Biochemical and molecular characerization of a levansucrase from Lactobacillus reuteri
| Autor: | Hijum, S.A.F.T. van, Szalowska, E., Maarel, M.J.E.C. van der, Dijkhuizen, L. |
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| Přispěvatelé: | TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO |
| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Předmět: |
Sucrose
Molecular biology Genetic code Bacterial strain Carboxy terminal sequence Bacterial enzyme Levan Cell growth In vivo study Calcium ion Carbohydrate Conformation Chemical analysis Enzyme activity Polymer Protein motif Bacteria (microorganisms) Priority journal Sequence Deletion Bacterial gene Hydrolysis Inulin Sequence analysis Recombinant Proteins Amino acid Bacterium isolation DNA Bacterial Lactobacillus reuteri Molecular Sequence Data Fructose Streptococcus salivarius Food technology Raffinose Enzyme synthesis Escherichia coli Amino Acid Sequence Enzyme purification Nutrition Base Sequence Sequence Homology Amino Acid Enzyme kinetics In vitro study Streptococcus High temperature Nonhuman Levansucrase Fructans Kinetics Lactobacillus Hexosyltransferases Genes Bacterial Bacterial cell Protein expression Cell anchorage Transferase Controlled study Nucleotide sequence |
| Zdroj: | Microbiology, 150, 621-630 |
| Popis: | Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with β(2→6) linkages] from sucrose or raffinose. Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene (lev), encoding a protein (Lev) consisting of 804 amino acids. Lev showed highest similarity with an inulosucrase of L. reuteri 121 [Inu; producing an inulin polymer with β(2→1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (LevΔ773His) that produced the same levan polymer [with only 2-3 % β(2→1→6) branching points] as L. reuteri 121 cells grown on raffinose. The low degree of branching of the L. reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius, having up to 30 % branches. Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro. Lev is strongly dependent on Ca2+ ions for activity. Unique properties of L. reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli, (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 °C, kinetics that are best described by the Hill equation. © 2004 SGM. Chemicals / CAS: amino acid, 65072-01-7; calcium ion, 14127-61-8; fructose, 30237-26-4, 57-48-7, 7660-25-5, 77907-44-9; inulin, 9005-80-5; levan, 50815-13-9, 9013-95-0; levansucrase, 9030-17-5; raffinose, 512-69-6; sucrose, 122880-25-5, 57-50-1; transferase, 9047-61-4; DNA, Bacterial; Fructans; Hexosyltransferases, EC 2.4.1.-; levansucrase, EC 2.4.1.10; Recombinant Proteins. Molecular Sequence Numbers: GENBANK: AF459437, AL162757, CAA05973, L08445, M18954, P11701, Q06447, Q55242, X02730, AF465251 |
| Databáze: | OpenAIRE |
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