Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase

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Abstract
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.