Se Jong Han

Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization.

An endochitinase was previously purified and the gene was cloned from the psychrophilic Antarctic bacterium, Sanguibacter antarcticus (KCTC 13143). In the present study, recombinant endochitinase, rChi21702, was expressed using a yeast expression system (Pichia pastoris) and codon optimization. The expressed rChi21702 was purified by Phenyl-Sepharose column chromatography. Optimal expression yielded 1-mg purified enzyme from 1-L bioreactor culture. When p-NP-(GlcNAc)(2) was used as a substrate, the specific activity of the enzyme was determined to be 20U/mg. In vitro assays and thin-layer chromatography demonstrated that the recombinant enzyme has endochitinase activity that produces diacetyl-chitobiose as a dominant end product when chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc)(2) are used as substrates. Optimal activity for rChi21702 was observed at 37 degrees C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10 degrees C and 44% activity at 0 degrees C. Taken together, the results indicate that rChi21702 has psychrotolerant endochitinase activity even after recombinant expression in yeast cells.

Selection of Extraction Solvent and Temperature Effect on Stability of the Algicidal Agent Prodigiosin

An organic solvent for extracting prodigiosin from culture broth was selected and a test to determine the long-term stability of prodigiosin was performed to develop prodigiosin as a biological control agent against Chattonella antiqua, a harmful alga that can cause red tides. Prodigiosin was extracted using nine solvents, and the extracts were analyzed by liquid chromatography-mass spectroscopy. Acetone was selected as the best organic solvent because of its high extraction efficiency and less processing time. Stability tests for prodigiosin were performed at various temperatures, and algicidal activity against C. antiqua was also tested. Ultimately, > 98% stability was sustained after 30 days at 4°C, whereas < 30% stability was maintained after 30 days at 37°C. Although prodigiosin was kept for 30 days in an optimum organic solvent, its stability was safely maintained and algicidal activity was sustained at 4°C. These results indicate that acetone is a very useful extraction and storage solvent for prodigiosin.

Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases

Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127–2,130 bp, encoding 708–709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3–72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.

Bioactivities of ethanol extract from the Antarctic freshwater microalga, Chloromonas sp.

Cancer is the principal cause of human death and occurs through highly complex processes that involve the multiple coordinated mechanisms of tumorigenesis. A number of studies have indicated that the microalgae extracts showed anticancer activity in a variety of human cancer cells and can provide a new insight in the development of novel anti-cancer therapy. Here, in order to investigate molecular mechanisms of anticancer activity in the Antarctic freshwater microalga, Chloromonas sp., we prepared ethanol extract of Chloromonas sp. (ETCH) and performed several in vitro assays using human normal keratinocyte (HaCaT) and different types of cancer cells including cervical, melanoma, and breast cancer cells (HeLa, A375 and Hs578T, respectively). We revealed that ETCH had the antioxidant capacity, and caused significant cell growth inhibition and apoptosis of cancer cells in a dose-dependent manner, whereas it showed no anti-proliferation to normal cells. In addition, ETCH had a significant inhibitory effect on cell invasion without the cytotoxic effect. Furthermore, ETCH-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved caspase-3 and p53, and by decrease in anti-apoptotic protein, Bcl-2 in ETCH-treated cancer cells. Taken together, this work firstly explored the antioxidant and anticancer activities of an Antarctic freshwater microalga, and ETCH could be a potential therapeutic candidate in the treatment of human cancer.

Enhancing Extracellular Lipolytic Enzyme Production In An Arctic Bacterium, Psychrobacter sp. ArcL13, By Using Statistical Optimization And Fed-Batch Fermentation

A strain isolated from seawater samples in the Chuckchi Sea and exhibiting extracellular lipolytic activity was identified using 16S rRNA gene sequence analysis as Psychrobacter sp. ArcL13. The lipolytic enzyme exhibited cold-active properties and high hydrolytic activity toward p-nitrophenyl caprylate (C8), p-nitrophenyl decanoate (C10), and sunflower oil. Statistical optimization of the medium components was performed to enhance the production of cold-active extracellular lipolytic activity. Glucose, yeast extract (YE), and NaCl were selected as the main efficient nutrient sources. Fed-batch fermentation using optimized medium with concentrated YE as the main feeding material showed a maximum lipolytic activity of 10.7 U/mL, which was a 21-fold increase in production over unoptimized flask culture conditions. The information obtained in the present study could prove applicable to the production of cold-active lipase on a large scale.