• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.102-108
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• 2006

The IscA gene, encoding a levansucrase of 424 amino acids (aa) residues, was cloned from the genomic DNA of Pseudomonas aurantiaca S-4380, and overexpressed in Escherichia coli. The recombinant levansucrase overexpressed in E. coli was then used to produce levan from sucrose. Levan crystals with 98% purity could be obtained from the reaction mixture with $62\%$ yield using an alcohol precipitation method. The molecular weight of the levan was $7\times10^5$ daltons. Methylation studies showed that the levan was branched: main linkage C-2,6; branched linkage C-2,1; and degree of branching $6\%$. Three bacterial levans from different strains were incubated with levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032, which produced $di-\beta-D-fructofuranose$ dianhydride IV (DFA IV); final conversion yields from the levans to DFA IV were $39\%$ in Zymomonas mobilis, $53\%$ in Serratia levanicum, and $59\%$ in P. aurantiaca S-4380 levansucrase. The levan from P. aurantiaca S-4380 levansucrase gave the highest conversion yield of levan to DFAIV so far reported.

• KSBB Journal
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• 제18권4호
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• pp.312-317
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• 2003

Glycine의 배양액 첨가에 의해 Pseudomonas aurantiaca S-4380 유래 재조합 levansucrase의 세포 외 분비가 촉진된다는 것을 알 수 있었다. 배지액에 존재하는 glycine 농도에 비례 의존적으로 균체 내에서 발현된 효소의 분비량이 증가되었고, 세포의 성장은 억제됨을 알 수 있었다. SDS-PAGE 분석결과 0.5％, 1.0％ glycine 첨가로 배양시간이 증가하면서 세포내의 단백질 배양액으로 분비됨을 알 수 있었다. Levansucrase를 분비시키기 위한 최적화된 조건은 pH을 6.9-7.0으로 조절하면서 1.0％ glycine을 배양초기 첨가하였을 때 배양말기에 단백질의 급격한 분해 없이 145 U/mL의 효소 활성도를 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제12권4호
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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.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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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.