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Literature summary extracted from

  • Park, A.R.; Kim, J.S.; Jang, S.W.; Park, Y.G.; Koo, B.S.; Lee, H.C.
    Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus (2017), Microb. Cell Fact., 16, 224 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
5.1.3.11 synthesis the enzyme mutant Y114E can be used for production of lactulose for the nutritional industry. Using Y114E, isomerization of lactose to lactulose is optimnized, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h from 200 g/l of lactose Caldicellulosiruptor saccharolyticus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.1.3.11 gene Csac_0294, recombinant expressison of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Caldicellulosiruptor saccharolyticus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
5.1.3.11 apoenzyme crystal structure comparisons and molecular replacement method using the structure of Ruminococcus albus enzyme (PDB ID 3WKF) as a search model, overview Caldicellulosiruptor saccharolyticus

Protein Variants

EC Number Protein Variants Comment Organism
5.1.3.11 additional information Y114X mutants are found to lose activity entirely, while the parental enzyme and the Y114E mutant enzyme show increased isomerization activity from lactose to lactulose at 65°C. Isomerase and epimerase activities of wild-type and mutant enzymes, overview Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114A site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114C site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114D site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114E site-directed mutagenesis, the mutation leads to increased release of a byproduct, lactulose, from lactose at 65°C, while its activity is low at 37°C. The Y114E mutation increases isomerization of lactose, while decreasing the epimerization of lactose Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114F site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114G site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114H site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114I site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114K site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114L site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114M site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114N site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114P site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114Q site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114R site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114S site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114T site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114V site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus
5.1.3.11 Y114W site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C Caldicellulosiruptor saccharolyticus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.1.3.11 beta-1,4-mannobiose Caldicellulosiruptor saccharolyticus
-
 4-O-beta-D-mannopyranosyl-D-glucose
-
 r
5.1.3.11 beta-1,4-mannobiose Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331
-
 4-O-beta-D-mannopyranosyl-D-glucose
-
 r
5.1.3.11 cellobiose Caldicellulosiruptor saccharolyticus
-
 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
5.1.3.11 cellobiose Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331
-
 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
5.1.3.11 lactose Caldicellulosiruptor saccharolyticus
-
 epilactose
-
 r
5.1.3.11 lactose Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331
-
 epilactose
-
 r

Organism

EC Number Organism UniProt Comment Textmining
5.1.3.11 Caldicellulosiruptor saccharolyticus A4XGA6
-
-
5.1.3.11 Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331 A4XGA6
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.1.3.11 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis Caldicellulosiruptor saccharolyticus

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
5.1.3.11 additional information
-
 isomerase and epimerase activities of wild-type and mutant enzymes, overview Caldicellulosiruptor saccharolyticus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.1.3.11 beta-1,4-mannobiose
-
 Caldicellulosiruptor saccharolyticus 4-O-beta-D-mannopyranosyl-D-glucose
-
 r
5.1.3.11 beta-1,4-mannobiose
-
 Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331 4-O-beta-D-mannopyranosyl-D-glucose
-
 r
5.1.3.11 cellobiose
-
 Caldicellulosiruptor saccharolyticus 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
5.1.3.11 cellobiose
-
 Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
5.1.3.11 lactose
-
 Caldicellulosiruptor saccharolyticus epilactose
-
 r
5.1.3.11 lactose
-
 Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331 epilactose
-
 r

Subunits

EC Number Subunits Comment Organism
5.1.3.11 More three-dimensional structure and substrate binding structure analysis, residues Tyr114 and Asn184 play an important role in binding epilactose, overview. The active site, with its triple histidine center, is located inside the (alpha/alpha)6 barrel structure of the catalytic domain Caldicellulosiruptor saccharolyticus

Synonyms

EC Number Synonyms Comment Organism
5.1.3.11 cellobiose 2-epimerase
-
 Caldicellulosiruptor saccharolyticus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.1.3.11 37
-
 assay at Caldicellulosiruptor saccharolyticus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.1.3.11 7.5
-
 assay at Caldicellulosiruptor saccharolyticus

General Information

EC Number General Information Comment Organism
5.1.3.11 additional information three-dimensional structure and substrate binding structure analysis, residues Tyr114 and Asn184 play an important role in binding epilactose, overview. The active site, with its triple histidine center, is located inside the (alpha/alpha)6 barrel structure of the catalytic domain. Two essential conserved histidine residues, His247 and His377, and a third histidine residue located at the bottom of the substrate binding site are required for catalysis. Y114 and N184 in the catalyt domain are located at the interface of the mannose moiety of epilactose in the enzyme-product complex Caldicellulosiruptor saccharolyticus
5.1.3.11 physiological function the enzyme reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified beta-1,4-linked oligosaccharides, including beta-1,4-mannobiose, cellobiose, and lactose Caldicellulosiruptor saccharolyticus