Ki-Bang Song

Optimal conditions for levan formation by an overexpressed recombinant levansucrase

SummaryA genetically modified levansucrase, which contained His-affinity tag in its C-terminal, was constructed by PCR reaction using two synthetic primers. This modified protein was produced up to 30 % in total cell protein of E. coli, and was purified by a one-step affinity chromatography. The optimum pH for levan production was pH 5 and the optimum temperature was 0 °C. The higher velocity of levan formation within shorter enzyme reaction times was achieved by increasing the levels of enzyme concentration. The optimal sucrose concentration for levan production was around 20 %. Under these conditions, more than 50 g levan/l was produced.

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l−1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 106), as determined by HPLC while high molecular weight levan (>6 × 106) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.

Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis

Levan, fructo-oligosaccharides and fructosyl derivatives were formed from sucrose using recombinant levansucrase from Rahnella aquatilis. Levan formation was optimal at 30 °C resulting 57 % of the theoretical yield. The more suitable substrate concentration for levan formation was 200 g sucrose/L. Oligosaccharides was accumulated selectively at high substrate concentration. The increase of levan and oligosaccharides formation was not achieved by adding water-miscible organic solvents. Alkyl fructosides were synthesized from various alcohols as fructosyl acceptors by R. aquatilis levansucrase.

Transcriptional analysis of levU operon encoding saccharolytic enzymes and two apparent genes involved in amino acid biosynthesis in Zymomonas mobilis.

Extracellular levansucrase (LevU) and sucrase (InvB) are two of the three saccharolytic enzymes involved in the sucrose metabolism of Zymomonas mobilis. The levU and invB genes were clustered with a 155bp interval on the chromosome. Both genes were transcribed constitutively at the basal level and the transcription of both genes was induced significantly when sucrose was added to the medium. These genes were transcribed as a bicistronic mRNA and the expression was modulated by a single promoter, which is located upstream of the levU gene. The transcriptional initiation site was mapped to -64bp from the translation start site of levU gene. These results indicated that two genes are most likely to constitute an operon. The glk operon, which encodes four glycolytic enzymes, was located close to the levU operon on the chromosome. Two apparent ORFs (ORF3 and 4) were found at the intervening sequence located between the glk and levU operons. These ORFs were transcribed divergently and showed high homology at the amino acid level with the bacterial global regulatory protein (Lrp) and aspartate racemase.

Levan production by use of the recombinant levansucrase immobilized on titanium-activated magnetite

Abstract Levansucrase from Zymomonas mobilis expressed in Escherichia coli was immobilized onto the surface of magnetite. Optimum conditions for the immobilization were, pH 4.0; immobilization reaction time of 30 min and 8 U of enzyme per gram of matrix. Immobilization yield was 75% under optimized conditions. The maximum production yield of levan by the immobilized levansucrase was about 42% on a fructose released basis with sucrose as substrate at 100 g/l. Thermal stability of the levansucrase increased by immobilization procedure. Furthermore, immobilized enzyme showed the less polymerizing activity in levan formation, producing the low-molecular weight (MW) levan. The molecular weight of levan can be controlled by using the immobilized levansucrase in high yield. Immobilized levansucrase retained 61% of the original activity after five repeated uses.

Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherichia coli

A levansucrase gene (lsrA) from Rahnella aquatilis ATCC33071 was isolated from a genomic library and the nucleotide sequence of the lsrA structural gene was determined. lsrA is composed of 1248 bp and encodes 415 amino acid residues with a calculated molecular mass of 45.9 kDa. Although the amino acid sequence of lsrA gene showed good conservation with the sequences of reported levansucrases and of the conserved regions thought to be implicated in the enzyme activity, comparison of the deduced amino acid sequences certified the dissimilarity of the proteins from Gram-negative and Gram-positive bacteria. The lsrA gene was expressed from its own promoter in Escherichia coli in an active form. The lsrA expression in E. coli-pRL1CPR was affected by the growth phase of cells: it was repressed in the early phase of growth, but was significantly stimulated during the entrance of cells into the late phase of growth. The growth-phase-dependent fashion of lsrA expression was altered in a constitutive-like fashion by the deletion of an upstream region of lsrA (pNd137), suggesting that the growth-phase dependent expression of lsrA was mediated by the deleted upstream region.

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Abstract Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8–1%, in which approximately 20–50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.

Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV

Aims:  To clone and overexpress a novel levan fructotransferase gene lftA from Arthrobacter ureafaciens K2032.

Immobilization of levan fructotransferase for the production of di-fructose anhydride from levan

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was immobilized on various carriers of which Chitopearl BCW2501 beads showed the higher activity of 320 U g−1 for the formation of di-fructose anhydride compounds. The immobilized enzyme retained about 60% of its initial activity after being used for 20 cycles.

Cloning and Sequence Analysis of the estA Gene Encoding Enzyme for Producing (R)-β-Acetylmercaptoisobutyric Acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70-100 amino acids upstream of the G-X-S-X-G consensus sequence.