• Title/Summary/Keyword: levansucrase

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Secretion and Localization of Pseudomonas auratiaca Levansucrase Expressed in Saccharomyces cerevisiae (Saccharomyces cerevisiae에서 발현된 Pseudomonas aurantiaca Levansucrase의 분비국재성)

  • 임채권;김광현;김철호;이상기;남수완
    • Microbiology and Biotechnology Letters
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    • v.32 no.3
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    • pp.206-211
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    • 2004
  • Levansucrase gene(lscA) from Pseudomonas aurantiaca was subcloned downstream of GAL1 promoter in pYES 2.0 and pYInu-AT [GAL10 promoter + exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-lscA and p YInu-lscA, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Sacchromayces cerevisiae SEY2102, and transformants with high activity of levansucrase were selected. When each yeast transform ants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 8.62 U/ml with the strain harboring pYES-lscA and 5.43 U/ml with the strain harboring pYInu-lscA. The levansucrase activity of 80% was detected in the periplasmic space and cytoplasm. The levansucrase activity in the medium of SEY2102/pYInu-lscA was 0.87 U/ml whereas that of SEY2102/pYES-lscA was 0.47 U/ml, which implying the exoinulinase signal sequence didn't enhance the secretion efficiency of levansucrase. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

Heterologous Expression and Optimized One-Step Separation of Levansucrase via Elastin-like Polypeptides Tagging System

  • Kang, Hye-Jin;Kim, Jin-Hee;Chang, Woo-Jin;Kim, Eung-Soo;Koo, Yoon-Mo
    • Journal of Microbiology and Biotechnology
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    • v.17 no.11
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    • pp.1751-1757
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    • 2007
  • Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

Expression and Secretion of Zymomonas mobilis Levansucrase in Saccharomyces cerevisiae. (Saccharomyces cerevisiae에서 Zymomonas mobilis 유래 Levansucrase의 발현과 분비)

  • 임채권;김이경;김광현;김철호;이상기;남수완
    • Journal of Life Science
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    • v.14 no.3
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    • pp.429-434
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    • 2004
  • Levansucrase gene (levU) from Zymomonas mobilis was subcloned downstream of GALl promoter in pYES 2.0 and pYInu-AT [GALl0 promoter+exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-levU and pYInu-levU, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Saccharomyces cerevisiae SEY2102, and then transformants showing high activity of levansucrase were selected. When each yeast transformants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 7.17 U/㎖ with the strain harboring pYES-levU and 6.61 U/㎖ with the strain harboring pYInu-levU. It was found that about 50% of levansucrase were detected in the medium and periplasmic space, and exoinulinase signal sequence didn't enhance the secretion efficiency. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

Effect of Glycine Supplement on Extracellular Secretion of Levansucrase form Pseudomonas aurantiaca S-4380 in Recombinant Escherichia coli (Glycine 첨가에 의한 Pseudomonas aurantiaca S-4380 유래 재조합 levansucrase 효소의 세포 외 분비촉진 효과)

  • 김승환;장은경;김인환;장기효;강순아;장병일
    • KSBB Journal
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    • v.18 no.4
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    • pp.312-317
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    • 2003
  • The addition of glycine up to 0.5% (w/v) to Luria broth (LB) media on the secretion of levansucrase in a recombinant strain Escherichia coli JM109/pUPLK1 was observed to enhance the release of periplasmic proteins from the cell to the broth, without significantly affecting the cell growth rate and protein productivity. However, the glycine concentration at 1 % (w/v), the cell density attainable at the stationary phase fell to about 50% and the extracellular activity of levansucrase corresponded to about 80% of the total (extracellular plus intracellular) activity and increased by 2.6-fold, comparing to the cells grown in the absence of glycine. The increased pH at stationary phase accelerated the degradation of levansucrase. Maximal extracellular activity was attained when 1 % glycine was supplemented at the onset of strain growth.

Secretory Production of Rahnella aquatilis ATCC 33071 Levansucrase Expressed in Escherichia coli

  • KANG , SOON-AH;LEE, JAE-CHEOL;PARK, YOUNG-MIN;LEE, CHAN;KIM, SEUNG-HWAN;CHANG, BYUNG-IL;KIM, CHUL-HO;SEO, JEONG-WOO;RHEE, SANG-KI;JUNG, SUNG-JE;KIM, SANG-MOO;PARK, SEONG-KYU;JANG, KI-HYO
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1232-1238
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    • 2004
  • To investigate the production and characteristics of thermostable levan sucrase from Rahnella aquatilis ATCC 33071, the levan sucrase gene from R. aquatilis was cloned and expressed in Escherichia coli without induction system. Expression of levansucrase gene in E. coli had no notable or detrimental effect on the growth of host strain, and the recombinant levan sucrase exhibited levan synthesis activity. Levansucrase was secreted to the periplasm in E. coli, and addition of $0.5\%$ glycine yielded further secretion of levansucrase to the growth medium and resulted in an increase of total levansucrase activity. Furthermore, the cellular levansucrase was evaluated for the production of levan by using toluene­permeabilized whole-cells. The levansucrase was thermostable at $37^{\circ}C$. The molecular size oflevan was $1{\times}\;10^{6}$ Da, as determined by HPLC, and the degree of polymerization of levan varied with incubation temperatures: Low incubation temperature was preferable for the production of high-molecular size levan. The present study demonstrated that the mass production of levan and levan oligosaccharides can be achieved by glycine supplementation to the growth medium or by toluene­permeabilized whole-cells.

Molecular Characterization of the Levansucrase Gene from Pseudomonas aurantiaca S-4380 and Its Expression in Escherichia coli

  • Jang, Eun-Kyung;Jang, Ki-Hyo;Isaac Koh;Kim, In-Hwan;Kim, Seung-Hwan;Kang, Soon-Ah;Kim, Chul-Ho;Ha, Sang-Do;Rhee, Sang-Ki
    • Journal of Microbiology and Biotechnology
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    • v.12 no.4
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    • pp.603-609
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    • 2002
  • DFA IV is di-D-fructose-2,6':6,2'-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca 5-4380, was identified to produce low-branched levan, and the levansucrase gene (lsch) from this bacterium was found to be composed of 1,275 Up coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5${\alpha}$ by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coii culture broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P.aurantiaca 5-4380 levansucrase.

Bacterial Surface Display of Levansucrase of Zymomonas mobilis Using Bacillus Subtilis Spore Display System (고초균 포자를 이용한 Zymomonas mobilis 유래의 levansucrase 표면 발현)

  • Kim, June-Hyung;Choi, Soo-Keun;Jung, Heung-Chae;Pan, Jae-Gu;Kim, Byung-Gee
    • KSBB Journal
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    • v.26 no.3
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    • pp.243-247
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    • 2011
  • Using Bacillus subtilis spore display system, with cotG as an anchoring motif, levansucrase from Zymomonas mobilis, was displayed on the outer surface of Bacillus subtilis spore. Flow cytometry of DB104 (pSDJH-cotG-levU) spore, proved the surface localization of CotG-LevU fusion protein on the spore compared to that of DB104. Enzymatic activity of DB104 (pSDJH-cotG-levU) spore showed more than 1.5 times higher levansucrase specific activity compared to that of the host spore, which is a remarkable increase of enzymatic activity considering the existence of sacA (sucrase) and sacB (levansucrase) in the Bacillus subtilis chromosome. The spore integrity, revealed by sporulation frequency test after heat and lysozyme treatment of spore, did not changed at all in spite of the CotG-LevU fusion protein incorporation into the spore coat layer during spore formation process. These data prove again that Bacillus subtilis spore could be considered as good live immobilization vehicle for efficient bioconversion process.

Cloning and Sequence Analysis of a Levansucrase Gene from Rahnella aquatilis ATCC15552

  • Kim, Hyun-Jin;Yang, Ji-Young;Lee, Hyeon-Gye;Cha, Jae-Ho
    • Journal of Microbiology and Biotechnology
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    • v.11 no.4
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    • pp.693-699
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    • 2001
  • An intracellular levansucrase gene, lscR from Rahnella aquatilis ATCC 15552, was cloned and its nucleotide sequence was determined. Nucleotide sequence analysis of this gene revealed a 1,238 bp open reading frame coding for a protein of 415 amino acids. The levansucrase was expressed by using a T7 promoter in Escherichia coli BL21 (DE3) and the enzyme activity was detected in the cytoplasmic fraction. The optimum pH and temperature of this enzyme for levan formation was pH 6 and $30^{\circ}C$, respectively. The deduced amino acid sequence of the lscR gene showed a high sequence similarity (59-89%) with Gram-negative levansucrses, while the level of similarity with Gram-positive enzymes was less than 42%. Multiple alignments of levansucrase sequences reported from Gram-negative and Gram-positive bacteria revealed seven conserved regions. A comparison of the catalytic properties and deduced amino acid sequence of lscR with those of other bacterial levansucrases strongly suggest that Gram-negative and Gram-positive levansucrases have an overall different structure, but they have a similar structure at the active site.

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Purification and Characterization of an Extracellular Levansucrase from Zymomonas mobilis ZM1(ATCC 10988). (Zymomonas mobilis ZM1이 생산하는 균체외 Levansucrase의 정제 및 특성)

  • 송기방;서정우;주현규;이상기
    • Microbiology and Biotechnology Letters
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    • v.26 no.4
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    • pp.309-315
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    • 1998
  • An extracellular levansucrase, which catalyzes the formation of levan from sucrose, from the culture broth of Zymomonas mobilis ZM1 was purified by conventional column purification methods. The final purification yield was 18.3 fold of the crude enzyme from Z. mobilis, with 16.5 % of the enzyme recovered in the preparation step. The molecular weight of the enzyme was estimated to be 91,000 by Superose 12 gel filtration, and 45,000 by SDS-PAGE, indicating that levansucrase is a dimer. The optimum pH for the enzyme activity was around pH 4.0 for sucrose hydrolysis, and was around pH 5.0 for levan formation. The enzyme was inhibited by some metal ions, such as Hg$\^$2+/ and Cu2$\^$2+/, and 50% of inhibition was observed with 5mM EDTA. The enzyme activity was enhanced by the presence of detergent Triton X-100, but inhibited by SDS completely The enzyme catalyzes the liberation of reducing sugars, oligosacccharides and the formation of fructose polymer(levan). The enzyme also catalyzes the transfructosylation reaction of fructose moiety from sucrose to various sugar acceptor molecules, including sugar alcohols.

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Characterization of Levan Hydrolysis Activity of Levansucrase from Zymomonas mobilis ATCC 10988 and Rahnella aquatilis ATCC 33071

  • Jang, Ki-Hyo;Kang, Soon-Ah;Kim, Chul-Ho;Lee, Jae-Cheol;Kim, Mi-Hyun;Son, Eun-Wha;Rhee, Sang-Ki
    • Food Science and Biotechnology
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    • v.16 no.3
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    • pp.482-484
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    • 2007
  • To investigate production and hydrolysis of levan, the levansucrase enzymes from Zymomonas mobilis ATCC 10988 and Rahnella aquatilis ATCC 33071 were used. The optimum temperature of R. aquatilis levansucrase for levan formation was $37^{\circ}C$, whereas that of Z. mobilis was $4^{\circ}C$, under the experimental conditions. Both levansucrases also catalyzed the reverse levan hydrolysis reaction. Levan hydrolysis reactions from both levansucrases were temperature dependent; high temperature ($20^{\circ}C$) was more favorable than low temperature ($4^{\circ}C$) by 4 times. Fructose was the only product from hydrolysis reaction by both levansucrases, showing that both levansucrases mediated the hydrolysis reaction of exo-enzyme acting. In both enzymes, initial levan hydrolysis activity was almost accounted to 1% of initial levan formation activity. The results allow the estimation of the fructose release rate in enzyme processing conditions.