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Literature summary for 4.1.2.10 extracted from

  • Nuylert, A.; Ishida, Y.; Asano, Y.
    Effect of glycosylation on the biocatalytic properties of hydroxynitrile lyase from the Passion Fruit, Passiflora edulis A comparison of natural and recombinant enzymes (2017), ChemBioChem, 18, 257-265 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
the enzyme is cloned and expressed in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence Passiflora edulis

Protein Variants

Protein Variants Comment Organism
N105Q the mutant enzyme shows much lower thermostability, pH stability, and organic solvent tolerance than the hydroxynitrile lyase from leaves and that expressed in Pichia pastoris. kcat of the mutant enzyme is 1.3fold lower than the kcat of the wild-type enzyme, kcat/Km of the mutant enzyme is 2.3fold lower than the kcat/Km of the wild type enzyme from leaves Passiflora edulis

General Stability

General Stability Organism
the presence of glycan can prevent the self-degradation of this enzyme at 10°C Passiflora edulis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
11.2
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase from leaves Passiflora edulis
13
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase expressed in Pichia pastoris Passiflora edulis
19.8
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris Passiflora edulis
26.1
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase expressed in Escherichia coli Passiflora edulis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
13000
-
hydroxynitrile lyase expressed in Escherichia coli, SDS-PAGE Passiflora edulis
13000
-
hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris, SDS-PAGE Passiflora edulis
14000
-
hydroxynitrile lyase from leaves, SDS-PAGE Passiflora edulis
14067
-
calculated from sequence, mature protein without signal peptide Passiflora edulis
17000
-
hydroxynitrile lyase expressed in Pichia pastoris, SDS-PAGE Passiflora edulis

Organism

Organism UniProt Comment Textmining
Passiflora edulis A0A1L7NZN4 var. flavicarpa
-

Posttranslational Modification

Posttranslational Modification Comment Organism
glycoprotein hydroxynitrile lyase from leaves and that expressed in Pichia pastoris are glycosylated, whereas that expressed in Escherichia coli is not. Predicted mature enzyme has a N-glycosylation site on the asparagine residue at 105 Passiflora edulis

Purification (Commentary)

Purification (Comment) Organism
-
Passiflora edulis

Source Tissue

Source Tissue Comment Organism Textmining
leaf
-
Passiflora edulis
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
54.1
-
25°C, pH 4, hydroxynitrile lyase expressed in Escherichia coli Passiflora edulis
133.6
-
25°C, pH 4, hydroxynitrile lyase from leaves Passiflora edulis
134
-
25°C, pH 4, hydroxynitrile lyase expressed in Pichia pastoris Passiflora edulis
140
-
25°C, pH 4, hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris Passiflora edulis

Storage Stability

Storage Stability Organism
10°C, 12 h, the stabilities of hydroxynitrile lyase from leaves and hydroxynitrile lyase expressed in Pichia pastoris are comparable, with remaining enzyme activities of 60-94%. The activities of hydroxynitrile lyase mutant N105Q expressed in Pichia pastor and hydroxynitrile lyase expressed in Escherichia coli vary between 70% and 0% in all biphasic systems apart from those with DEE and DIPE. The nonglycosylated hydroxynitrile lyase expressed in Escherichia coli and hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris both lose 50% of their activity in aqueous buffer with shaking at 1500 rpm at 10°C Passiflora edulis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cyanide + benzaldehyde the glycosylated enzyme efficiently performs transcyanation of (R)-mandelonitrile with a 98% enantiomeric excess in a biphasic system with diisopropyl ether Passiflora edulis (R)-mandelonitrile
-
?

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
the hydroxynitrile lyase from leaves and that expressed in Pichia pastoris show much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme expressed in Escherichia coli and the deglycosylated mutant N105Q expressed in Pichia pastoris Passiflora edulis
30 50 1 h, stable, hydroxynitrile lyase from leaves, hydroxynitrile lyase expressed in Pichia pastoris, hydroxynitrile lyase expressed in Escherichia coli, hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris Passiflora edulis
55
-
1 h, 10% loss of activity, hydroxynitrile lyase from leaves and hydroxynitrile lyase expressed in Pichia pastoris Passiflora edulis
60
-
1h, hydroxynitrile lyase from leaves loses 20% of its activity. Hydroxynitrile lyase expressed in Pichia pastoris loses 40% of its activity. Hydroxynitrile lyase expressed in Escherichia coli and hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris completely lose activity Passiflora edulis
70
-
1 h, hydroxynitrile lyase from leaves loses 60% of its activity, Hydroxynitrile lyase expressed in Pichia pastoris, completely loses activity Passiflora edulis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
38.4
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyaseexpressed in Escherichia coli Passiflora edulis
39.7
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris Passiflora edulis
50.9
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase from leaves Passiflora edulis
52.1
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase expressed in Pichia pastoris Passiflora edulis

pH Stability

pH Stability pH Stability Maximum Comment Organism
3.5 10 activities of all the hydroxynitrile lyase are broadly stable over the range from pH 3.5 to 10.0, although the activity of hydroxynitrile lyase expressed in Escherichia coli is reduced to 60% and 70% at pH 7.0 and 10.0, respectively Passiflora edulis

pI Value

Organism Comment pI Value Maximum pI Value
Passiflora edulis calculated from sequence, mature protein without signal peptide
-
5.2

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.47
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase expressed in Escherichia coli Passiflora edulis
2
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase mutant N105Q expressed in Pichia pastoris Passiflora edulis
4
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase expressed in Pichia pastoris Passiflora edulis
4.56
-
benzaldehyde 25°C, pH 4, hydroxynitrile lyase from leaves Passiflora edulis