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Literature summary for 3.2.1.40 extracted from

  • Xu, L.; Liu, X.; Yin, Z.; Liu, Q.; Lu, L.; Xiao, M.
    Site-directed mutagenesis of alpha-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis ba94 (2016), Appl. Microbiol. Biotechnol., 100, 10385-10394 .
    View publication on PubMed

Application

Application Comment Organism
synthesis a wild-type alpha-L-rhamnosidase from Alternaria sp. L1 can synthesize rhamnose-containing chemicals through reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor Alternaria sp. L1

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type enzyme and mutant enzymes in Pichia pastoris Alternaria sp. L1

Protein Variants

Protein Variants Comment Organism
R548A complete loss of reverse hydrolytic activity Alternaria sp. L1
W261A complete loss of reverse hydrolytic activity Alternaria sp. L1
W261Y mutant enzyme with improved product yield in synthesis of rhamnose-containing chemicals through reverse hydrolysis reaction with rhamnose as glycosyl donor. Reverse hydrolysis is accelerated with 43.7% in relative yield compared to the wild-type enzyme. Based on 3D structural modeling, it is supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp257. Mutant W261Y also exhibits a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme Alternaria sp. L1
W555A complete loss of reverse hydrolytic activity Alternaria sp. L1
Y302F mutant enzyme with improved product yield in synthesis of rhamnose-containing chemicals through reverse hydrolysis reaction with rhamnose as glycosyl donor. Reverse hydrolysis is accelerated with 33.3% in relative yield compared to the wild-type enzyme Alternaria sp. L1
Y316F mutant enzyme with improved product yield in synthesis of rhamnose-containing chemicals through reverse hydrolysis reaction with rhamnose as glycosyl donor. Reverse hydrolysis is accelerated with 19.8% in relative yield compared to the wild-type enzyme Alternaria sp. L1

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.69
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme Y316F Alternaria sp. L1
1.31
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme W261Y Alternaria sp. L1
1.47
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, wild-type enzyme Alternaria sp. L1
2.01
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme Y302F Alternaria sp. L1
2.11
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme R548A Alternaria sp. L1
3.12
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme W555A Alternaria sp. L1

Organism

Organism UniProt Comment Textmining
Alternaria sp. L1
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl alpha-L-rhamnopyranoside + H2O
-
Alternaria sp. L1 4-nitrophenol + alpha-L-rhamnopyranose
-
?

Synonyms

Synonyms Comment Organism
RhaL1
-
Alternaria sp. L1

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0246
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme W261Y Alternaria sp. L1
0.0498
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme Y316F Alternaria sp. L1
0.16
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme W555A Alternaria sp. L1
0.55
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme Y302F Alternaria sp. L1
1.77
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme R548A Alternaria sp. L1
3.07
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, wild-type enzyme Alternaria sp. L1

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0188
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme W261Y Alternaria sp. L1
0.0505
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme W555A Alternaria sp. L1
0.0722
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme Y316F Alternaria sp. L1
0.27
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme Y302F Alternaria sp. L1
0.84
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, mutant enzyme R548A Alternaria sp. L1
2.09
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.5, 55°C, wild-type enzyme Alternaria sp. L1