Cloned (Comment) | Organism |
---|---|
functional recombinant expression of wild-type and engineered enzyme in Escherichia coli strain BL21(DE3), improvement of cloning and constitutive expression of soluble active mTG, overview. Usage of constitutive vector pET9a and a synthetic construct encoding the C-terminally His-tagged mTG thermostable variant S2P. Further increase in expression levels may be possible by coexpressing periplasmic secretory proteins, since secretion into the periplasm is frequently the limiting factor for production | Streptomyces mobaraensis |
Protein Variants | Comment | Organism |
---|---|---|
additional information | the S2P variant is generated by random mutagenesis of the wild-type enzyme, and found to be more thermostable, able to withstand incubation at 60°C, and more active than the wild-type enzyme. The synthetic operon construct (based on GenBank ID KX775947) consists of two parts: first a gene encoding the pro-domain crucial for proper folding of the enzyme and second the gene encoding the mTG thermostable variant S2P, with a C-terminal His-tag. Each part is paired with a preceding PelB secretory sequence. The Km value is 3fold lower for the mutant S2P as compared to the wild-type. Conversely, the turnover number is higher for the wild-type enzyme, although the enzymatic efficiency is 2fold higher for the mutant. The mutant unfolds at a slightly higher temperature (56.3°C vs. 55.8°C) indicating improved thermostability although not statistically significant | Streptomyces mobaraensis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics. The Km value is 3fold lower for the mutant S2P as compared to the wild-type. Conversely, the turnover number is higher for the wild-type enzyme, although the enzymatic efficiency is 2fold higher for the mutant | Streptomyces mobaraensis | |
4.2 | - |
Z-Gln-Gly | recombinant mutant S2P, pH 7.2, 37°C | Streptomyces mobaraensis | |
11.6 | - |
Z-Gln-Gly | recombinant wild-type enzyme, pH 7.2, 37°C | Streptomyces mobaraensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Streptomyces mobaraensis | transglutaminase catalyzes the acyl transfer reaction between gamma-carboxyamide groups (acyl donor) and primary amines (acyl acceptor). In proteins, it is able to crosslink the gamma-carboxyamide of glutamine and the primary epsilon-amine in lysine | ? | - |
- |
|
protein glutamine + alkylamine | Streptomyces mobaraensis | - |
protein N5-alkylglutamine + NH3 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces mobaraensis | P81453 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Streptomyces mobaraensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | transglutaminase catalyzes the acyl transfer reaction between gamma-carboxyamide groups (acyl donor) and primary amines (acyl acceptor). In proteins, it is able to crosslink the gamma-carboxyamide of glutamine and the primary epsilon-amine in lysine | Streptomyces mobaraensis | ? | - |
- |
|
additional information | the enzymatic transamidation reaction between a gamma-glutamyl donor (Z-Gln-Gly) and hydroxylamine releasing ammonia is coupled to the glutamate dehydrogenase (GDH)-catalyzed reductive amination of 2-oxoglutarate. The activity of GDH is dependent on NADH as a cofactor, whose disappearance can be monitored at 340 nm. NADH concentration was calculated based on a calibration curve, which in turn is used to calculate activity of mTG | Streptomyces mobaraensis | ? | - |
- |
|
protein glutamine + alkylamine | - |
Streptomyces mobaraensis | protein N5-alkylglutamine + NH3 | - |
? | |
Z-Gln-Gly + hydroxylamine | - |
Streptomyces mobaraensis | Z-N5-hydroxyglutaminyl-Gly + NH3 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
microbial transglutaminase | - |
Streptomyces mobaraensis |
MTG | - |
Streptomyces mobaraensis |
TGase | - |
Streptomyces mobaraensis |
transglutaminase | - |
Streptomyces mobaraensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Streptomyces mobaraensis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
45.5 | 56.3 | melting temperature of enzyme mutant S2P | Streptomyces mobaraensis |
55.8 | - |
melting temperature of wild-type enzyme | Streptomyces mobaraensis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.6 | - |
Z-Gln-Gly | recombinant mutant S2P, pH 7.2, 37°C | Streptomyces mobaraensis | |
0.85 | - |
Z-Gln-Gly | recombinant wild-type enzyme, pH 7.2, 37°C | Streptomyces mobaraensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.2 | - |
assay at | Streptomyces mobaraensis |
General Information | Comment | Organism |
---|---|---|
physiological function | microbial transglutaminase (mTG) is a robust enzyme catalyzing the formation of an isopeptide bond between glutamine and lysine residues. Transglutaminase catalyzes the acyl transfer reaction between gamma-carboxyamide groups (acyl donor) and primary amines (acyl acceptor). In proteins, it is able to crosslink the gamma-carboxyamide of glutamine and the primary epsilon-amine in lysine | Streptomyces mobaraensis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.073 | - |
Z-Gln-Gly | recombinant wild-type enzyme, pH 7.2, 37°C | Streptomyces mobaraensis | |
0.143 | - |
Z-Gln-Gly | recombinant mutant S2P, pH 7.2, 37°C | Streptomyces mobaraensis |