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

Protein Eng. 1990 Jan;3(3):181-91. doi: 10.1093/protein/3.3.181.


Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-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 Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-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 alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Calcium / metabolism
  • Geobacillus stearothermophilus / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Structure
  • Mutation*
  • Protein Conformation
  • Sequence Homology, Nucleic Acid
  • Structure-Activity Relationship
  • alpha-Amylases / genetics
  • alpha-Amylases / metabolism*


  • alpha-Amylases
  • Calcium