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Literature summary extracted from

  • Moon, K.O.; Choi, K.H.; Kang, H.Y.; Oh, J.I.; Jang, S.B.; Park, C.S.; Lee, J.H.; Cha, J.
    Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase (2008), J. Microbiol. Biotechnol., 18, 1064-1069.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.2.16 expressed in Escherichia coli as a His-tagged fusion protein Microbacterium sp.

Protein Variants

EC Number Protein Variants Comment Organism
4.2.2.16 A264G relative hydrolytic activity: 20.4% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.
4.2.2.16 additional information residues that are critical to the intramolecular fructosyl transfer reaction of LFTase are analysed. An error-prone PCR mutagenesis process is carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan are screened by the dinitrosalicylic acid (DNS) method. Selected mutants reveal two major products: one is a di-D-fructose-2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity N85S substitution is responsible for the strong enhancement of the hydrolytic activity Microbacterium sp.
4.2.2.16 N238K relative hydrolytic activity: 23.6% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.
4.2.2.16 N85S relative hydrolytic activity: 34.8% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.
4.2.2.16 N85S/A264G relative hydrolytic activity: 49.9% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.
4.2.2.16 N85S/D268E relative hydrolytic activity: 30% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.
4.2.2.16 N85S/N224K/Q229E/T253N relative hydrolytic activity: 37% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.
4.2.2.16 P313R/F518L relative hydrolytic activity: 15.8% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7% Microbacterium sp.

Organism

EC Number Organism UniProt Comment Textmining
4.2.2.16 Microbacterium sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.2.16 after Ni-NTA chromatogrphy the enzyme solution is further purified on a Hi-Prep 16/60 Sephacryl S-200 high resolution column using fast-performance liquid chromatography Microbacterium sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.2.16 levan
-
 Microbacterium sp. 5 di-D-fructose-2,6':2',6 dianhydride + ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.2.16 levan fructotransferase
-
 Microbacterium sp.
4.2.2.16 LFTase
-
 Microbacterium sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.2.16 37
-
 assay at Microbacterium sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.2.16 7
-
 assay at Microbacterium sp.