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Information on EC 1.4.1.1 - alanine dehydrogenase and Organism(s) Bacillus subtilis and UniProt Accession Q08352
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1.4.1.1 alanine dehydrogenaseSpecify your search results
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xanthomonas
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demographic
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shower
The taxonomic range for the selected organisms is: Bacillus subtilis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
scald, alanine dehydrogenase, l-alanine dehydrogenase, 40 kda antigen, l-aladh, (s)alanine dehydrogenase, shealadh, af1665, haadh1, rv2780, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
(S)alanine dehydrogenase
-
-
-
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(S)alanine:NAD oxidoreductase
-
-
-
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40 kDa antigen
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-
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ALADH
alanine oxidoreductase
-
-
-
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alpha-alanine dehydrogenase
-
-
-
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dehydrogenase, alanine
-
-
-
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L-alanine dehydrogenase
-
-
-
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NAD-dependent alanine dehydrogenase
-
-
-
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NAD-linked alanine dehydrogenase
-
-
-
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NADH-dependent alanine dehydrogenase
-
-
-
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TB43
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-
-
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ALADH
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
L-alanine + H2O + NAD+ = pyruvate + NH3 + NADH + H+
predominantly ordered kinetic mechanism in which NAD+ adds before L-Ala, and ammonia, pyruvate, and NADH are released in that order
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
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oxidation
-
-
-
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reduction
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-
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oxidative deamination
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-
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reductive amination
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PATHWAY SOURCE
PATHWAYS
BRENDA
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SYSTEMATIC NAME
IUBMB Comments
L-alanine:NAD+ oxidoreductase (deaminating)
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CAS REGISTRY NUMBER
COMMENTARY
9029-06-5
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
3-fluoropyruvate + NH3 + NADH + H+
3-fluoro-L-alanine + H2O + NAD+
-
-
-
-
?
4-methyl-2-oxopentanoate + NH3 + NADH
2-amino-4-methylpentanoate + NAD+ + H2O
-
-
-
-
?
L-Ile + H2O + NAD+
2-oxo-3-methylpentanoate + NH3 + NADH
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5% of the activity with L-Ala
-
-
?
L-Val + H2O + NAD+
3-methyl-2-oxobutanoate + NH3 + NADH
-
9% of the activity with L-Ala
-
-
?
pyruvate + NH3 + NADPH + H+
L-Ala + H2O + NADP+
-
NADPH is a poor cofactor for wild-type
-
-
?
L-2-aminobutanoate + H2O + NAD+
2-oxobutanoate + NH3 + NADH + H+
-
13.3% of the activity with L-Ala
-
r
L-Ala + H2O + NAD+
pyruvate + NH3 + NADH
-
the physiological role is to catabolize L-Ala to pyruvate and NH3, inducible by L-Ala, D-Ala and 11 other D-amino acids. The enzyme catabolizes L-Ala, and thereby limits the amount of L-Ala available to alanine racemase for the synthesis of D-Ala
-
-
?
additional information
?
-
one-pot preparation of D-amino acids through biocatalytic deracemization using alanine dehydrogenase and omega-transaminase. AlaDH produces pyruvate from L-alanine with NAD+, NOX oxidizes NADH to NAD+, and reductive amination of the resulting pyruvate back to the amino acid in an enantiomerically opposite form by D-selective omega-transaminase (omega-TA) using isopropylamine as an amino donor cosubstrate
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-
-
additional information
?
-
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Aspecific enzyme with regard to stereochemistry of the hydrogen transfer to NAD+
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-
?
additional information
?
-
-
the enzyme is required for normal sporulation
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-
?
additional information
?
-
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transfer of hydride is a partially limiting step and the reaction rate is limited by the release of product NADH. The fluorine constituent doesn't cause a significant change in the area of bonds that are being converted, and deuteriated solvent present in the reaction medium only slightly affects the conversion of [E-S] complex into [E-P] complex
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-Ala + H2O + NAD+
pyruvate + NH3 + NADH
-
the physiological role is to catabolize L-Ala to pyruvate and NH3, inducible by L-Ala, D-Ala and 11 other D-amino acids. The enzyme catabolizes L-Ala, and thereby limits the amount of L-Ala available to alanine racemase for the synthesis of D-Ala
-
-
?
additional information
?
-
-
the enzyme is required for normal sporulation
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADH
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12940, 349549, 349555, 349556, 349559, 349560, 349562, 349563, 349568, 349580, 349585, 349587, 349590, 349593, 349597, 349598, 349599, 705545, 742134
NAD+
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-
12940, 349549, 349555, 349556, 349559, 349560, 349562, 349563, 349568, 349580, 349585, 349587, 349590, 349593, 349597, 349598, 349599, 684793
NAD+
-
affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2,4,6-Trinitrobenzenesulfonic acid
-
inactivation follows pseudo first-order kinetics with a 1:1 stoichiometric ratio between the reagent and the enzyme subunit. Partial protection by each of the substrates, NADH or pyruvate. Complete protection only in presence of the ternary complex enzyme-NADH-pyruvate
3-(2-pyridyldithio)propionate
-
inactivation follows pseudo first-order kinetics with a 1:1 stoichiometric ratio between the reagent and the enzyme subunit. Partial protection by each of the substrates, NADH or pyruvate. Complete protection only in presence of the ternary complex enzyme-NADH-pyruvate
5'-(p-(fluorosulfonyl)-benzoyl)adenosine
-
inactivation follows pseudo-first-order kinetics, complete inactivation of the enzyme can not be obtained even at high reagent concentration
Zn2+
-
allosteric competitive inhibitor, reversible, inducing conformational change through the intersubunit interaction, positive cooperative binding of the substrate in presence of Zn2+
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
amination: maximal turnover is about 650000 per min, deamination: maximal turnover is 54000 per min
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45.2
enzymatic activity of AlaDH expressed in Escherichia coli JM109 at 42.1°C
48.3
enzymatic activity of AlaDH expressed in Escherichia coli JM109 at 30.1°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.2 - 10
-
pH 7.2: about 10% of activity maximum, pH 10.0: about 30% of activity maximum, reductive amination
8 - 11
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pH 8: about 35% of activity maximum, pH 11: about 75% of activity maximum, oxidative deamination
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
L-alanine dehydrogenase is a NADH-dependent enzyme that catalyzes the reversible reductive amination of pyruvate using ammonia as amine source
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D196A
-
alterating cofactor specificity from NADH to NADPH, 10fold decrease in activity with NADH, 4fold increase in activity with NADPH
D196A/L197R
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alterating cofactor specificity from NADH to NADPH, almost the same activity with NADPH as the wild-type enzyme for NADH
D196A/L197R/N198S/R201A
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alterating cofactor specificity from NADH to NADPH, loss of activity with NADH, 5fold increase in activity with NADPH
D196A/L197R/R201A
-
alterating cofactor specificity from NADH to NADPH, loss of activity with NADH, 2fold increase in activity with NADPH
additional information
additional information
biocatalytic deracemization of aliphatic amino acids into D-enantiomers by running cascade reactions: (1) stereoinversion of L-alanine to a D-form by L-alanine dehydrogenase and omega-transaminase and (2) regeneration of NAD+ by NADH oxidase. Under the cascade reaction conditions containing 100 mM isopropylamine and 1 mM NAD+, complete deracemization of 100 mM DL-alanine is achieved after 24 h with 95% reaction yield of D-alanine (over 99% enantiomeric excess, 52% isolation yield). AlaDH produces pyruvate from L-alanine with NAD+, NOX oxidizes NADH to NAD+, and reductive amination of the resulting pyruvate back to the amino acid in an enantiomerically opposite form by D-selective omega-transaminase (omega-TA) using isopropylamine as an amino donor cosubstrate. Method evaluation, overview
additional information
the purified enzyme is immobilized on porous agarose microbeads activated with glyoxyl groups (aliphatic aldehydes). 85% of the offered enzyme is immobilized on these microbeads and the enzyme recovers 45% of its initial reduction amination activity upon the multivalent and irreversible attachment. The immobilized enzyme shows 13fold increased thermostability compared to the soluble enzyme. The optimally immobilized enzyme is also stabilized against acidic pH. The enzyme works as heterogeneous biocatalyst for the synthesis of L-[13N]alanine using pyruvate and [13N]NH4OH obtaining a radiochemical yield of over 95% in 20 minutes. This immobilized enzyme is reused for up to 5 cycles keeping the maximum yield. Immobilization method optimization, overview
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
63
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, PEG-succinimide-immobilized or PEG-p-nitrophenylchloroformate-immobilized AlaDH in 50 mM phosphate buffer, 15 days, no loss of activity
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
the GST-AlaDH fusion protein is released by cleaving with PreScission protease
recombinant GST-tagged enzyme from Escherichia coli strain JM109 by glutathione affinity chrmatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
AlaDH gene is cloned into a pGEX6p-1 and expressed in Escherichia coli JM109. AlaDH gene is then cloned intoa vector pNZ8148 to generate a vector pNZ8148/ald. The same AlaDH gene is placed downstream of the lactate dehydrogenase (ldh) promoter from Streptococcus thermophilus to generate pNZ273/ldhp/AlaDH. The pNZ8148/AlaDH and pNZ273/ldhp/AlaDH are introduced separately in Lactococcus lactis NZ9000. As a result of over-expressed AlaDH, the production of alanine detected by HPLC in Lactococcus lactis NZ9000 carrying pNZ273/ldhp/ald reaches 52 mg/ml, an approximately 26fold increase compared to the parent strain Lactococcus lactis NZ9000, but not in Lactococcus lactis NZ9000 carrying pNZ8148/ALaDH
gene AlaDH, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
gene ald, functional heterologous expression in Escherichia coli strain JM109, as GST-tagged protein, and in Lactococcus lactis strain NZ9000
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
heterologous expression of the Bacillus subtilis AlaDH in Lactococcus lactis using the promoter of lactate dehydrogenase from Streptococcus thermophilus leads to a better alanine production in the recombinant strain
synthesis
the enzyme can be used as heterogeneous biocatalyst for reductive aminations of 2-oxoacids
nutrition
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Lactococcus lactis strain NZ9000 expressing the enzyme from Bacillus subtilis can be used in imporvement of dairy fermentation for developing healthy yogurts with sweet taste or other fermented dairy foods
synthesis
-
alterating of cofactor specificity from NADH to NADPH via protein engineering for application as a regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases. Mutant D196A/L197R can be applied in a coupled oxidation/transamination reaction of the dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans omega-aminotransferase, allowing recycling of NADP+ and L-Ala. Recycling factors of 33 for NADP+ and 13 for L-Ala are observed
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nitta, Y.; Yasuda, Y.; Tochikubo, K.; Hachisuka, Y.
L-Amino acid dehydrogenases in Bacillus subtilis spores
J. Bacteriol.
117
588-592
1974
Bacillus subtilis
Weiss, P.M.; Chen, C.Y.; Cleland, W.W.; Cook, P.F.
Use of primary deuterium and 15N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases
Biochemistry
27
4814-4822
1988
Bacillus subtilis
Yoshida, A.; Freese, E.
Enzymic properties of Alanine dehydrogenase of Bacillus subtilis
Biochim. Biophys. Acta
96
248-262
1965
Bacillus subtilis
Itoh, N.; Morikawa, R.; Itoh, N.; Morikawa, R.
Crystallization and properties of L-alanine dehydrogenase from Streptomyces phaeochromogenes
Agric. Biol. Chem.
47
2511-2519
1983
Streptomyces phaeochromogenes, Bacillus subtilis, Lysinibacillus sphaericus, Streptomyces flaveus, Micromonospora melanosporea, Streptomyces bobili, Streptomyces olivochromogenes, Streptomyces roseochromogenus, Kitasatospora albolonga, Streptomyces albus, Streptomyces aureus, Streptomyces caespitosus, Streptomyces clavuligerus, Streptomyces coelicolor, Streptomyces griseoluteus, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces lydicus, Streptomyces roseus, Streptomyces ruber
-
Grimshaw, C.E.; Cleland, W.W.
Kinetic mechanism of Bacillus subtilis L-alanine dehydrogenase
Biochemistry
20
5650-5655
1981
Bacillus subtilis
Grimshaw, C.E.; Cook, P.F.; Cleland, W.W.
Use of isotope effects and pH studies to determine the chemical mechanism of Bacillus subtilis L-alanine dehydrogenase
Biochemistry
20
5655-5661
1981
Bacillus subtilis
Alizade, M.A.; Bressler, R.; Brendel, K.
Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis
Biochim. Biophys. Acta
397
5-8
1975
Bacillus subtilis
Sawa, Y.; Tani, M.; Murata, K.; Shibata, H.; Ochiai, H.
Purification and characterization of alanine dehydrogenase from a cyanobacterium, Phormidium lapideum
J. Biochem.
116
995-1000
1994
Bacillus subtilis, Phormidium lapideum
Vali, Z.; Kilar, F.; Lakatos, S.; Venyaminov, S.A.; Zavodszky, P.
L-Alanine dehydrogenase from Thermus thermophilus
Biochim. Biophys. Acta
615
34-47
1980
Bacillus subtilis, Thermus thermophilus
Keradjopoulos, D.; Holldorf, A.W.
Thermophilic character of enzymes from extreme halophilic bacteria
FEMS Microbiol. Lett.
1
179-182
1977
Bacillus cereus, Bacillus subtilis, Halobacterium salinarum, Bacillus cereus T
-
Williamson, D.H.
L-Alanin
Methods Enzym. Anal. , 3rd Ed. (Bergmeyer, H. U. , ed. )
2
1724-1727
1974
Bacillus subtilis
-
Delforge, D.; Devreese, B.; Dieu, M.; Delaive, E.; Van Beeumen, J.; Remacle, J.
Identification of lysine 74 in the pyruvate binding site of alanine dehydrogenase from Bacillus subtilis. Chemical modification with 2,4,6-trinitrobenzenesulfonic acid, n-succinimidyl 3-(2-pyridyldithio)propionate, and 5'-(p-(fluorosulfonyl)benzoyl)adenosine
J. Biol. Chem.
272
2276-2284
1997
Bacillus subtilis
Kim, S.J.; Lee, W.Y.; Kim, K.H.
Unusual allosteric property of L-alanine dehydrogenase from Bacillus subtilis
J. Biochem. Mol. Biol.
31
25-30
1998
Bacillus subtilis
-
Galkin, A.; Kulakova, L.; Ashida, H.; Sawa, Y.; Esaki, N.
Cold-adapted alanine dehydrogenases from two antarctic bacterial strains: gene cloning, protein characterization, and comparison with mesophilic and thermophilic counterparts
Appl. Environ. Microbiol.
65
4014-4020
1999
Vibrio proteolyticus, Geobacillus stearothermophilus, Bacillus subtilis, Carnobacterium sp., Shewanella sp., Carnobacterium sp. St2, Shewanella sp. Ac10
Siranosian, Kathryn Jaacks; Ireton, Keith; Grossman, Alan D.
Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis
J. Bacteriol.
175
6789-6796
1993
Bacillus subtilis
Berberich, Robert; Kaback, Michael; Freese, Ernst.
D-Amino acids as inducers of L-alanine dehydrogenase in Bacillus subtilis
J. Biol. Chem.
243
1008-1013
1968
Bacillus subtilis
-
Brunhuber, N.M.W.; Blanchard, J.S.
The biochemistry and enzymology of amino acid dehydrogenases
Crit. Rev. Biochem. Mol. Biol.
29
415-467
1994
Anabaena cylindrica, Geobacillus stearothermophilus, Bacillus cereus, Bacillus subtilis, Bacillus japonicum, Bacillus licheniformis, Lysinibacillus sphaericus, Thermus thermophilus, Halobacterium salinarum, Mycobacterium tuberculosis, Pseudomonas sp., Kitasatospora aureofaciens
Forde, J.; Oakey, L.; Jennings, L.; Mulcahy, P.
Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies
Anal. Biochem.
338
102-112
2005
Bacillus subtilis
Baysal, S.H.; Uslan, A.H.; Pala, H.H.; Tuncoku, O.
Encapsulation of PEG-urease/PEG-AlaDH within sheep erythrocytes and determination of the systems activity in lowering blood levels of urea in animal models
Artif. Cells Blood Substit. Immobil. Biotechnol.
35
391-403
2007
Bacillus subtilis
Ye, W.; Huo, G.; Chen, J.; Liu, F.; Yin, J.; Yang, L.; Ma, X.
Heterologous expression of the Bacillus subtilis (natto) alanine dehydrogenase in Escherichia coli and Lactococcus lactis
Microbiol. Res.
165
268-275
2009
Bacillus subtilis, no activity in Lactococcus lactis, Bacillus subtilis natto
Ye, W.; Huo, G.; Chen, J.; Liu, F.; Yin, J.; Yang, L.; Ma, X.
Heterologous expression of the Bacillus subtilis (natto) alanine dehydrogenase in Escherichia coli and Lactococcus lactis
Microbiol. Res.
165
268-775
2009
Bacillus subtilis (Q08352), Bacillus subtilis
Szymanska-Majchrzak, J.; Palka, K.; Kanska, M.
Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase
Appl. Radiat. Isot.
123
21-25
2017
Bacillus subtilis
Lerchner, A.; Jarasch, A.; Skerra, A.
Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity
Biotechnol. Appl. Biochem.
63
616-624
2016
Bacillus subtilis
Han, S.; Shin, J.
One-pot preparation of D-amino acids through biocatalytic deracemization using alanine dehydrogenase and omega-transaminase
Catal. Lett.
148
3678-3684
2018
Bacillus subtilis (Q08352), Bacillus subtilis 168 (Q08352)
-
da Silva, E.; Gomez-Vallejo, V.; Llop, J.; Lopez-Gallego, F.
Structural, kinetic and operational characterization of an immobilized L-aminoacid dehydrogenase
Process Biochem.
57
80-86
2017
Bacillus subtilis (Q08352), Bacillus subtilis 168 (Q08352)
-
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