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

Liisa Holm, Anu Koivula, Päivi Lehtovaara, Ari Hemminki, Jonathan Knowles

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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.
Original languageEnglish
Pages (from-to)181-191
JournalProtein Engineering
Volume3
Issue number3
DOIs
Publication statusPublished - 1990
MoE publication typeA1 Journal article-refereed

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Geobacillus stearothermophilus
Mutagenesis
Amylases
alpha-Amylases
Bacilli
Mutation
Molecular Models
Enzyme activity
Enzymes
Escherichia coli
Catalyst activity
Catalytic Domain
Amino acids
Amino Acids
Substrates

Cite this

Holm, Liisa ; Koivula, Anu ; Lehtovaara, Päivi ; Hemminki, Ari ; Knowles, Jonathan. / Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha- amylase. In: Protein Engineering. 1990 ; Vol. 3, No. 3. pp. 181-191.
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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.",
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Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha- amylase. / Holm, Liisa; Koivula, Anu; Lehtovaara, Päivi; Hemminki, Ari; Knowles, Jonathan.

In: Protein Engineering, Vol. 3, No. 3, 1990, p. 181-191.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha- amylase

AU - Holm, Liisa

AU - Koivula, Anu

AU - Lehtovaara, Päivi

AU - Hemminki, Ari

AU - Knowles, Jonathan

PY - 1990

Y1 - 1990

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AB - 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.

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