• Microbiology and Biotechnology Letters
• /
• v.19 no.3
• /
• pp.222-227
• /
• 1991

Rhizobium fredii USDA193 is one of the causal organism for root nodule formation in soybean (peking). Previously we cloned the pectate lyase gene (SY1) of R. fredii USDA193. The $pel^-$ mutants (SY1$\Omega$ and SY1$\Omega$1) of SY1 were obtained using the in vitro insertional omega mutagenesis of RpelB (of Rhizobium pel) and fill-in reaction of RpelE (of Rhizobium pel) gene respectively, and we constructed two mutants (R, fredii USDA193$\Omega$ and R. fredii USDA193$\Omega$1) in pectate lyase function by marker-exchange with pe1B::$\Omega$ and R. fredii USDA193 strain (rif). The pectate lyase activity of two pel- mutant of R. fredii USDA193 was determined by spectrophotometric method. However, all pectate lyase activity of these mutants was not lost upon the mutagenesis by marker-exchange. This suggests that other pectate lyase genes may be present on the plasmid or the chromosome of R. fredii. As yet we do not have evidence linking RpelB and RpelE genes of R. fredii directly to the early nodulation process.

• Microbiology and Biotechnology Letters
• /
• v.29 no.2
• /
• pp.72-77
• /
• 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
• /
• v.17 no.1 s.81
• /
• pp.132-136
• /
• 2007

When alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was produced as aggregated insoluble particles known as inclusion bodies. In order to produce a soluble and active form of alginate lyase, E. coli cells fore cotransformed with the plasmids designed to permit coexpression of aly together with molecular chaperones such as DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results revealed that the coexpression of aly together with DnaK/DnaJ/GrpE chaperone had a marked effect on the production of this protein as a soluble and active form, presumably through facilitating correct folding of alginate lyase protein. The optimal concentration of L-arabinose for the induction of DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/ml. When DnaK/DnaJ/GrpE chaperone was coexpressed, about 34% in the total alginate lyase was produced in the soluble fraction. By addition of 10% cetylpyridinium chloride, a clear zone around the colony coexpressing aly and DnaK/DnaJ/GrpE chaperone was formed, indicating that the alginate in the medium was hydrolyzed by active alginate lyase enzyme.

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

• Korean Journal Plant Pathology
• /
• v.10 no.3
• /
• pp.163-168
• /
• 1994

Erwinia rhapontici causes soft-rot disease in a number of plants such as rhubarb, onion, hyacinth and garlic. Pectate lyase (Pel) depolymerizes pectin and other polygalacturonates, which is though to play a role in bacterial invasion of plants. Pel activity was not detected in E. rhapontici cultured in a minimal salts medium containing glycerol, polygalacturonate, or citrus pectin as a carbon source. However, when sterilized potato tuber and Chinese cabbage slices were added to minimal salts polygalacturonate (0.5%) medium, E. rhapontici produced pectate lyase enzyme. Also Pel activity was consistently detected from macerated potato tubers, Chinese cabbage leaves, lettuce leaves and celery petioles tissue. Pel in the extract of macerated Chinese cabbage caused by E. rhapontici strain 1, resulted in electrolyte loss, tissue maceration and cell death of potato tuber tissue. These results indicate that E. rhapontici produces pectate lyase only in the presence of non-diffusible plant components, and that this enzyme probably contributes to its pathogenicity.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.29 no.5
• /
• pp.637-642
• /
• 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
• /
• v.25 no.5
• /
• pp.681-686
• /
• 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.

• Korean Journal of Microbiology
• /
• v.26 no.3
• /
• pp.270-277
• /
• 1988

Microorganisms isolated from soil (150 strains), fungi (39 strains), yeasts (9 strains) and Streptomyces species (39 strains) were assayed for L-phenylalanine ammonia-lyase(PAL) activity. 17 strains of fungi and 46 strains of soil isolates were proved to produce PAL, Aspergillus panamensis, Penicillium varioti and 11 soil isolates showed comparatively large PAL activity. When PAL activity was assayed with cell-free extracts of these 13 strains and 7 strains of Rhodotorula and Rhodosporidium geni, Rhodosporidium toruloides (IFO 0559) showed the highest PAL activity with 0.333 units per g of the wet cell weight.

• Korean Journal of Microbiology
• /
• v.24 no.2
• /
• pp.194-197
• /
• 1986

Malonate kinase and isocitrate lyase were induced in Acinetobacter calcoaceticus grown on malonate as a sole carbon source but repressed by succinate. The induction of those two enzymes was stimulated by dibutyryl cyclic adenosine 3', 5'-monophosphate, indicating that the expression of their genes for those enzymes is dependent on cyclic adenosine 3', 5'-monophosphate.

• Microbiology and Biotechnology Letters
• /
• v.20 no.6
• /
• pp.655-662
• /
• 1992

Pectate lyase produced by recombinant strain containing pectate lyase gene from alkalitolerant Bacillus sp. YA-14 was succesively purified with 257.6 purification folds and a 10.2% yields by the affinity method, eM-cellulose column chromatography followed by gel filtration on Sephadex G-I00 column. The optimal pH and temperature for pectate lyase activity were 10.0 and $60^{\circ}C$, respectively. The enzyme was stable between pH 4.0 and 10.0, and up to $50^{\circ}C$. The molecular weight of this enzyme was estimated to be 43,000 daltons by SDS-PAGE. Amino acid analysis showed that the enzyme contained more polar and basic amino acids, especially serine, glycine and tyrosine, than that of various pectate lyase from other strains. The N-terminal amino acid sequence was Ala-Asp-Leu-Gly-His-Gln-Thr.