• Journal of Life Science
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• v.20 no.11
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• pp.1582-1588
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• 2010

A new alginate lyase gene of marine bacterium Streptomyces sp. M3 had been previously cloned in pColdI vector and transformed into E. coli BL21 (DE3). In this study, M3 lyase protein without signal peptide was overexpressed by induction with IPTG and purified with Ni-Sepharose affinity chromatography. The absorbance at 235 nm of the reaction mixture and TLC analysis showed that M3 alginate lyase was a polyG-specific lyase. When M3 lyase was assayed with substrate for 10 min, optimum pH and optimum temperature were pH 9 and $60^{\circ}C$. For the effect of 1mM metal ion on M3 lyase activity, $Ca^{++}$ and $Mn^{++}$ ions increased the alginate degrading activity by two-fold, whereas $Hg^{++}$ and $Zn^{++}$ ions inhibited the lyase activity completely. $Mg^{++}$, $Co^{++}$, $Na^+$, $K^+$, and $Ba^{++}$ did not show any strong effects on alginate lyase activity.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.5
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• pp.637-642
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• 1996

We isolated a marine bacterium, Pseudomonas sp,, which could produce the enzyme of alginate lyase, and cloned the alginate lyase gene in Escherichia coli. The cloned DNA was overexpressed with approximately $50\%$ amount of total proteins. In addition, the expressed proteins were not secreted into the medium, and most of them existed in the cytoplasm by the soluble form, but not observed any inclusion body by TIM. For the optimum enzyme activity, temperature was $20^{\circ}C$, pH was 7.0, and Km and Vmax values of the enzyme were $0.4\%$ and 625 units/mg, respectively.

• 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.

• Journal of Life Science
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• v.19 no.11
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• pp.1522-1528
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• 2009

A marine bacterium was isolated from brown seaweeds for its ability to degrade alginate. Analysis of 16S ribosomal DNA sequence revealed that the strain belongs to Streptomyces like strain ALG-5 which was reported previously. New alginate lyase gene of Streptomyces sp. M3 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. The consensus sequences of N-terminal YXRSELREM and C-terminal YFKAGXYXQ were conserved in the M3 alginate lyase amino acid sequences. The homology model for the M3 alginate lyase showed a characteristic structure of $\beta$-jelly roll fold main domain like alyPG from Corynebacterium sp. ALY-1. The homogenate of the recombinant E. coli with the alginate lyase gene showed more degrading activity for polyguluronate block than polymannuronate block. The results from the multiple alignments and the homology modeling elucidated in the M3 alginate lyase can be classified into family PL-7.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.681-686
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• 2015

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℃ (1,370 U/mg). It was a cold-adapted alginate lyase, which showed 54.5% and 72.1% of maximum activity at 15℃ and 20℃, 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.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1671-1682
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• 2018

Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that $30^{\circ}C$ with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.

• Microbiology and Biotechnology Letters
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• v.29 no.2
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• pp.72-77
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• 2001

Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.

• Journal of Life Science
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• v.25 no.2
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• pp.130-135
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• 2015

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At $25^{\circ}C$ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, $25^{\circ}C$ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

• 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.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.118-123
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• 2001

The alginate lyase A1-III gene of Sphingomonas species A1 is composed of 1,077 nucleotides, encoding a protein (359 amino acids) with a molecular mass of 40,322 Da. Recombinant A1-III expressed in Escherichia coli exhibited the same full enzymatic activity as native A1-III. In order to identify the critical residue for activity, a site-directed mutation was introduced into the A1-III gene (H192A, His192->Ala). Recombinant A1-III (H192A) exhibited a significant decrease in enzyme activity (one-thirty thousandth of that of A1-III), without any conformational change, as detected by the CD spectra in the far UV region. Also, the chemical modification of wild-type A1-III with methyl 4-nitro benzene sulfonate resulted in a 40% decrease from the initial activity, whereas the same modification of A1-III (H192A) produced no change in the activity. The role of His192 on the catalytic process was also explored based on a model of A1-III docked with mannuronic acid into the active site.