Abstract: Using overlap extension PCR, an A154C and G155C double site mutant named A154C/G155C was constructed based on the thermoacidophilic amylase gene BD5088 from Thermococcus sp. The recombinant plasmid pETBD5088C2 containing A154C/G155C was transformed into E. coli BL21 (DE3) for gene expression. The recombinant A154C/G155C amylase obtained showed a wider range of reaction pH, especially for acidic pH, than the amylase from BD5088. The half-life time of thermostability for the mutant at 100℃ was 22 min, almost doubling when compared with BD5088 (40 min). In addition, after heating for 60 min, A154C/G155C maintained 40% of its original amylase activity compared with 20% for BD5088, indicating an increase in thermostability. Moreover, the amylase activity of A154C/G155C increased at medium temperature or 90℃ and nearly doubled at 65℃. These results together with three-dimensional structure analysis indicate that mutation of the positions 154 and 155 to Cys in BD5088 is of great importance for maintaining the thermostability of amylase, has considerable effect on its enzymatic characteristics and may be involved in the formation of disulfide bonds.

Key words: thermoacidophilic amylase, site-directed mutagenesis, thermostability, disulfide bond