• Journal of Life Science
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• v.22 no.1
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• pp.7-15
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• 2012

A polyguluronate-specific lyase from Streptomyces sp. strain M3 has been previously cloned and characterized. In this study, the M3 alginate lyase gene in the pColdI vector was mutated by site-directed mutagenesis and random mutagenesis to enhance the alginate degrading activity. Six mutants were obtained: Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu, and Gly194Cys. Phe99Leu and Lys191Glu mutants completely lost their alginate lyase activity, whereas the alginate degrading activity of Gly194Cys mutant increased by nearly 10 fold. The 3-D protein structure of M3 alginate lyase, which was constructed using the Swiss-Model automodeler, was also compared to the crystal structure of another alginate lyase. A mutated glycine residue was positioned between Gly193 and Tyr195 of the C-terminal conserved sequence, YFKAGXYXQ. A phenylalanine residue (at position 99) and a glycine residue (at position 194) mutated in this study were distant from the active site, but the degrading activity was strongly affected by their mutation.

• Fisheries and Aquatic Sciences
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• v.15 no.3
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• pp.259-263
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• 2012

To isolate an alginate-degrading bacterium, we conducted a single colony isolation using a solid medium containing alginate as the sole carbon source. A marine bacterium Y-4 capable of degrading alginate was isolated from seawater. The strain was identified to be Pseudoalteromonas sp., based on morphological, biochemical, 16S rDNA homology, and phylogenetic analyses. Moreover, Pseudoalteromonas sp. Y-4 exhibited alginate lyase activity in the presence of 4% alginate even though many known alginate-degrading bacteria degrade in the range of 0.5-1% alginate. The optimum culture conditions for the Y-4 strain were 2% alginate, pH 8.0, and 3% NaCl at $30^{\circ}C$. The highest alginate lyase activity was also observed under the same conditions. To our knowledge, this is the first reported isolation of a marine bacterium degrading high concentrations of alginate.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.531-535
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• 1998

The gene encoding alginate lyase was isolated from a library constructed with the vector, pUC19, and expressed in Escherichia coli. The nucleotide sequence of the cloned alginate lyase gene (ALY) from Pseudomonas sp. W7 was determined. The nucleotide sequence revealed a 1,035 bp open reading frame (ORF), encoding 345 amino acid residues with a calculated molecular mass of 37,478 Da. The N-terminal amino acid sequences (15 residues) of purified alginate lyase corresponded to that of the deduced amino acid sequence.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.414-419
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• 2006

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasm ids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

• Journal of Life Science
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• v.25 no.12
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• pp.1393-1398
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• 2015

In this study, we investigated the extraction condition of alginate from Laminaria japonica, the enzymatic degradation of the extracted alginate, and the inhibitory activity of the degraded alginate on the differentiation of 3T3-L1 preadipocytes. The optimal conditions for the efficient extraction, precipitation, and recovery of alginate from the brown seaweed L. japonica were 1% for Na₂CO₃ concentration, 80℃ for extraction temperature, and ethanol for precipitation solvent. In the enzymatic reaction for the production of low-molecular-weight alginate (LMWA) by using alginate lyase from Flavobacterium sp., the initial concentration of Laminaria alginate was 3%. The low-molecular-weight degree from alginate was independent with the enzyme concentration, and the optimal concentration of alginate lyase was found to be 5 unit/ml. Through the enzymatic reaction with 5 unit/ml of alginate lyase at 37℃ for 3 hr, the viscosity and molecular weight of LMWA were 4.5 cp and 307 kDa, respectively. Treatment with LMWA significantly suppressed the accumulation of lipid droplet and triglyceride in 3T3-L1 preadipocytes with a dose-dependent manner. Therefore, it seems that LMWA treatment could inhibit the differentiation of 3T3-L1 preadipocytes. These results indicate that LMWA or the degraded alginate produced by alginate lyase enzyme can be useful for the development of anti-obesity biosubstances.

• Applied Chemistry for Engineering
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• v.23 no.1
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• pp.100-104
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• 2012

Oligosaccharides production showed various biological activities in vivo like functional foods and industrial materials utilized available within many practical applications which have obtained from the degradation of alginate. Alginate is rich in the main component of seaweeds especially the brown algae. We investigated what degrading alginate from seaweeds to make alginate oligosaccharides can utilize in various fields using enzyme secreting Erwinia tasmaniensis. In this study, we observed an optimal culture condition of E. tasmaniensis, and characteristics of alginate lyase secreting E. tasmaniensis. These bacteria, E. tasmaniensis, were isolated from Nuruk. In this case, a suitable growth factor for E. tasmaniensis was culture it for 36 h in broth media on concentration of 1.0% (w/v) alginate. The enzyme showed the highest level of alginate lyase activity when cultured on broth media containing 1.0% (w/v) sodium alginate for 72 h. Optimal condition of pH, temperature and duration time for alginate lyase activity were found to be pH 6.0, $20^{\circ}C$ and 60 min, respectively.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.350-354
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• 2009

A Gram-negative, alginate lyase-producing bacterium was isolated from the Haeundae Coast, Korea. The isolated strain N7151-6 produced alginate lyase. The optimal temperature and pH for growth were found to be $30^{\circ}C$ and pH 8.0, respectively. This strain can be grown at the NaCl concentration of 0-7% (w/v). Analysis of 16S rDNA sequence and physiological profiling indicated that the strain N7151-6 belonged to Pseudomonas sp. The enzyme alginate lyase produced by Pseudomonas sp. N7151-6 was partially purified by ultrafiltration (MWCO= 30 kDa). The optimum pH and temperature for the activity of the purified enzyme were found to be 7.0 and $30^{\circ}C$, respectively. The enzyme was stable at the pH range of 5.0-9.0 and temperature range of $23-30^{\circ}C$. The total activity of alginate lyase produced was reached about 110 unit/L.

• Journal of Life Science
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• v.23 no.12
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• pp.1420-1427
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• 2013

A novel acetylalginate esterase (AcAlgE) gene was previously cloned and characterized from Sphingomonas sp. MJ-3. In this study, the synergistic effects of MJ-3 AcAlgE, and KS-408 alginate lyase on the degradation of acetylalginate from Pseudomonas aeruginosa were investigated by using high-field 1H-NMR and an FPLC-equipped peptide column. The alginate lyase coupled assay of AcAlgE showed that degradation of high molecular weight acetylalginate was more difficult than degradation of acid hydrolyzed acetylalginate. The degradation of acetylalginate by alginate lyase was easier after AcAlgE was used to remove the acetyl group from acetylalginate. This result showed that the recombinant AcAlgE enhanced the degradation of acetylalginate by alginate lyase.

• Fisheries and Aquatic Sciences
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• v.2 no.1
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• pp.93-97
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• 1999

The promoter region of alginate lyase gene (aly) from Pseudomonas sp. W7 was used for the high expression of gilthead seabream (Sparus aurata) growth hormone (GH) gene in Esherichia coli. PCR product encoding the premature segment of the growth hormone. was cloned to the downstream of aly promoter. GH was overexpressed With 46 ammo acid of alginate lyase as fusion protein. GH was immunoreactive and production of GH was repressed with supplementation of $0.4\%$ glucose into culture media.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.92-95
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• 1995

Halophilic Pseudomonas sp.W7 isolated from laver in the southem sea of Korea showed alginate lyase activity. Gene (aly) encoding alginate lyase was cloned in E.coli JM83 and the N-terminal amino acid sequence of the enzyme was determined after purificaion. The recombinant enzyme has been shown to have a molecular weight of about 40kDa after 12% SDS-polyacrylamide gel electrophoresis.