Fermentation optimization and enzyme characterization of a new ι-Carrageenase from Pseudoalteromonas carrageenovora ASY5

Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 35­40°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.

Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg²⁺, Mn²⁺, K⁺; Ca²⁺, Na⁺, Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Sr²⁺ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

Molecular cloning, expression and characterization of alpha-amylase gene from a marine bacterium Pseudoalteromonas sp. MY-1.

A gene (amyA) encoding an extracellular alpha-amylase from a marine bacterium Pseudoalteromnonas sp. MY-1 was cloned and expressed in Escherichia coli. It comprised an open-reading-frame of 2,007 base pairs and encoded a protein of 669 amino acids with a predicted molecular weight of 73,770 daltons and a pI of 5.15. The entire amino acid sequence of amyA gene showed 86% similarity to the alpha-amylase preproprotein from Pseudoalteromonas haloplanktis. It consisted of a signal peptide, alpha-amylase catalytic domain and an amy C domain. The recombinant amylase was purified to homogeneity and biochemically characterized. The enzyme revealed maximum activity at pH 7.0 and 40 degrees C. The enzyme hydrolyzed soluble starch and some maltooligosaccharides to several oligosaccharides, and maltose was the common product from different substrates.

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.