Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzyme in Pichia pastoris | Trichoderma reesei |
Protein Variants | Comment | Organism |
---|---|---|
R171K | the mutant displays retained activity on polymeric galactomannan but reduced activity on oligosaccharides due to an increase of Km. While the wild-type enzyme produces mannobiose as dominant product from mannotetraose, the R171K mutant enzyme produces mannotriose and mannose. The preferred productive binding mode of mannotetraose is shifted from subsite -2 to +2 in the wild-type to subsite -3 to +1 in the R171K mutant. The wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot. Wild-type and mutant enzyme show the ability to perform alcoholysis reactions with methanol and butanol, forming new beta-linked glycoconjugates. It appears that the wild-type enzyme produces mainly mannobiose conjugates using mannotetetraoses substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding | Trichoderma reesei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.05 | - |
mannohexaose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
0.08 | - |
Mannopentaose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
0.25 | - |
4-methylumbelliferyl mannotrioside | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
0.31 | - |
Mannotetraose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
0.34 | - |
mannohexaose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
0.46 | - |
4-methylumbelliferyl mannotrioside | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
0.6 | - |
locust bean gum | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
1.3 | - |
locust bean gum | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
1.7 | - |
Mannopentaose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
10.6 | - |
Mannotetraose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trichoderma reesei | Q99036 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-methylumbelliferyl mannotrioside + H2O | - |
Trichoderma reesei | 4-methylumbelliferone + mannotriose | - |
? | |
locust bean gum + H2O | - |
Trichoderma reesei | ? | - |
? | |
mannohexaose + H2O | - |
Trichoderma reesei | ? | - |
? | |
mannopentaose + H2O | - |
Trichoderma reesei | ? | - |
? | |
mannotetraose + H2O | the wild-type enzyme produces mannobiose as dominant product from mannotetraose. The R171K mutant enzyme produces mannotriose and mannose | Trichoderma reesei | ? | - |
? | |
additional information | the wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot Wild-type and mutant enzyme show the ability to perform alcoholysis reactions with methanol and butanol, forming new beta-linked glycoconjugates. It appears that the wild-type enzyme produces mainly mannobiose conjugates using mannotetetraoses substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding | Trichoderma reesei | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
endo-1,4-beta-D-mannanase | - |
Trichoderma reesei |
TrMan5A | - |
Trichoderma reesei |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
4-methylumbelliferyl mannotrioside | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
0.03 | - |
4-methylumbelliferyl mannotrioside | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
2 | 8 | mannohexaose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
3 | - |
Mannotetraose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
6 | - |
Mannotetraose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
11 | - |
Mannopentaose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
13 | - |
Mannopentaose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
20 | - |
mannohexaose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
240 | - |
locust bean gum | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
260 | - |
locust bean gum | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.02 | - |
4-methylumbelliferyl mannotrioside | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
0.12 | - |
4-methylumbelliferyl mannotrioside | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
0.28 | - |
Mannotetraose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
6.5 | - |
Mannopentaose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
19.4 | - |
Mannotetraose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
82.4 | - |
mannohexaose | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
163 | - |
Mannopentaose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
200 | - |
locust bean gum | pH 40°C, pH 4.5, mutant enzyme R171K | Trichoderma reesei | |
400 | - |
locust bean gum | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei | |
400 | - |
mannohexaose | pH 40°C, pH 4.5, wild-type enzyme | Trichoderma reesei |