Sung Gu Lee

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.

Proteomic and transcriptomic investigations on cold-responsive properties of the psychrophilic Antarctic bacterium Psychrobacter sp. PAMC 21119 at subzero temperatures.

Psychrobacter sp. PAMC 21119, isolated from Antarctic permafrost soil, grows and proliferates at subzero temperatures. However, its major mechanism of cold adaptation regulation remains poorly understood. We investigated the transcriptomic and proteomic responses of this species to cold temperatures by comparing profiles at -5°C and 20°C to understand how extreme microorganisms survive under subzero conditions. We found a total of 2,906 transcripts and 584 differentially expressed genes (≥ twofold, P <0.005) by RNA-seq. Genes for translation, ribosomal structure and biogenesis were upregulated, and lipid transport and metabolism was downregulated at low temperatures. A total of 60 protein spots (≥ 1.8 fold, P < 0.005) showed differential expression on two-dimensional gel electrophoresis and the proteins were identified by mass spectrometry. The most prominent upregulated proteins in response to cold were involved in metabolite transport, protein folding and membrane fluidity. Proteins involved in energy production and conversion, and heme protein synthesis were downregulated. Moreover, isoform exchange of cold-shock proteins was detected at both temperatures. Interestingly, pathways for acetyl-CoA metabolism, putrescine synthesis and amino acid metabolism were upregulated. This study highlights some of the strategies and different physiological states that Psychrobacter sp. PAMC 21119 has developed to adapt to the cold environment in Antarctica.

Crystallization and preliminary X‐ray crystallographic analysis of an ice‐binding protein (FfIBP) from Flavobacterium frigoris PS1. Addendum

An addendum to the article by Do et al. [(2012) Acta Cryst. F68, 806–809].

Draft Genome Sequence of Paenisporosarcina sp. Strain TG-14, a Psychrophilic Bacterium Isolated from Sediment-Laden Stratified Basal Ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica

The psychrophilic bacterium Paenisporosarcina sp. TG-14 was isolated from sediment-laden stratified basal ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica. Here we report the draft genome sequence of this strain, which may provide useful information on the cold adaptation mechanism in extremely variable environments.

Draft Genome Sequence of Moritella dasanensis Strain ArB 0140, a Psychrophilic Bacterium Isolated from the Arctic Ocean

The psychrophilic bacterium Moritella dasanensis strain ArB 0140 was isolated near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Here we report a 4.89-Mb draft genome sequence of Moritella dasanensis ArB 0140, which could provide comprehensive information on a psychrophilic mechanism in extreme environments.

Possible Roles of Antarctic Krill Proteases for Skin Regeneration

Antarctic krill has a strong proteolytic enzyme system, which comes from a combination of several proteases. This powerful activity can be easily detected by krill’s superior post mortem autolysis. Mammalian skin consists of epidermis and dermal connective tissue, and functions as a barrier against threatening environments. A clot in a wound site of the skin should be removed for successful skin regeneration. Epithelial cells secrete proteases to dissolve the clot. In previous studies Antarctic krill proteases were purified and characterized. The proteolytic enzymes from Antarctic krill showed higher activity than mammalian enzymes. It has been suggested that these krill clean up the necrotic skin wound to induce a natural healing ability. The enzymes exhibited additional possibilities for several other biomedical applications, including dental plaque controlling agent and healing agent for corneal alkali burn. Considering that these versatile activities come from a mixture of several enzymes, discovering other proteolytic enzymes could be another feasible way to enhance the activity if they can be used together with krill enzymes. Molecular cloning of the krill proteases should be carried out to study and develop the applications. This review introduces possible roles of the unique Antarctic krill proteases, with basic information and suggestion for the development of an application to skin regeneration.

Crystal structure and modeling of the tetrahedral intermediate state of methylmalonate-semialdehyde dehydrogenase (MMSDH) from Oceanimonas doudoroffii

The gene product of dddC (Uniprot code G5CZI2), from the Gram-negative marine bacterium Oceanimonas doudoroffii, is a methylmalonate-semialdehyde dehydrogenase (OdoMMSDH) enzyme. MMSDH is a member of the aldehyde dehydrogenase superfamily, and it catalyzes the NADdependent decarboxylation of methylmalonate semialdehyde to propionyl-CoA. We determined the crystal structure of OdoMMSDH at 2.9 Å resolution. Among the twelve molecules in the asymmetric unit, six subunits complexed with NAD, which was carried along the protein purification steps. OdoMMSDH exists as a stable homodimer in solution; each subunit consists of three distinct domains: an NAD-binding domain, a catalytic domain, and an oligomerization domain. Computational modeling studies of the OdoMMSDH structure revealed key residues important for substrate recognition and tetrahedral intermediate stabilization. Two basic residues (Arg103 and Arg279) and six hydrophobic residues (Phe150, Met153, Val154, Trp157, Met281, and Phe449) were found to be important for tetrahedral intermediate binding. Modeling data also suggested that the backbone amide of Cys280 and the side chain amine of Asn149 function as the oxyanion hole during the enzymatic reaction. Our results provide useful insights into the substrate recognition site residues and catalytic mechanism of OdoMMSDH.

Draft Genome Sequence of Pseudomonas pelagia CL-AP6, a Psychrotolerant Bacterium Isolated from Culture of Antarctic Green Alga Pyramimonas gelidicola

ABSTRACT Pseudomonas pelagia CL-AP6, isolated from a culture of the Antarctic green alga Pyramimonas gelidicola, is a psychrotolerant bacterium. Here, we report the draft genome sequence of this strain, which may provide insights into the mutualistic interaction between microalgae and bacteria in sea ice, as well as the cold adaptation mechanisms of bacteria.

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.