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Literature summary for 3.2.1.1 extracted from

  • Liu, Y.H.; Lu, F.P.; Li, Y.; Wang, J.L.; Gao, C.
    Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations (2008), Appl. Microbiol. Biotechnol., 80, 795-803.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Bacillus subitilis WB600 Bacillus licheniformis

Protein Variants

Protein Variants Comment Organism
L134R amyl, from recombinant Bacillus subtilis, when the pH is reduced from 6.5 to 4.5, compared with wild-type, Km increases but turnover number decreases, the mutants show an inverse trend, which results in catalytic efficiency (kcat/Km) increased. When the pH is 6.5, the kcat/Km is about 1.5times that of the mutants L134R, S320A, and L134R/S320A. In contrast, the kcat/Km of L134R and S320A are about 8.6- and 7.6times higher than that of the wild-type at pH 4.5. No significant difference on the kcat/Km of the mutants L134R, S320A, and L134R/S320A is shown when the pH is 5.5 and 6.5, respectively Bacillus licheniformis
L134R/S320A amyd, from recombinant Bacillus subtilis, when the pH is reduced from 6.5 to 4.5, compared with wild-type, Km increases but turnover number decreases, the mutants show an inverse trend, which results in catalytic efficiency (kcat/Km) increased. The highest kcat/Km with pH 4.5 approximately 14times that of the wild-type is observed in the double mutant. No significant difference on the kcat/Km of the mutants L134R, S320A, and L134R/S320A is shown when the pH is 5.5 and 6.5, respectively Bacillus licheniformis
S320A amy2, from recombinant Bacillus subtilis, when the pH is reduced from 6.5 to 4.5, compared with wild-type, Km increases but turnover number decreases, the mutants show an inverse trend, which results in catalytic efficiency (kcat/Km) increased. When the pH is 6.5, the kcat/Km is about 1.5times that of the mutants L134R, S320A, and L134R/S320A. In contrast, the kcat/Km of L134R and S320A are about 8.6- and 7.6times higher than that of the wild-type at pH 4.5. No significant difference on the kcat/Km of the mutants L134R, S320A, and L134R/S320A is shown when the pH is 5.5 and 6.5, respectively Bacillus licheniformis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
starch 0.60 mg/ml, double mutant, kcat/Km: 1560 ml/mg/s, L134R/S320A, 25°C, pH 6.0 Bacillus licheniformis
additional information
-
starch 0.61 mg/ml, mutant S329A, kcat/Km: 1870 ml/mg/s, 25°C, pH 6.0 Bacillus licheniformis
additional information
-
starch 0.64 mg/ml, mutant L134R, kcat/Km: 1670 ml/mg/s, 25°C, pH 6.0 Bacillus licheniformis
additional information
-
starch 0.86 mg/ml, wild-type, kcat/Km: 1920 ml/mg/s, 25°C, pH 6.0 Bacillus licheniformis
0.01
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, kcat/Km: L134R/S320A, 680000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
0.0113
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 740000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
0.0117
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 710000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
0.012
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 770000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
0.137
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 1910000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis
0.147
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 1420000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
0.171
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 1350000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis
0.185
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 680000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
0.186
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 1210000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis
0.19
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 1100000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
0.193
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 1070000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
0.2
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 1230000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
0.201
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 920000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
0.201
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 870000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
0.205
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 880000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
0.281
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 140000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis

Organism

Organism UniProt Comment Textmining
Bacillus licheniformis P06278 wild-type; CICC 10181
-
Bacillus licheniformis CICC 10181 P06278 wild-type; CICC 10181
-

Purification (Commentary)

Purification (Comment) Organism
wild-type, and recombinant mutants L134R, S320A, and L134R/S320A Bacillus licheniformis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside + H2O
-
Bacillus licheniformis p-nitrophenol + 4,6-ethyliden-[G7]-alpha-D-maltoheptaoside
-
?
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside + H2O
-
Bacillus licheniformis CICC 10181 p-nitrophenol + 4,6-ethyliden-[G7]-alpha-D-maltoheptaoside
-
?
starch + H2O
-
Bacillus licheniformis ?
-
?
starch + H2O
-
Bacillus licheniformis CICC 10181 ?
-
?

Synonyms

Synonyms Comment Organism
alpha amylase
-
Bacillus licheniformis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at, determination of catalytic properties on the substrates Bacillus licheniformis
37
-
assay at, determination of catalytic properties and pH study Bacillus licheniformis
70
-
assay at Bacillus licheniformis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
100
-
around 60% of the enzyme activity is still detectable in wild-type and mutant enzymes after incubating in a boiling bath for 60 min Bacillus licheniformis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
starch double mutant, L134R/S320A, kcat/Km: 1560 ml/mg/s, L134R/S320A, 25°C, pH 6.0 Bacillus licheniformis
additional information
-
starch mutant L134R, kcat/Km: 1670 ml/mg/s, 25°C, pH 6.0 Bacillus licheniformis
additional information
-
starch mutant S329A, kcat/Km: 1870 ml/mg/s, 25°C, pH 6.0 Bacillus licheniformis
additional information
-
starch wild-type, kcat/Km: 1920 ml/mg/s, 25°C, pH 6.0 Bacillus licheniformis
7.86
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 680000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
8.14
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 710000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
8.32
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 740000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
8.73
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 770000 1/s/M, 25°C, pH 6.0 Bacillus licheniformis
34
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 140000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis
175
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 880000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
175
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 870000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
186
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 920000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
187
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 1230000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
201
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 1070000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
205
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside wild-type, kcat/Km: 1420000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
209
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 1100000 1/s/M, 37°C, pH 6.5 Bacillus licheniformis
219
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant S329A, kcat/Km: 1210000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis
233
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 680000 1/s/M, 37°C, pH 5.5 Bacillus licheniformis
234
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside mutant L134R, kcat/Km: 1350000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis
266
-
4,6-ethylidene-[G7]-p-nitrophenyl-[G1]-alpha-D-maltoheptaoside double mutant, L134R/S320A, kcat/Km: L134R/S320A, 1910000 1/s/M, 37°C, pH 4.5 Bacillus licheniformis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
assay at Bacillus licheniformis
7
-
optimum Bacillus licheniformis

pH Range

pH Minimum pH Maximum Comment Organism
5.5 9.5 4000 U/ml at pH 5.5, above 4000 U/ml at pH 10.0, pH-optimum: 10000 U/ml Bacillus licheniformis

pH Stability

pH Stability pH Stability Maximum Comment Organism
4.5
-
the mutant L134R/S320A shows a stronger activity in acidic conditions, In contrast, the wild-type is sensitive to acidic pH. The double mutant is more effective in increasing enzyme stability against acidic pH than L134R and S320A Bacillus licheniformis