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Hee Sook Kim – One of the best experts on this subject based on the ideXlab platform.

  • Homology Modeling and Active Sites of PolyMG-specific Alginate Lyase from Stenotrophomonas maltophilia KJ-2
    Journal of Life Science, 2014
    Co-Authors: Hee Sook Kim

    Abstract:

    Alginates are linear acidic polysaccharides composed with (1-4)-linked α-L-guluronic acid and β-Dmannuronic acid. Alginate can be degraded by diverse Alginate Lyases, which cleave the Alginate using a β-elimination reaction and produce unsaturated uronate oligomers. A gene for a polyMG-specific Alginate Lyase possessing a novel structure was previously identified and cloned from Stenotrophomonas maltophilia KJ-2. Homology modeling of KJ-2 polyMG-specific Alginate Lyase showed it belongs to the PL6 family, whereas three Azotobacter vinelandii polyMG Lyases belong to the PL7 family of polysaccharide Lyases. From H-NMR spectra data, KJ-2 polyMG Lyase preferably degraded the M-β(1-4)-G glycosidic bond than the G-α(1-4)-M glycosidic bond. Seventeen mutants were made by site-directed mutagenesis, and Alginate Lyase activity was analyzed. Lys220Ala, Arg241Ala, Arg241Lys, and Arg265Ala lost Alginate Lyase activity completely. Arg155Ala, Gly303Glu, and Tyr304Phe also lost the activity by 60.7-80.1%. These results show that Arg155, Lys220, Arg241, Arg265, Gly303, and Tyr304 are important residues for catalytic activity and substrate binding.

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  • molecular cloning purification and characterization of a novel polymg specific Alginate Lyase responsible for Alginate mg block degradation in stenotrophomas maltophilia kj 2
    Applied Microbiology and Biotechnology, 2012
    Co-Authors: Su In Lee, Eun Yeol Lee, Sung Hee Choi, Hee Sook Kim

    Abstract:

    A gene for a polyMG-specific Alginate Lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant Alginate Lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 °C. Interestingly, the recombinant Alginate Lyase was expected to have a similar catalytic active site of chondroitin B Lyase but did not show chondroitin Lyase activity. In the test of substrate specificity, the recombinant Alginate Lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded Alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific Alginate Lyase preferably degraded the glycosidic bond in M–G linkage than that in G–M linkage. The KJ-2 polyMG-specific Alginate Lyase can be used in combination with other Alginate Lyases for a synergistic saccharification of Alginate.

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  • Effect of polyhistidine-tagging site on the stability of recombinant Alginate Lyase from Streptomyces sp. ALG-5
    Journal of Pharmaceutical Investigation, 2012
    Co-Authors: Heesub Song, Eun Ji Park, Young Hee Shin, Hee Sook Kim, Dong Hee Na

    Abstract:

    The purpose of this study was to investigate the effect of polyhistidine (His)-tagging site on the stability of Alginate Lyase from a marine bacterium Streptomyces species ALG-5 by the combined use of microchip electrophoresis and enzymatic depolymerizing activity assay. In microchip electrophoresis, C-terminally His-tagged Alginate Lyase (C-His-AL) was more stable than N-terminally His-tagged Alginate Lyase (N-His-AL) after the incubation in 50 mM potassium phosphate buffer (pH 7.0) at 37°C for 14 days. When the enzymatic depolymerizing activity of the same samples was measured, the activity of C-His-AL was not significantly changed for 14 days, whereas N-His-AL showed substantially declined activity after incubation. Consequently, this study demonstrated that the C-terminally His-tagging is more efficient than N-terminally His-tagging for preparing stable ALG-5 Alginate Lyase.

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Eun Yeol Lee – One of the best experts on this subject based on the ideXlab platform.

  • molecular cloning purification and characterization of a novel polymg specific Alginate Lyase responsible for Alginate mg block degradation in stenotrophomas maltophilia kj 2
    Applied Microbiology and Biotechnology, 2012
    Co-Authors: Su In Lee, Eun Yeol Lee, Sung Hee Choi, Hee Sook Kim

    Abstract:

    A gene for a polyMG-specific Alginate Lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant Alginate Lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 °C. Interestingly, the recombinant Alginate Lyase was expected to have a similar catalytic active site of chondroitin B Lyase but did not show chondroitin Lyase activity. In the test of substrate specificity, the recombinant Alginate Lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded Alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific Alginate Lyase preferably degraded the glycosidic bond in M–G linkage than that in G–M linkage. The KJ-2 polyMG-specific Alginate Lyase can be used in combination with other Alginate Lyases for a synergistic saccharification of Alginate.

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  • molecular identification of a polym specific Alginate Lyase from pseudomonas sp strain ks 408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in Alginate
    Canadian Journal of Microbiology, 2011
    Co-Authors: Natania Kam, Eun Yeol Lee, Yoo Jung Park, Hee Sook Kim

    Abstract:

    An Alginate Lyase gene of a newly isolated Pseudomonas sp. strain KS-408 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. A partial protein sequence of KS-408 Alginate Lyase was homology-modeled on the basis of the crystal structure of A1-III Alginate Lyase from Sphingomonas sp. strain A1. The proposed 3-D structure of KS-408 Alginate Lyase shows that Asn-198, His-199, Arg-246, and Tyr-253 residues are conserved for the catalytic active site. The recombinant KS-408-1F (with signal peptide) and KS-408-2F (without signal peptide) Alginate Lyases with the (His)6 tag consist of 393 (44.5 kDa) and 372 (42.4 kDa) amino acids with isoelectric points of 8.64 and 8.46, respectively. The purified recombinant KS-408 Alginate Lyase was very stable when it was incubated at 40 °C for 30 min. Alginate oligosaccharides produced by the KS-408-2F Alginate Lyase were purified on a Bio-Gel P2 column and analyzed by thin-layer chromatography, fast-protein liquid chromatography, …

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  • Alginate Lyase: Structure, property, and application
    Biotechnology and Bioprocess Engineering, 2011
    Co-Authors: Hee Sook Kim, Choul-gyun Lee, Eun Yeol Lee

    Abstract:

    Alginate is a linear polysaccharide in which β-D-mannuronate (M) and its epimer, α-L-guluronate (G), are covalently (1–4)-linked in different sequences. Alginate is mainly used as a food additive to modify food texture due to its high viscosity and gelling property. Alginate Lyase can degrade Alginate by cleaving the glycosidic bond through a β-elimination reaction, generating oligomer with 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end. Alginate oligosaccharides have been shown to stimulate the growth of human endothelial cells and the secretion of cytotoxic cytokines from human macrophage. Alginate can be converted into unsaturated monosaccharide by saccharification process using endolytic and exolytic Alginate Lyases, thus Alginate Lyases have potential as key biocatalyst for application of Alginate as a renewable source for biochemicals and biofuels in near future. In this paper, structures and functions of various Alginate Lyases are reviewed. Prospects on future applications of Alginate Lyases are also discussed.

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Feng Han – One of the best experts on this subject based on the ideXlab platform.

  • characterization of a new endo type polysaccharide Lyase pl family 6 Alginate Lyase with cold adapted and metal ions resisted property
    International Journal of Biological Macromolecules, 2018
    Co-Authors: Shan Gao, Zhelun Zhang, Luyao Tang, Yulong Tan, Feng Han

    Abstract:

    Abstract Alginate Lyase played an important role in brown algae degradation, and its enzymatic degradation products showed various biological activities. Although many Alginate Lyases have been characterized, the enzymes with special characterizations are still rather rare. In this study, a new Alginate Lyase gene, tsaly6A , has been cloned from marine bacterium Thalassomonas sp. LD5, and expressed in Escherichia coli . The deduced Alginate Lyase, TsAly6A, belonged to the polysaccharide Lyase (PL) family 6 and showed the highest amino acid identity (63%) with an exo-type oligoAlginate Lyase AlyGC. However, this study showed that TsAly6A was an endo-type enzyme yielding Alginate trisaccharides (64.5%) as the main products. Compared with other Alginate Lyases, TsAly6A showed high trisaccharide-yielding levels. Meanwhile, TsAly6A showed the specific activity of 15,960 U/μmol at its optimal pH (pH 8.0) and temperature (35 °C). In addition, TsAly6A was a cold-adapted, salt-activated and metal ions-resisted Alginate Lyase, which will enable it to perform high activity in the solution containing various ions. Its cold-adaptation, metal ions-tolerance and high trisaccharides yields make TsAly6A an excellent candidate for industrial applications.

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  • cloning expression and characterization of a cold adapted and surfactant stable Alginate Lyase from marine bacterium agarivorans sp l11
    Journal of Microbiology and Biotechnology, 2015
    Co-Authors: Xuemei Yang, Lan Zhang, Feng Han

    Abstract:

    The purpose of this study was to find a cold-adapted and surfactant-stable Alginate Lyase as a candidate for biotechnological and industrial applications. The gene for a new Alginate Lyase, AlyL1, from Agarivorans sp. L11 was cloned and expressed in Escherichia coli. The recombinant AlyL1 was most active at 40°C (1,370 U/mg). It was a cold-adapted Alginate Lyase, which showed 54.5% and 72.1% of maximum activity at 15°C and 20°C, respectively. AlyL1 was an alkaliphilic enzyme and most active at pH 8.6. In addition, it showed high stability in the presence of various surfactants at a high concentration (from 0.1% to 1% (w/v)). AlyL1 was an endo-type Alginate Lyase that degraded both polyM and polyG blocks, yielding disaccharides and trisaccharides as the main products. This is the first report of the cloning and functional expression of a cold-adapted and surfactant-stable Alginate Lyase. AlyL1 might be an interesting candidate for biotechnological and industrial applications.

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  • family 13 carbohydrate binding module of Alginate Lyase from agarivorans sp l11 enhances its catalytic efficiency and thermostability and alters its substrate preference and product distribution
    Fems Microbiology Letters, 2015
    Co-Authors: Xuemei Yang, Mengmeng Bao, Feng Han

    Abstract:

    The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in Alginate Lyases has not been elucidated. A new Alginate Lyase gene, alyL2 , was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The Alginate Lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide Lyase (PL7) module. To better understand the function of CBM13 in Alginate Lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7–6.6 times those of AlyL2-CM at 30–50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in Alginate Lyase provides new insights into the degradation of Alginate by marine microorganisms.

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