步揭示蛋白质的耐热机制，对含有耐热碱性磷酸酯酶（ＦＤ－ＴＡＰ）的表达质粒ｐＴＡＰ５０３Ｆ进行了随机诱变，用菌落原位显色法从约５０００个转化子中筛选到４个耐热性下降的突变型克隆，并对其中１个克隆（ＴＡＰＭ３）进行了部分酶学性质、ＤＮＡ和氨基酸序列的研究。酶学性质研究表明，与野生型相比，该突变型酶的耐热性有较大幅度的下降，而热激活性无明显改变。 ＤＮＡ序列分析表明在１２３９位ＴＡＰＭ３发生Ｇ→Ａ转换，导致４２７位的甘氨酸变为丝氨酸（Ｇ４２７５），这种突变对酶的耐热性、米氏常数、活化能影响较为明显。这说明只须１个氨基酸置换就会对蛋白质耐热性变化起巨大作用，并且氨基酸残基侧链的大小、电荷等能使蛋白质结构松散的性质，会导致蛋白质耐热性下降。To reveal the mechanism of protein thermostability, we used in vivo random mutagenesis to generate variants of PTAP503F which contained thermostable alkaline phosphatase (FD-TAP). After screening about 5 000 clones, we obtained 4 temperature-sensitive mutants. The study of enzymatic properties of one mutant (TAPM3) showed that the thermostability of the mutant enzyme descended a lot compared to the wild type, while the thermoactivity remained stable. DNA sequencing showed that the G-A transition in position 1 239 resulted in the substitution from glysine to serine in position 427. This mutation conspicuously affected thermostability, Michaelis constant and energy of activation. This suggests that only one substitution of amino acid will make great changes in thermostability and other properties, meanwhile, side-chain size, charge of residues and so on, which loosen the structure of protein, will result in the descent of thermostability.

关 键 词：耐热碱性磷酸酯酶 随机诱变 蛋白质 耐热机制 

分 类 号：Q55[生物学—生物化学]