Literature summary for 3.2.1.40 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 .

Search for more literature for 3.2.1.40

Application

Application	Comment	Organism
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
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

Protein Variants	Comment	Organism
D594C	increased thermal stability at 70°C	Aspergillus terreus
D594P	strongly decreased thermal stability at 70°C	Aspergillus terreus
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
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
G827I	slightly increased thermal stability at 70°C	Aspergillus terreus
G827K	increased thermal stability at 70°C	Aspergillus terreus
G827L	increased thermal stability at 70°C	Aspergillus terreus
G827M	increased thermal stability at 70°C	Aspergillus terreus

KM Value [mM]

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

Organism

Organism	UniProt	Comment	Textmining
Aspergillus terreus	I0AZ41	-	-

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
4-nitrophenyl alpha-L-rhamnopyranoside + H2O	-	Aspergillus terreus	4-nitrophenol + alpha-L-rhamnopyranose	-	?
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]

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

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
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]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
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]

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

pH Optimum

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

pH Range

pH Minimum	pH Maximum	Comment	Organism
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]

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

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Release 2023.1 (January 2023)