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Literature summary extracted from

  • Hirata, A.; Adachi, M.; Utsumi, S.; Mikami, B.
    Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type (2004), Biochemistry, 43, 12523-12531.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.2 expression of wild-type and mutant enzymes in Escherichia coli strain XL1-Blue Bacillus cereus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.2 purified recombinant wild-type and mutant T47M/Y164E/T328N, hanging drop vapour diffusion method, 18°C, 0.005 ml of 15 mg/ml protein in 0.05 M sodium acetate is mixed with 0.005 ml mother liquor containing 15% PEG 6000, 5% saturated ammonium sulfate, 0.1 M phosphate, pH 6.5, crystallization of mutants Y164E and Y164F in the same way except for usage of 0.1 M sodium acetate buffer, pH 4.6, instead of phosphate buffer, X-ray diffraction structure determination and analysis at 1.72-1.95 A resolution, active site structure modelling Bacillus cereus

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.2 additional information engineering of the enzyme's pH optimum, conversion of the pH optimum from the bacterial type with pH 6.7 to the higher-plant type with pH 5.4, from soybean enzyme, overview Bacillus cereus
3.2.1.2 T47M/Y164E/T328N site-directed mutagenesis, the mutation of Y164 leads to disruption of the hydrogen bonding around the catalytic site, the mutant enzyme shows a shifted pH optimum and a 88% decreased kcat compared to the wild-type enzyme Bacillus cereus
3.2.1.2 Y164E site-directed mutagenesis, the mutation of Y164 leads to disruption of the hydrogen bonding around the catalytic site, the mutant enzyme shows a shifted pH optimum and a 59% decreased kcat compared to the wild-type enzyme Bacillus cereus
3.2.1.2 Y164F site-directed mutagenesis, the mutation of Y164 leads to disruption of the hydrogen bonding around the catalytic site, the mutant enzyme shows a shifted pH optimum and a 64% decreased kcat compared to the wild-type enzyme Bacillus cereus
3.2.1.2 Y164H site-directed mutagenesis, the mutation of Y164 leads to disruption of the hydrogen bonding around the catalytic site, the mutant enzyme shows a shifted pH optimum and a 97% decreased kcat compared to the wild-type enzyme Bacillus cereus
3.2.1.2 Y164Q site-directed mutagenesis, the mutation of Y164 leads to disruption of the hydrogen bonding around the catalytic site, the mutant enzyme shows a shifted pH optimum and a 83% decreased kcat compared to the wild-type enzyme Bacillus cereus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.2 additional information
-
 additional information kinetics, recombinant wild-type andmutant enzymes Bacillus cereus
3.2.1.2 0.47
-
 amylopectin 37°C, recombinant mutant T47M/Y164E/T328N Bacillus cereus
3.2.1.2 0.73
-
 amylopectin 37°C, recombinant wild-type enzyme Bacillus cereus
3.2.1.2 0.92
-
 amylopectin 37°C, recombinant mutant Y164F Bacillus cereus
3.2.1.2 1.22
-
 amylopectin 37°C, recombinant mutant Y164Q Bacillus cereus
3.2.1.2 1.24
-
 amylopectin 37°C, recombinant mutant Y164E Bacillus cereus
3.2.1.2 2.63
-
 amylopectin 37°C, recombinant mutant Y164H Bacillus cereus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.1.2 amylopectin + H2O Bacillus cereus
-
 maltose + ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.2 Bacillus cereus P36924
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.2 recombinant wild-type and mutant enzymes from Escherichia coli strain XL1-Blue Bacillus cereus

Reaction

EC Number Reaction Comment Organism Reaction ID
3.2.1.2 (alpha-D-glucopyranosyl-(1-4))n-alpha-D-glucopyranose + H2O = (alpha-D-glucopyranosyl-(1-4))n-2-alpha-D-glucopyranose + alpha-D-glucopyranosyl-(1-4)-beta-D-glucopyranose Glu367 is important in catalysis, the plant enzyme shows a reaction mechanism different from the bacterial enzyme, overview Bacillus cereus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.2 amylopectin + H2O
-
 Bacillus cereus maltose + ?
-
 ?
3.2.1.2 amylopectin + H2O from potato Bacillus cereus maltose + ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.1.2 BCB
-
 Bacillus cereus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.2 37
-
 assay at Bacillus cereus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.2 92
-
 amylopectin 37°C, recombinant mutant Y164H Bacillus cereus
3.2.1.2 325
-
 amylopectin 37°C, recombinant mutant T47M/Y164E/T328N Bacillus cereus
3.2.1.2 453
-
 amylopectin 37°C, recombinant mutant Y164Q Bacillus cereus
3.2.1.2 988
-
 amylopectin 37°C, recombinant mutant Y164F Bacillus cereus
3.2.1.2 1129
-
 amylopectin 37°C, recombinant mutant Y164E Bacillus cereus
3.2.1.2 2739
-
 amylopectin 37°C, recombinant wild-type enzyme Bacillus cereus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.2 additional information
-
 comparison of pH-dependency/pH-profiles of recombinant wild-type and mutant enzymes Bacillus cereus
3.2.1.2 4.2
-
 mutant Y164Q Bacillus cereus
3.2.1.2 4.6
-
 mutant Y164E Bacillus cereus
3.2.1.2 4.8
-
 mutants Y164H and Y164F Bacillus cereus
3.2.1.2 6
-
 mutant T47M/Y164E/T328N Bacillus cereus
3.2.1.2 6.7
-
 wild-type enzyme Bacillus cereus