Literature summary for 3.2.1.2 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.

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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
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
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
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
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]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Bacillus cereus	P36924	-	-

Purification (Commentary)

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

Reaction

Reaction	Comment	Organism	Reaction ID
(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)

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

Synonyms

Synonyms	Comment	Organism
BCB	-	Bacillus cereus

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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