Toward the Synthesis and Improved Biopotential of an N-methylated Analog of a Proline-Rich Cyclic Tetrapeptide from Marine Bacteria.

An N-methylated analog of a marine bacteria-derived natural proline-rich tetracyclopeptide was synthesized by coupling the deprotected dipeptide fragments Boc-l-prolyl-l-N-methylleucine-OH and l-prolyl-l-N-methylphenylalanine-OMe. A coupling reaction was accomplished utilizing N,N'-Dicyclohexylcarbodidimde (DCC) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC·HCl) as coupling agents and Triethylamine (TEA) or N-methylmorpholine (NMM) as the base in the presence of the racemization suppressing agent. This was followed by the cyclization of the linear tetrapeptide fragment under alkaline conditions. The structure of the synthesized cyclooligopeptide was confirmed using quantitative elemental analysis, FTIR (Fourier-transform infrared spectroscopy), ¹H NMR (Nuclear magnetic resonance spectroscopy), 13C NMR, and mass spectrometry. From the bioactivity results, it was clear that the newly synthesized proline-rich tetracyclopeptide exhibited better anthelmintic potential against Megascoplex konkanensis, Pontoscotex corethruses, and Eudrilus eugeniae at a concentration of 2 mg/mL as well as improved antifungal activity against pathogenic dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at a concentration of 6 µg/mL, as compared to non-methylated tetracyclopeptide. Moreover, N-methylated tetracyclopeptide displayed significant activity against pathogenic Candida albicans.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5¨¬C, and showed 0.867 unit of o-nitrophenyl ¥â-D-galactopyranoside(ONPG) hydrolyzing activity at 4¨¬C. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20¨¬C, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48 percent), C12:0 3OH (9.21 percent), C16:0 (41.83 percent), C17:0 ¥ø8 (7.24 percent) and C18:1 ¥ø7 (7.04 percent). All suthese results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20¨¬C and 7.8, respectively. The enzyme was stable at 4¨¬C for 7 days, but its activity decreased to about 50 percent of initial activity at 37¨¬C in 7 days.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme.

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5°C, and showed 0.867 unit of o-nitrophenyl ß-D-galactopyranoside(ONPG) hydrolyzing activity at 4°C. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20°C, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48%), C12:0 3OH (9.21%), C16:0 (41.83%), C17:0 ω8 (7.24%) and C18:1 ω7 (7.04%). All these results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20°C and 7.8, respectively. The enzyme was stable at 4°C for 7 days, but its activity decreased to about 50% of initial activity at 37°C in 7 days.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5ºC, and showed 0.867 unit of o-nitrophenyl -D-galactopyranoside(ONPG) hydrolyzing activity at 4ºC. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20ºC, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48%), C12:0 3OH (9.21%), C16:0 (41.83%), C17:0 8 (7.24%) and C18:1 7 (7.04%). All suthese results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20ºC and 7.8, respectively. The enzyme was stable at 4ºC for 7 days, but its activity decreased to about 50% of initial activity at 37ºC in 7 days.

Molecular cloning, characterization and enzymatic properties of a novel βeta-agarase from a marine isolate Psudoalteromonas sp. AG52

An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A β-agarase gene which has 96.8 percent nucleotide identity to Aeromonas β-agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant β-agarase (rAgaA) was overexpressed in Escherichia coli and purified as a fusion protein. The optimal temperature and pH for activity were 55 ºC and 5.5, respectively. The enzyme had a specific activity of 105.1 and 79.5 unit/mg toward agar and agarose, respectively. The pattern of agar hydrolysis demonstrated that the enzyme is an endo-type β-agarase, producing neoagarohexaose and neoagarotetraose as the final main products. Since, Pseudoalteromonas sp. AG52 encodes an agaA gene, which has greater identity to Aeromonas β-agarase, the enzyme could be considered as novel, with its unique bio chemical characteristics. Altogether, the purified rAgaA has potential for use in industrial applications such as development of cosmetics and pharmaceuticals.

Molecular cloning, characterization and enzymatic properties of a novel ßeta-agarase from a marine isolate Psudoalteromonas SP. AG52.

An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A ß-agarase gene which has 96.8% nucleotide identity to Aeromonas ß-agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant ß-agarase (rAgaA) was overexpressed in Escherichia coli and purified as a fusion protein. The optimal temperature and pH for activity were 55 °C and 5.5, respectively. The enzyme had a specific activity of 105.1 and 79.5 unit/mg toward agar and agarose, respectively. The pattern of agar hydrolysis demonstrated that the enzyme is an endo-type ß-agarase, producing neoagarohexaose and neoagarotetraose as the final main products. Since, Pseudoalteromonas sp. AG52 encodes an agaA gene, which has greater identity to Aeromonas ß-agarase, the enzyme could be considered as novel, with its unique bio chemical characteristics. Altogether, the purified rAgaA has potential for use in industrial applications such as development of cosmetics and pharmaceuticals.

Molecular cloning, characterization and enzymatic properties of a novel eta-agarase from a marine isolate Psudoalteromonas sp. AG52

An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A -agarase gene which has 96.8% nucleotide identity to Aeromonas -agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant -agarase (rAgaA) was overexpressed in Escherichia coli and purified as a fusion protein. The optimal temperature and pH for activity were 55 ºC and 5.5, respectively. The enzyme had a specific activity of 105.1 and 79.5 unit/mg toward agar and agarose, respectively. The pattern of agar hydrolysis demonstrated that the enzyme is an endo-type -agarase, producing neoagarohexaose and neoagarotetraose as the final main products. Since, Pseudoalteromonas sp. AG52 encodes an agaA gene, which has greater identity to Aeromonas -agarase, the enzyme could be considered as novel, with its unique bio chemical characteristics. Altogether, the purified rAgaA has potential for use in industrial applications such as development of cosmetics and pharmaceuticals.

Extracellular proteases from the Antarctic marine Pseudoalteromonas sp. P96-47 strain / Proteasas extracelulares de la cepa marina antártica Pseudoalteromonas sp. P96-47

The extracellular protease-production capacity of 33 bacterial isolates taken from marine biotopes in King George Island, Antarctica, was evaluated in liquid cultures. The P96-47 isolate was selected due to its high production capacity and was identified as Pseudoalteromonas sp. The optimal growth temperature was 20 °C and the optimal for protease production was 15 °C. Proteases were purified from culture supernatants, developing a multiple-band profile in zymograms. They were classified as neutral metalloproteases and worked optimally at 45 °C with an Eact of 47 kJ/ mol. Their stability was higher at neutral pH, retaining more than 80% of activity at pH 6-10 after 3 h incubation at 4 °C. After 90 min incubation at 40 and 50 °C, the percentages of residual activities were 78% and 44%. These results contribute to the basic knowledge of Antarctic marine proteases and also help evaluate the probable industrial applications of P96-47 proteases.

La capacidad productora de proteasas extracelulares de 33 aislamientos bacterianos tomados de biotopos marinos en la Isla Rey Jorge, Antártida, fue evaluada en cultivo líquido. El aislamiento P96-47 fue seleccionado debido a su alta capacidad productora y fue identificado como Pseudoalteromonas sp. La temperatura óptima de crecimiento fue de 20 °C y la de producción de 15 °C. Las proteasas fueron purificadas a partir del sobrenadante de cultivo, y en los zimogramas desarrollaron un perfil de múltiples bandas. Estas proteasas fueron clasificadas como metaloproteasas neutras y se observó que trabajan óptimamente a 45 °C, con una Eact de 47 kJ/ mol. Su estabilidad fue superior a pH neutro y retuvieron más del 80% de su actividad a pH 6-10 después de 3 h de incubación a 4 °C. Luego de 90 min de incubación a 40 y 50 °C, las actividades residuales fueron 78% y 44%, respectivamente. Los resultados que se presentan en este trabajo contribuyen al conocimiento básico de las proteasas marinas antárticas y también a evaluar las probables aplicaciones industriales de las proteasas de P96-47.