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.