Literature summary for 3.2.1.1 extracted from

Khemakhem, B.; Ali, M.B.; Aghajari, N.; Juy, M.; Haser, R.; Bejar, S.
Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation (2009), Biotechnol. Bioeng., 102, 380-389.

Activating Compound

Activating Compound	Comment	Organism	Structure
Detergents	the mutant enzyme AmyUS100DELTAIG/M197A is found to be very stable in the presence of solid detergents (commercial detergents). The activity is increased in somes cases. Incubation of the enzyme at 40°C with Ariel, Skip, Dixan, Det, NewDet, Nadhif,m and OMO increases the activity between 10 and 50%. The activity of the engineered enzyme persists at 60°C of the majority of the commercial solid detergents. The incubation with the liquid detergents Lav+ and Nadhif increases the activity by 10-20%, respectively. The presence of the Dinol detergent, the activity decreases to 97 and 91% at 40 and 60°C, respectively	Geobacillus stearothermophilus

Application

Application	Comment	Organism
detergent	AmyUS100DELTAIG is designed to improve the thermostability of the thermoactive and thermostable maltohexaose forming alpha-amylase produced in Geobacillus stearothermophilus sp. US100, AmyUS100	Geobacillus stearothermophilus

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli ER2566	Geobacillus stearothermophilus

Protein Variants

Protein Variants	Comment	Organism
M197A	site-directed mutagenesis. Studies of its catalytic properties show no effect on the thermostability, pH activity/stability, calcium demand and chelator resistance. Specific activity is decreased from 1000 to 845 U/mg. The profile of starch hydrolysis is affected. As a result hereof AmyUS100DELTAIG/M197A produces in majority maltose and maltotriose as major products compared to maltohexaose and maltopentaose produced by the wild-type and the AmyUS100DELTAIG variant. The mutant retains 85% of its original activity. 70% of the mutantM197A activity is retained after 60 min of treatment at 60°C in the presence of 1.8 M H2O2, whereas AmyUS100DELTAIG is totally inactivated. These results confirm the importance of Met197 in the oxidative sensibility, situated in the cavity of the active site	Geobacillus stearothermophilus

Inhibitors

Inhibitors	Comment	Organism
EDTA	AmyUS100DELTAG retains 40 and 25% of its original activity at 40 and 60°C, respectively, when incubated with 200 mM of EDTA, compared to 12 and 0% for AmyUS100	Geobacillus stearothermophilus
linear alkylbenzene sulfonate	mutant M197A, concentration of 10% incubation at 60°C for 1 h, 71% residual relative activity, assay at 80°C and pH 5.6	Geobacillus stearothermophilus
SDS	mutant M197A, concentration of 10% incubation at 60°C for 1 h, 45% residual relative activity, assay at 80°C and pH 5.6	Geobacillus stearothermophilus
Triton X-100	mutant M197A, concentration of 10% incubation at 60°C for 1 h, 71% residual relative activity, assay at 80°C and pH 5.6	Geobacillus stearothermophilus
Tween-20	mutant M197A, concentration of 10% incubation at 60°C for 1 h, 94% residual relative activity, assay at 80°C and pH 5.6	Geobacillus stearothermophilus
Tween-80	mutant M197A, concentration of 10% incubation at 60°C for 1 h, 88% residual relative activity, assay at 80°C and pH 5.6	Geobacillus stearothermophilus

Organism

Organism	UniProt	Comment	Textmining
Geobacillus stearothermophilus	P06279	sp. US100	-

Oxidation Stability

Oxidation Stability	Organism
activity of AmyUS100DELTAIG and Termamyl300 (commercial amylase used in detergent) are instantaneously decreased in the presence of 1.8 M H2O2, whereas AmyUSDELTAIG/M197A retains 85% of its original activity. 70% of the mutantM197A activity is retained after 60 min of treatment at 60°C. Termamyl300 retains 20% of its original activity and AmyUS100DELTAIG is totally inactivated. These results confirm the importance of Met197 in the oxidative sensibility, situated in the cavity of the active site	Geobacillus stearothermophilus

Organism

activity of AmyUS100DELTAIG and Termamyl300 (commercial amylase used in detergent) are instantaneously decreased in the presence of 1.8 M H2O2, whereas AmyUSDELTAIG/M197A retains 85% of its original activity. 70% of the mutantM197A activity is retained after 60 min of treatment at 60°C. Termamyl300 retains 20% of its original activity and AmyUS100DELTAIG is totally inactivated. These results confirm the importance of Met197 in the oxidative sensibility, situated in the cavity of the active site

Geobacillus stearothermophilus

Purification (Commentary)

Purification (Comment)	Organism
recombinant protein	Geobacillus stearothermophilus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	decreased from 1000 to 845 U/mg for AmyUS100DELTAIG to the mutant M197A, assay at 80°C and pH 5.6	Geobacillus stearothermophilus

Storage Stability

Storage Stability	Organism
spry-dried enzyme, after 6 months in liquid detergent, 90% of its original activity retained. After 1 year the activity decreases to 61%. The activity of the enzyme in solid detergent is almost invariant. In presence of commercial proteases salvinase and alcalase the enzyme activity decreases by 22 and 18%, respectively, after 6 months	Geobacillus stearothermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
starch + H2O	-	Geobacillus stearothermophilus	maltohexaose + ?	-	?

Synonyms

Synonyms	Comment	Organism
AmyUS100	variant of the most thermoactive and thermostable maltohexaose forming alpha-amylase produced in Geobacillus stearothermophilus sp. US100	Geobacillus stearothermophilus
AmyUS100DELTAIG	recombinant protein	Geobacillus stearothermophilus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
80	-	assay at	Geobacillus stearothermophilus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5.6	-	assay at	Geobacillus stearothermophilus

pH Range

pH Minimum	pH Maximum	Comment	Organism
4	9	AmyUS100 and mutant M197A enzyme	Geobacillus stearothermophilus