Information on EC 4.1.2.38 - benzoin aldolase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.1.2.38
-
RECOMMENDED NAME
GeneOntology No.
benzoin aldolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-hydroxy-1,2-diphenylethanone = 2 benzaldehyde
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
reversal of an aldol condensation
SYSTEMATIC NAME
IUBMB Comments
2-hydroxy-1,2-diphenylethanone benzaldehyde-lyase (benzaldehyde-forming)
A thiamine-diphosphate protein.
CAS REGISTRY NUMBER
COMMENTARY hide
122097-01-2
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(E)-2-oxo-4(pyridin-3-yl)-3-but-3-enoic acid
(E)-3-(pyridine-3-yl)acrylaldehyde
show the reaction diagram
-
-
-
?
(R)-benzoin
benzaldehyde + benzaldehyde
show the reaction diagram
2-furaldehyde + 2-furaldehyde
(R)-2,2'-furoin
show the reaction diagram
-
-
i.e. (R)-1,2-di(furan-2-yl)-2-hydroxyketone
-
?
2-furaldehyde + 2-furaldehyde
2,2'-furoin
show the reaction diagram
-
-
-
-
r
2-hydroxy-1,2-diphenylethanone
benzaldehyde
show the reaction diagram
2-hydroxypropiophenone
?
show the reaction diagram
-
-
-
-
?
3,5-dimethoxy-benzaldehyde
(R)-3,3',5,5'-tetramethoxy-benzoin
show the reaction diagram
-
-
-
-
?
3,5-dimethoxy-benzaldehyde + 3,5-dimethoxy-benzaldehyde
(R)-3,3',5,5'-tetramethoxy-benzoin
show the reaction diagram
-
-
-
-
?
3,5-dimethoxybenzaldehyde + 3,5-dimethoxybenzaldehyde
3,3',5,5'-tetramethoxybenzoin
show the reaction diagram
-
-
-
-
r
3-chlorobenzaldehyde + acetaldehyde
(R)-(3-chlorophenyl)-2-hydroxy-1-propanone
show the reaction diagram
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-
-
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r
3-ethoxybenzaldehyde
(R)-1,2-di(3-ethoxyphenyl)-2-hydroxyethanone
show the reaction diagram
-
-
-
-
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3-methoxybenzaldehyde
(R)-3,3'-dimethoxybenzoin
show the reaction diagram
-
-
-
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r
3-methoxybenzaldehyde + 2-chlorobenzaldehyde
(R)-3-methoxy-2'-chlorobenzoin
show the reaction diagram
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-
-
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r
3-methylbenzaldehyde
(R)-2-hydroxy-1,2-di(3-tolyl)-ethanone
show the reaction diagram
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-
-
-
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4-ethoxybenzaldehyde
(R)-1,2-di(4-ethoxyphenyl)-2-hydroxyethanone
show the reaction diagram
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-
-
-
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4-methoxybenzaldehyde
(R)-1,2-di-(4-methoxy-phenyl)-2-hydroxy-ethan-1-one
show the reaction diagram
-
-
-
-
?
acetaldehyde
(R)-acetoin
show the reaction diagram
-
-
-
-
?
acetaldehyde + 3-chlorobenzaldehyde
(R)-1-(3-chloro-phenyl)-2-hydroxy-propan-1-one
show the reaction diagram
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-
-
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r
anisoin
4-methoxybenzaldehyde + 4-methoxybenzaldehyde
show the reaction diagram
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-
-
-
?
benzaldehyde + (R)-2,2-dimethyl-1,3-dioxolan-4-carbaldehyde
acyloin + benzoin
show the reaction diagram
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-
-
-
?
benzaldehyde + 2,2-dimethoxyacetaldehyde
3,3-dimethoxy-2-hydroxy-1-phenyl-propanone
show the reaction diagram
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-
-
-
?
benzaldehyde + 2-methylbutanal
(2R,3S)-2-hydroxy-3-methyl-1-phenylpentan-1-one + 1-hydroxy-3-methyl-1-phenylpentan-2-one
show the reaction diagram
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63% (2R,3S)-2-hydroxy-3-methyl-1-phenylpentan-1-one, 85% syn product, plus 37% 1-hydroxy-3-methyl-1-phenylpentan-2-one, 40% syn product
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?
benzaldehyde + 2-methylpentanal
(2R,3S)-2-hydroxy-3-methyl-1-phenylhexan-1-one + 1-hydroxy-3-methyl-1-phenylhexan-2-one
show the reaction diagram
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50% (2R,3S)-2-hydroxy-3-methyl-1-phenylhexan-1-one, more than 99% syn poduct, plus 50% 1-hydroxy-3-methyl-1-phenylhexan-2-one
-
?
benzaldehyde + 2-methylpropanal
(2R)-2-hydroxy-3-methyl-1-phenylbutan-1-one + 1-hydroxy-3-methyl-1-phenylbutan-2-one
show the reaction diagram
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50% (2R)-2-hydroxy-3-methyl-1-phenylbutan-1-one, more than 99% enantiomeric excess, plus 50% 1-hydroxy-3-methyl-1-phenylbutan-2-one, 93% enantiomeric excess
-
?
benzaldehyde + acetaldehyde
(R)-2-hydroxy-1-phenyl-propanone
show the reaction diagram
benzaldehyde + acetaldehyde
(R)-2-hydroxy-1-phenylpropanone
show the reaction diagram
benzaldehyde + acetaldehyde
2-hydroxy-1-phenylpropanone
show the reaction diagram
-
-
-
-
?
benzaldehyde + benzaldehyde
(R)-benzoin
show the reaction diagram
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immobilized BAL mediated reaction, conversion is low
i.e. 2-hydroxy-1,2-diphenylethan-1-one
-
?
benzaldehyde + benzaldehyde
benzoin
show the reaction diagram
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-
-
-
?
benzaldehyde + dimethoxy acetaldehyde
(R)-2-hydroxy-3,3-dimethoxy-1-phenylpropane-1-one
show the reaction diagram
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-
-
-
?
benzaldehyde + methoxy acetaldehyde
(R)-2-hydroxy-3-methoxy-1-phenylpropane-1-one
show the reaction diagram
-
-
-
-
?
benzofuran-2-carbaldehyde
(R)-1,2-di(benzo-furan-2-yl)-2-hydroxyethanone
show the reaction diagram
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-
-
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benzoin
benzaldehyde + benzaldehyde
show the reaction diagram
Benzoylformate
?
show the reaction diagram
-
-
-
-
?
benzoylformic acid
?
show the reaction diagram
-
-
-
?
cyclohexylaldehyde + acetaldehyde
1-cyclohexyl 2-hydroxypropanone
show the reaction diagram
-
-
-
-
?
dimethoxy acetaldehyde + benzaldehyde
(R)-2-hydroxy-3,3-dimethoxy-1-phenyl-propan-1-one
show the reaction diagram
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-
-
-
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furan-2-aldehyde + 2,2-dimethoxyacetaldehyde
3,3-dimethoxy-2-hydroxy-1-furan-2-yl-propanone
show the reaction diagram
-
-
-
-
?
furan-3-carbaldehyde
(R)-1,2-di(3-furanyl)-2-hydroxyethanone
show the reaction diagram
-
-
-
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-
furan-3-carbaldehyde + furan-3-carbaldehyde
(R)-1,2-di(3-furanyl)-2-hydroxyethanone
show the reaction diagram
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-
-
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furan-carbaldehyde
(R)-1,2-difuryl-2-hydroxy-ethan-1-one
show the reaction diagram
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-
-
-
?
isovaleraldehyde
(R)-5-hydroxy-2,7-dimethyloctan-4-one
show the reaction diagram
-
-
-
-
?
methoxy acetaldehyde + 4-methoxybenzaldehyde
(R)-2-hydroxy-3-methoxy-1-(4-methoxy-phenyl)-propan-1-one
show the reaction diagram
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-
-
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r
methyl benzoylphosphonate
?
show the reaction diagram
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-
-
-
?
n-butanal
(R)-5-hydroxyoctan-4-one
show the reaction diagram
-
-
-
-
?
n-pentanal
(R)-6-hydroxydecan-5-one
show the reaction diagram
-
-
-
-
?
p-anisaldehyde + dimethoxy acetaldehyde
(R)-2-hydroxy-3,3-dimethoxy-1-(4-methoxyphenyl)propan-1-one
show the reaction diagram
-
immobilized BAL mediated reaction
-
-
?
phenylpyruvic acid
?
show the reaction diagram
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-
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?
propanal
4-hydroxyhexan-3-one
show the reaction diagram
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-
-
-
?
propanal + propanal
(S)-propioin
show the reaction diagram
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-
-
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?
thiophene-3-carbaldehyde
(R)-2-hydroxy-1,2-di(3-thienyl)ethanone
show the reaction diagram
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-
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additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-hydroxy-1,2-diphenylethanone
benzaldehyde
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thiamine diphosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
-
less effective than Mg2+ or Mn2+ or Ca2+
Ca2+
-
1 mM, absolute requirement of a divalent cation, may be substituted by Mn2+ or Mg2+
Cu2+
-
less effective than Mg2+ or Mn2+ or Ca2+
Mn2+
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1 mM, absolute requirement for a divalent cation, may be substituted by Mg2+ or Ca2+
additional information
-
Co2+, Zn2+, Ni2+, Fe2+, Al2+ at 1 mM are ineffective. KCl, NaCl, NH4Cl at 1-160 mM: no influence
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-Acetoxy-1,2-bis(4'-methoxyphenyl)-ethanone
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0.2 mM: 50% inhibition
Benzoin
-
product inhibition
Desoxyanisoin
-
0.4 mM: 50% inhibition
desoxybenzoin
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0.9 mM: 50% inhibition
Mg2+
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inhibitory above 7.5 mM
propanal
thiamine diphosphate
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more than 0.5 mM: inhibition, requirement for 0.01 mM
Urea
-
4 mM: inactivation
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
benzaldehyde
-
no activity with 200 mg m/L benzaldehyde. Reaction conducted with 50 mg benzaldehyde results in a 90% conversion after 45 min
dimethyl sulfoxide
-
highest activity between 20% and 30% dimethyl sulfoxide (v/v) with a peak at 25%. Records of pH-activity profiles at different dimethyl sulfoxide concentrations and constant ionic strength (100 mM) reveals a strong influence of the cosolvent on the activity of BAL, and indicates a cross effect on the optimal pH with a shift of profiles to the alkaline milieu. Correlation between the dependency on pH and dimethyl sulfoxide concentration is due to interaction of dimethyl sulfoxide with the amino acid side chain Glu50 in the catalytic site of the biocatalyst
galactose
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isopropyl-beta-D-thiogalactopyranoside addition is not required to induce recombinant protein production as galactose, one of the monomers of trisaccharide raffinose present in the beet molasses (1.2%), induces the lac promoter
thiamine diphosphate
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the cofactor is fixed at the two ends in separate domains, suspending a comparatively mobile thiazolium ring between them
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.975 - 1.205
(R)-3,3',5,5'-tetramethoxy-benzoin
0.06
(R)-3,3'-dimethoxybenzoin
-
in 50 mM potassium phosphate buffer (pH 8), containing 0.5 mM thiamine diphosphate and 0.5 mM MgCl2
0.3 - 5
2-hydroxypropiophenone
0.005 - 0.05
3,5-dimethoxy-benzaldehyde
2.1
3-Methoxybenzaldehyde
-
in 50 mM potassium phosphate buffer (pH 8), containing 0.5 mM thiamine diphosphate and 0.5 mM MgCl2
6
acetaldehyde
-
in TEA buffer (35 mM, pH 8) containing 0.35 mM thiamine diphosphate, 0.35 mM MgSO4, and 30% (v/v) DMSO, at 20C
0.0325
Anisoin
-
37C, pH 8
2.2 - 10
benzaldehyde
0.009 - 0.2
Benzoin
415
propanal
-
in aqueous solution
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6 - 4.9
2-hydroxypropiophenone
45.44 - 46.15
3,5-dimethoxy-benzaldehyde
6.6 - 66.9
Benzoin
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2
2-Acetoxy-1,2-bis(4'-methoxyphenyl)-ethanone
-
37C, pH 8
0.4
Desoxyanisoin
-
37C, pH 8
0.9
desoxybenzoin
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37C, pH 8
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.22
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enzyme-polyelectrolyte multilayer capsules
1.09
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propanal carboligation
10.9
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native enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8.5
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broad
8.5
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activity assay
9
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highest carboligation activity
9.5
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highest carboligation activity, 10fold increase in activity compared to pH 7.0
additional information
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increase of the optimal pH of BAL with increasing concentrations of dimethyl sulfoxide
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
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no activity at pH 6 and below, maximal activity at pH 7.5-8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
activity assay
25
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activity assay
30
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activity assay
35
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maximal activity for the benzoin lyase reaction
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53000
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x * 53000, SDS-PAGE
54000
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4 * 54000, gel filtration
58920
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monomer, calculated from sequence
59800
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His-tagged BAL subunit, SDS-PAGE and MALDI-TOF-MS
80000
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gel filtration
216000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 53000, SDS-PAGE
homotetramer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cocrystallization of mutant A28S with inhibitors methyl benzoylphosphonate and benzoylphosphonate. The introduced serine residue of the mutant is phosphorylated by benzoylphosphonate
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hanging drop vapour diffusion method, structure of the recombinant seleno-L-methionine-labelled enzyme with bound cofactor thiamine diphosphate at 2.6 A resolution
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in 0.1 M Tris-HCl pH 7.5, 0.2 M potassium isothiocyanate, 10% (w/v) polyethylene glycol 1000, and 10% polyethylene glycol 8000
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the structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate is determined to 2.49 A
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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in potassium phosphate, Tris, and imidazole buffers, pH values below 6 lead to complete inactivation after about one day, while the enzyme shows some activity when kept above pH 8
678578
7
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maximum of enzyme stability is achieved at pH 7
682234
10
-
sharp decrease in activity above pH 10
681480
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
a concentration of 1 mM MgSO4 and 0.01 mM thiamine diphosphate is sufficient to keep BAL stable at 25C
30 - 40
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at 30 and 40C in aqueous solution, the relative activity increases slightly during the first measurements, and after that the decrease is very low. At 50C, a strong exponential decrease is observed. No significant differences of half-life between 30C and 40C with 16 and 18 h, respectively, whereas at 50C, a strong decrease of the half-life is observed with 0.4 h
37
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reaction temperature shall not exceed 37C, in the presence of 5 mM MgSO4 and 0.1 mM thiamine diphosphate the enzyme's activity is reduced by only 15% over 3 h at 37C, however, a concentration of 1 mM MgSO4 and 0.01 mM thiamine diphosphate leads to more than 40% inactivation within 3 h at 37C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme entrapped in polyvinyl alcohol and suspended in hexane is stable in hexane for 1 week at 4C and for more than 1 month at -20C
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the half-life of BAL in the presence of the investigated solvents, but without formation of distinct solvent phases is 17.3 h, 5.6 h and 3.2 h for saturation with methyl-tert-butylether, 2-octanone, and toluene, respectively, in non-reactive monophasic media. With MTBE, 2-octanone, and toluene, very similar half-lives of 1.5 h, 1.4 h and 2.0 h are found for unmodified BAL under process conditions in reactive biphasic media. The initial activity of BAL decreases to approximately 60%, when methoxy-poly(ethylene) glycol (mPEG)750 is attached, and to 70%, when a longer-chain mPEG2000 is used. Overall, the modification with mPEG leads to good stabilization of BAL under non-reactive conditions. The strongest effect is achieved against toluene, where the stability of mPEG2000-BAL surpasses the stability of unmodified BAL by factor 18
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1-octanol
-
log P value 3.0, half life 60 h
cyclohexane
-
log P value 2.7, half life 10 h
dibutyl ether
-
log P value 1.4, half life 670 h
dichloromethane
-
log P value 1.5, half life 3 h
diethyl ether
-
log P value 0.8, half life 240 h
diisopropyl ether
-
log P value 2.6, half life 50 h
dithiothreitol
-
positive effect on the enzyme stability
Ethyl acetate
-
log P value 0.7, half life 12 h
heptane
-
log P value 3.3, half life 5 h
isohexane
-
log P value 3.9, half life 15 h
tert-butyl methyl ether
-
log P value 1.0, half life 750 h
tetrahydrofuran
-
log P value 0.4, half life 0 h
toluene
-
log P value 2.3, half life 1 h
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, purified enzyme, stable for at least 2 months
-
30C, in cofactor-free potassium phosphate buffer at pH 7.0, 3 h, 65% loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by Ni2+-NTA affinity column, to purity
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immobilized metal ion chromatography, after which the enzyme shows only 35-45% of its activity, however, activity is completely regained after incubation in the presence of Mg2+ and thiamine diphosphate
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Ni-Sepharose column chromatography
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nickel-affinity HiTrap agarose column chromatography and Sephadex S-200 gel filtration
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strain biovar I, partial
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using Ni2+-affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed from pRSETA::balHIS in Escherichia coli BL21(DE3)pLySs
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expressed in Escherichia coli
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expressed in Escherichia coli BL21 (DE3) cells
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expressed in Escherichia coli BL21(DE3)pLySs carrying pRSETA::balHIS
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expressed in Escherichia coli BL21(DE3)pLysS containing the pUC19-BAL-HIS construct
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expressed in Escherichia coli from SG13009prep4 (pBAL-His6)
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expressed in Escherichia coli SG13009 cells
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expressed in Escherichia coli SG13009 pRep4 cells
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expressed in Escherichia coli strain K12 (ATCC 10798)
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expressed in Escherichia coli strain SG13009
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expressed in Escherichia coli strain SG13009 containing plasmid pBALhis6
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expression in Escherichia coli BL21 (DE3) pLySs and Escherichia coli JM109 carrying pRSETA::bal. Expression in Escherichia coli K12, Escherichia coli BL21 (DE3) pLySs and Escherichia coli JM109 carrying modified pUC18::bal
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expression in Escherichia coli cells
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expression in Escherichia coli; expression in Pseudomonas putida
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for expression in Escherichia coli cells
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the plasmid pKK-BAL-His is constructed for expression of wild-type BAL in Escherichia coli BL21DE3pLysS cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A480I
-
reduced lyase activity
F484I
-
reduced lyase activity
H286A
-
reduced lyase activity
H29A
-
the role of His29 in cofactor activation and catalysis is delineated
W163A
-
the role of Trp163 in cofactor activation and catalysis is delineated
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis