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Literature summary extracted from

  • Ge, L.; Li, D.; Wu, T.; Zhao, L.; Ding, G.; Wang, Z.; Xiao, W.
    B-factor-saturation mutagenesis as a strategy to increase the thermostability of alpha-L-rhamnosidase from Aspergillus terreus (2018), J. Biotechnol., 275, 17-23 .
    View publication on PubMed

Application

EC Number Application Comment Organism
3.2.1.40 food industry alpha-L-rhamnosidase is an important enzyme with applications in the food industries because it can release terminal L-rhamnose residues from various natural products. The D594Q and G827K/D594Q mutant enzymes are more suitable for the industrial processes of isoquercitrin preparation than the wild-type enzyme Aspergillus terreus
3.2.1.40 pharmacology alpha-L-rhamnosidase is an important enzyme with applications in the pharmaceutical industries because it can release terminal L-rhamnose residues from various natural products. The D594Q and G827K/D594Q mutant enzymes are more suitable for the industrial processes of isoquercitrin preparation than the wild-type enzyme Aspergillus terreus

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.40 D594C increased thermal stability at 70°C Aspergillus terreus
3.2.1.40 D594P strongly decreased thermal stability at 70°C Aspergillus terreus
3.2.1.40 D594Q half-life at 70°C is prolonged by 2.1fold. Analysis of the 3D structure shows that in the thermostable variants the number of hydrogen bonds and salt bridges is increased, explaining the enhanced thermostability. The KM value for 4-nitrophenyl-alpha-L-rhamnopyranoside decreases by 4.0%. The kcat/KM value increases by 15.5%. The mutant enzyme exhibits markedly improved isoquercitrin yield at 70°C that increases by 13.5% Aspergillus terreus
3.2.1.40 D594Q/G827K half-life at 70°C is prolonged by 2.3fold. Analysis of the 3D structure shows that in the thermostable variants the number of hydrogen bonds and salt bridges is increased, explaining the enhanced thermostability. The KM value for 4-nitrophenyl-alpha-L-rhamnopyranoside decreases by 3.8%. The kcat/KM value increases by 9.2%. The mutant enzyme exhibits markedly improved isoquercitrin yield at 70°C that increases by 11.0% Aspergillus terreus
3.2.1.40 G827I slightly increased thermal stability at 70°C Aspergillus terreus
3.2.1.40 G827K increased thermal stability at 70°C Aspergillus terreus
3.2.1.40 G827L increased thermal stability at 70°C Aspergillus terreus
3.2.1.40 G827M increased thermal stability at 70°C Aspergillus terreus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.40 0.457
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, mutant enzyme D594Q Aspergillus terreus
3.2.1.40 0.458
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, mutant enzyme D594Q/G827K Aspergillus terreus
3.2.1.40 0.476
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, wild-type enzyme Aspergillus terreus

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.40 Aspergillus terreus I0AZ41
-
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.2.1.40 454
-
 substrate: 4-nitrophenyl alpha-L-rhamnopyranoside, pH 6.5, 65°C, wild-type enzyme Aspergillus terreus
3.2.1.40 486
-
 substrate: 4-nitrophenyl alpha-L-rhamnopyranoside, pH 6.5, 65°C, mutant enzyme D594Q/G827K Aspergillus terreus
3.2.1.40 504
-
 substrate: 4-nitrophenyl alpha-L-rhamnopyranoside, pH 6.5, 65°C, mutant enzyme D594Q Aspergillus terreus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.40 4-nitrophenyl alpha-L-rhamnopyranoside + H2O
-
 Aspergillus terreus 4-nitrophenol + alpha-L-rhamnopyranose
-
 ?
3.2.1.40 rutin + H2O the yield of isoquercitrin for the wild-type enzyme reached the maximum at 4 h. However, the yield of isoquercitrin for the mutants D594Q and G827K/D594Q enzyme reached the maximum at 6 h. The D594Q and G827K/D594Q mutant enzymes produce 13.5% and 11.0% more isoquercitrin than the wild-type, respectively Aspergillus terreus isoquercitrin + alpha-L-rhamnose
-
 ?

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.40 65
-
 wild-type enzyme, mutant enzyme D594Q and mutant enzyme G827K/D594Q Aspergillus terreus

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.2.1.40 65 75 65°C: temperature-optimum of wild-type enzyme and mutant enzymes D594Q and G827K/D594Q. At 70 °C, the wild type enzyme retains 88.4% of its maximum activity, whereas the D594Q and G827K/D594Q mutant enzymes retain 96.6% and 98.5% of their maximum activities, respectively. At 75°C, the wild-type enzyme retains 48.5% of its maximum activity, whereas the D594Q and G827K/D594Q mutant enzymes retain 71.2% and 81.1% of their maximum activities, respectively Aspergillus terreus

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.2.1.40 70
-
 half-life: 19 min (wild-type enzyme), 39.8 min (mutant enzyme D594Q), 43.1 min (mutant enzyme D594Q/G827K) Aspergillus terreus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.40 412
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, wild-type enzyme Aspergillus terreus
3.2.1.40 434
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, mutant enzyme D594Q/G827K Aspergillus terreus
3.2.1.40 458
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, mutant enzyme D594Q Aspergillus terreus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.40 6.5
-
 wild-type enzyme, mutant enzyme D594Q and mutant enzyme G827K/D594Q Aspergillus terreus

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.2.1.40 5 7.5 wild-type enzyme and mutant enzymes D594Q and G827K/D594Q retain more than 90% relative activity over the pH range 5.0-7.5 Aspergillus terreus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.40 867
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, wild-type enzyme Aspergillus terreus
3.2.1.40 947
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, mutant enzyme D594Q/G827K Aspergillus terreus
3.2.1.40 1001
-
 4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 65°C, mutant enzyme D594Q Aspergillus terreus