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Literature summary for 3.2.1.133 extracted from

  • Jones, A.; Lamsa, M.; Frandsen, T.P.; Spendler, T.; Harris, P.; Sloma, A.; Xu, F.; Nielsen, J.B.; Cherry, J.R.
    Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25 (2008), J. Biotechnol., 134, 325-333.
    View publication on PubMed
Search for more literature for 3.2.1.133

Application

Application Comment Organism
food industry Novamyl is currently used in the baking industry as an anti-staling agent in breads at neutral or near neutral pH. It is rapidly inactivated during the baking process. Relative to the Novamyl wild-type the variants exhibit more than 10°C increase in thermal stability at pH 4.5, improving the anti-staling effect Bacillus sp. (in: Bacteria)
food industry wild-type enzyme is not thermostable at low pH as encountered in recipes such as sourdough and rye dough, recombined variants increase thermal stability more than 10°C at pH 4.0-4.5, application test is performed with wheat flour dough at pH 5.5-5.9 and sourdough at pH 4.0-4.3 baked at 230°C for 22 min, firmness and elasticity tested at days 1, 3, and 7 after baking and vacuum packing Bacillus sp. (in: Bacteria)

Cloned(Commentary)

Cloned (Comment) Organism
Novamyl libraries are iniatially made in Escherichia coli, then shuttled and expressed in the screen host Bacillus subtilis. The variants NM319, NM326, NM398, NM404, and NM447 are grown in fermenters, and the expressed enzymes are purified and tested for anti-staling properties in standard bread pH 5.5-5.9 and/or bread at sourdough (pH 4.0-4.3) Bacillus sp. (in: Bacteria)
shuffled libraries of Novamyl are produced in in Escherichia coli, shuttled to screening host B. subtilis strain A164 delta 5, plasmid pHP13amp Bacillus sp. (in: Bacteria)

Protein Variants

Protein Variants Comment Organism
D30A/K40R/D261G Novamyl variant NM398, similar to wild-type at pH 4.0 Bacillus sp. (in: Bacteria)
D30A/K40R/D261G variant NM398, medium thermotolerance due to D261G mutation Bacillus sp. (in: Bacteria)
F188L/D261G/T288P Novamyl variant NM447, outperform wild-type Novamyl in bread at pH 4.3. Wild-type Novamyl requires a nearly 30fold protein dosage increase over NM447 to obtain anti-staling bread properties. NM447 appears to have a broad pH functionality profile and performs better than wild-type Novamyl in standard bread to pH 5.9. It seems to be be generally tehrmally stabilized, both at pH 4.0 and 5.0. Bacillus sp. (in: Bacteria)
F188L/D261G/T288P variant NM447, most thermotolerant, probably due to F188L mutation, but reduced activity, medium thermotolerance due to D261G mutation, improved anti-staling performance in application test, about 70% activity retained after incubation at 80°C at pH 4.3 for 25 min Bacillus sp. (in: Bacteria)
additional information mutagenesis is induced by error-prone PCR, mutagenic fragments screened for thermostable variants (80°C) at low pH 4.3, most thermostable variants show a decreased activity to the wild-type enzyme Bacillus sp. (in: Bacteria)
N115D/F188L Novamyl variant NM319, similar to wild-type at pH 4.0, at pH 5.5 the variant outperform wild-type Novamyl, significantly more thermally stable that wild-type Bacillus sp. (in: Bacteria)
N115D/F188L variant NM319, most thermotolerant, probably due to F188L mutation, but reduced activity, improved anti-staling performance in application test, about 40% activity retained after incubation at 80°C at pH 4.3 for 25 min Bacillus sp. (in: Bacteria)
T142A Novamyl variant NM326, similar to wild-type at pH 4.0 Bacillus sp. (in: Bacteria)
T142A variant NM326, inducing 20% activity increase and 50% thermal stability increase Bacillus sp. (in: Bacteria)
T142A/D261G/N327S/K425E/K520R/N5951 variant NM404, medium thermotolerance due to D261G mutation Bacillus sp. (in: Bacteria)
T142A/D261G/N327S/K425E/K520R/N595I Novamyl variant NM404, similar to wild-type at pH 4.0 Bacillus sp. (in: Bacteria)

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
amylopectin + H2O Bacillus sp. (in: Bacteria)
-
 maltose + maltotriose wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P ?
amylopectin + H2O Bacillus sp. (in: Bacteria) TS-25
-
 maltose + maltotriose wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P ?

Organism

Organism UniProt Comment Textmining
Bacillus sp. (in: Bacteria)
-
-
-
Bacillus sp. (in: Bacteria)
-
 TS-25
-
Bacillus sp. (in: Bacteria) TS-25
-
-
-
Bacillus sp. (in: Bacteria) TS-25
-
 TS-25
-

Purification (Commentary)

Purification (Comment) Organism
mutagenic PCR fragment purification with Quiaquick Gel Extract kit, and agarose electrophoresis, then cloned into plasmid pHP13amp, fermentation in bioreactors, enzyme variants purified on alpha-cyclodextrin coupled to agarose column, corroborated by SDS-PAGE Bacillus sp. (in: Bacteria)
recombinant protein, the variants NM319, NM326, NM398, NM404, and NM447 are grown in fermenters and the expressed enzymes are purified Bacillus sp. (in: Bacteria)

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
amylopectin + H2O
-
 Bacillus sp. (in: Bacteria) maltose + maltotriose wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P ?
amylopectin + H2O
-
 Bacillus sp. (in: Bacteria) maltose + maltotriose wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% matotriose of mutant F188L/D261G/T288P ?
amylopectin + H2O
-
 Bacillus sp. (in: Bacteria) TS-25 maltose + maltotriose wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P ?
amylopectin + H2O
-
 Bacillus sp. (in: Bacteria) TS-25 maltose + maltotriose wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% matotriose of mutant F188L/D261G/T288P ?

Synonyms

Synonyms Comment Organism
maltogenic alpha-amylase
-
 Bacillus sp. (in: Bacteria)
NM319 Novamyl variant Bacillus sp. (in: Bacteria)
NM326 Novamyl variant Bacillus sp. (in: Bacteria)
NM398 Novamyl variant Bacillus sp. (in: Bacteria)
NM404 Novamyl variant Bacillus sp. (in: Bacteria)
NM447 Novamyl variant Bacillus sp. (in: Bacteria)
Novamyl
-
 Bacillus sp. (in: Bacteria)
Novamyl trade name Bacillus sp. (in: Bacteria)

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
 assay at Bacillus sp. (in: Bacteria)

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 NM404 and NM398 has modest improvements in thermal stability, whereas NM326 shows no significant improvement in thermal stability Bacillus sp. (in: Bacteria)
64
-
 Tm of Novamyl wild-type at pH 4.0 Bacillus sp. (in: Bacteria)
75
-
 Tm of variant NM447 at pH 4.0 Bacillus sp. (in: Bacteria)
80
-
 the variants NM447 and NM319 retain 67 and 40% specific activity following a 25-min incubation, respectively Bacillus sp. (in: Bacteria)
83
-
 Tm of Novamyl wild-type at pH 5.0 Bacillus sp. (in: Bacteria)
88
-
 TD of variant NM447 at pH 4.0, an increase of 10°C relative to the wild-type Novamyl Bacillus sp. (in: Bacteria)
95
-
 Tm of variant NM447 at pH 5.0 Bacillus sp. (in: Bacteria)

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4
-
 or pH 5.0, assay at Bacillus sp. (in: Bacteria)
5
-
 or pH 4.0, assay at Bacillus sp. (in: Bacteria)