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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。
In order to further reveal the mechanism of protein heat tolerance, the expression plasmid pTAP503F containing heat-resistant alkaline phosphatase (FD-TAP) was randomly mutagenized and screened from about 5000 transformants by colony in situ colorimetry to 4 A mutant clones with decreased heat resistance were selected and one of the clones (TAPM3) was subjected to partial enzymatic properties, DNA and amino acid sequence. Enzymatic properties of the study showed that compared with the wild-type, the mutant enzyme has a more significant decrease in heat resistance, and no significant changes in heat-activated. DNA sequence analysis showed that G → A transition occurred at TAPM3 at position 1239, resulting in the conversion of glycine at position 427 to serine (G4275). This mutation had obvious effect on heat resistance, Michaelis constant and activation energy of the enzyme. This shows that only a single amino acid substitution will have a great effect on protein thermostability changes, and the size of the amino acid residue side chains, charges and other properties that make the protein structure loose, will lead to protein heat resistance decreased.