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.
Original language | English |
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Pages (from-to) | 181-191 |
Journal | Protein Engineering |
Volume | 3 |
Issue number | 3 |
DOIs | |
Publication status | Published - 1990 |
MoE publication type | A1 Journal article-refereed |