Mutations that cover the sequence of Bacillus stearothermophilus α-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant α-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Taka-amylase A and a consensus alignment of mammalian, plant, and bacterial α-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central α/β barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.
Holm, L., Koivula, A., Lehtovaara, P., Hemminki, A., & Knowles, J. (1990). Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha- amylase. Protein Engineering, 3(3), 181-191. https://doi.org/10.1093/protein/3.3.181