Literature summary for 5.3.1.4 extracted from

Xu, Z.; Li, S.; Feng, X.; Zhan, Y.; Xu, H.
Function of aspartic acid residues in optimum pH control of L-arabinose isomerase from Lactobacillus fermentum (2014), Appl. Microbiol. Biotechnol., 98, 3987-3996.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3) cells	Limosilactobacillus fermentum

Crystallization (Commentary)

Crystallization (Comment)	Organism
sitting drop vapor diffusion method, using either 0.1 M Bis-Tris (pH 6.0), 25% (w/v) PEGMME5000, or 0.1 M Bis-Tris (pH 6.4 or 6.6), 23% (w/v) PEG3350, 0.2 or 0.3 M NaCl	Limosilactobacillus fermentum

Protein Variants

Protein Variants	Comment	Organism
D268E	the mutant shows 58% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D268K	the mutant shows 48% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D268K/D269K	the mutant shows 65% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D268K/D269K/D299K	the mutant with 115% activity compared to the wild type enzyme exhibits significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0). With the addition of borate, D-galactose is isomerized into D-tagatose by this mutant at pH 5.0, resulting in a high conversion rate of 62%	Limosilactobacillus fermentum
D268K/D299K	the mutant shows 42% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D268R	the mutant shows 88% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D269E	the mutant shows 62% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D269K	the mutant shows 130% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D269K/D299K	the mutant shows 84% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D269R	the mutant shows 88% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D299E	the mutant shows 109% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D299K	the mutant shows 90% activity compared to the wild type enzyme	Limosilactobacillus fermentum
D299R	the mutant shows 68% activity compared to the wild type enzyme	Limosilactobacillus fermentum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
46	-	D-galactose	mutant enzyme D268K/D269K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
51	-	D-galactose	mutant enzyme D268K/D269K/D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
55	-	D-galactose	mutant enzyme D269K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
56	-	D-galactose	mutant enzyme D268K/D269K/D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
56	-	D-galactose	mutant enzyme D269K/D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
61	-	D-galactose	mutant enzyme D269K/D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
63	-	D-galactose	wild type enzyme, at pH 6.0 and 65°C	Limosilactobacillus fermentum
74	-	D-galactose	mutant enzyme D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
78	-	D-galactose	mutant enzyme D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
82	-	D-galactose	mutant enzyme D299R, at pH 5.0 and 65°C	Limosilactobacillus fermentum
84	-	D-galactose	mutant enzyme D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
84	-	D-galactose	mutant enzyme D299R, at pH 6.0 and 65°C	Limosilactobacillus fermentum
93	-	D-galactose	mutant enzyme D299R, at pH 4.0 and 65°C	Limosilactobacillus fermentum
94	-	D-galactose	mutant enzyme D268K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
94	-	D-galactose	mutant enzyme D269K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
96	-	D-galactose	mutant enzyme D269K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
108	-	D-galactose	mutant enzyme D269K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
116	-	D-galactose	wild type enzyme, at pH 5.0 and 65°C	Limosilactobacillus fermentum
117	-	D-galactose	mutant enzyme D268K/D269K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
125	-	D-galactose	mutant enzyme D268K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
129	-	D-galactose	mutant enzyme D268K/D269K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
137	-	D-galactose	mutant enzyme D268K/D269K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
166	-	D-galactose	mutant enzyme D268K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
201	-	D-galactose	mutant enzyme D268K/D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
246	-	D-galactose	mutant enzyme D268K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
287	-	D-galactose	mutant enzyme D268K/D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mn2+	required	Limosilactobacillus fermentum

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
55000	-	6 * 55000, SDS-PAGE	Limosilactobacillus fermentum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-arabinose	Limosilactobacillus fermentum	-	L-ribulose	-	?
L-arabinose	Limosilactobacillus fermentum CGMCC2921	-	L-ribulose	-	?

Organism

Organism	UniProt	Comment	Textmining
Limosilactobacillus fermentum	D9ILD9	-	-
Limosilactobacillus fermentum CGMCC2921	D9ILD9	-	-

Purification (Commentary)

Purification (Comment)	Organism
nickel-affinity column chromatography and Source 15Q column chromatography	Limosilactobacillus fermentum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
1	2	mutant enzyme D299E, at pH 7.0 and 65°C	Limosilactobacillus fermentum
4.6	-	mutant enzyme D268K/D299K, at pH 5.5-6.0 and 65°C	Limosilactobacillus fermentum
5.3	-	mutant enzyme D268K, at pH 6.5-7.0 and 65°C	Limosilactobacillus fermentum
6.4	-	mutant enzyme D268E, at pH 6.5-7.0 and 65°C	Limosilactobacillus fermentum
6.8	-	mutant enzyme D268E, at pH 7.5 and 65°C	Limosilactobacillus fermentum
7.1	-	mutant enzyme D268K/D269K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
7.5	-	mutant enzyme D299R, at pH 5.0-5.5 and 65°C	Limosilactobacillus fermentum
9.2	-	mutant enzyme D269K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
9.7	-	mutant enzyme D268R, at pH 5.5 and 65°C	Limosilactobacillus fermentum
9.7	-	mutant enzyme D269R, at pH 6.0 and 65°C	Limosilactobacillus fermentum
9.9	-	mutant enzyme D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
11	-	wild type enzyme, at pH 6.5 and 65°C	Limosilactobacillus fermentum
12.7	-	mutant enzyme D268K/D269K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
14.3	-	mutant enzyme D269K, at pH 5.0-5.5 and 65°C	Limosilactobacillus fermentum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-galactose	-	Limosilactobacillus fermentum	D-tagatose	39% conversion after 48 h at pH 6.0 and 65°C	?
D-galactose	-	Limosilactobacillus fermentum CGMCC2921	D-tagatose	39% conversion after 48 h at pH 6.0 and 65°C	?
L-arabinose	-	Limosilactobacillus fermentum	L-ribulose	-	?
L-arabinose	-	Limosilactobacillus fermentum CGMCC2921	L-ribulose	-	?

Subunits

Subunits	Comment	Organism
homohexamer	6 * 55000, SDS-PAGE	Limosilactobacillus fermentum

Synonyms

Synonyms	Comment	Organism
L-AI	-	Limosilactobacillus fermentum

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
60	-	the wild type shows a half-life time of 36 h at pH 5.0 and 60°C	Limosilactobacillus fermentum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.5	-	-	Limosilactobacillus fermentum

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
4	6	the wild type shows half-life times of 8 h, 16 h and 36 h at pH values of 4.0, 5.0 and 6.0, respectively	Limosilactobacillus fermentum

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.02	-	D-galactose	wild type enzyme, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.035	-	D-galactose	mutant enzyme D268K/D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.05	-	D-galactose	mutant enzyme D268K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.055	-	D-galactose	mutant enzyme D268K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.057	-	D-galactose	mutant enzyme D268K/D269K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.065	-	D-galactose	mutant enzyme D268K/D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.067	-	D-galactose	mutant enzyme D268K/D269K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.07	-	D-galactose	mutant enzyme D268K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.078	-	D-galactose	mutant enzyme D268K/D269K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.09	-	D-galactose	mutant enzyme D268K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.1	-	D-galactose	mutant enzyme D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.103	-	D-galactose	mutant enzyme D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.105	-	D-galactose	mutant enzyme D269K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.108	-	D-galactose	mutant enzyme D268K/D269K/D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.108	-	D-galactose	mutant enzyme D299R, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.113	-	D-galactose	mutant enzyme D268K/D269K/D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.113	-	D-galactose	mutant enzyme D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.117	-	D-galactose	mutant enzyme D269K/D299K, at pH 4.0 and 65°C	Limosilactobacillus fermentum
0.118	-	D-galactose	mutant enzyme D299R, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.12	-	D-galactose	mutant enzyme D268K/D269K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.127	-	D-galactose	mutant enzyme D299R, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.13	-	D-galactose	wild type enzyme, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.135	-	D-galactose	mutant enzyme D269K/D299K, at pH 6.0 and 65°C	Limosilactobacillus fermentum
0.152	-	D-galactose	mutant enzyme D269K/D299K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.165	-	D-galactose	mutant enzyme D269K, at pH 5.0 and 65°C	Limosilactobacillus fermentum
0.173	-	D-galactose	mutant enzyme D269K, at pH 6.0 and 65°C	Limosilactobacillus fermentum