Cloning and Expression of Randomly MutatedBacillus subtilisα-Amylase Genes in HB101

The aim of this study was to isolate and express the randomly mutatedα-amylase gene from B.subtilis strain 168. BS168F:5′-gtgtcaagaatgtttgc-3′and BS168R:3′-gttttgttaaaagatga-5′primers were used to amplify the amylase gene using the followingcycle in error-prone PCR method: 94°C for30 s, 40°C for 2 min, and72°C for 2 min in 30 cycles that werefollowed with 72°C for 2 min as a post cycle.E. coli XL1 blue was used as host for plasmidconstruction. Amylase enzyme activity assay was performed usingcontinuous spectrophotometric procedures. Results of sequencing showedthat sequence was cloned from the first ATG and with the correct openreading frame. Having confirmed the integrity of the insert, the genewas ligated into expression vector pET-15b and then further confirmedusing digestion analysis. Amylase activity showed 3 clones with higherenzymatic activity compared with the wild type. Error-prone PCR methodproduced a mutated gene that provides amylase activity much higherthan that of wild type. Sequencing the mutated genes should shed lighton the important region of the genes that could be manipulated infuture studies