Literature summary for 4.2.2.16 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.

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Cloned(Commentary)

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

Protein Variants

Protein Variants	Comment	Organism
A264G	relative hydrolytic activity: 20.4% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.
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.
N238K	relative hydrolytic activity: 23.6% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.
N85S	relative hydrolytic activity: 34.8% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.
N85S/A264G	relative hydrolytic activity: 49.9% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.
N85S/D268E	relative hydrolytic activity: 30% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.
N85S/N224K/Q229E/T253N	relative hydrolytic activity: 37% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.
P313R/F518L	relative hydrolytic activity: 15.8% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%	Microbacterium sp.

Organism

Organism	UniProt	Comment	Textmining
Microbacterium sp.	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

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

Synonyms

Synonyms	Comment	Organism
levan fructotransferase	-	Microbacterium sp.
LFTase	-	Microbacterium sp.

Temperature Optimum [°C]

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

pH Optimum

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

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Release 2023.1 (January 2023)