• Journal of Life Science
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• v.27 no.5
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• pp.584-590
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• 2017

Antifreeze proteins (AFPs) bind to ice crystals and inhibit their growth. AFPs are essential for the survival of organisms living in subzero environments. Type I AFP (AFP37) isolated from winter flounder is an ${\alpha}$-helical peptide of 37 residues long. In this study, we attempted to develop short AFP fragments with higher activity and solubility. We designed and synthesized N-terminal 15 and 21 residue-long AFPs, designated AFP15 and 21. Also dimerized AFP15 and 21, designated dAFP15N and dAFP21N, respectively, were generated through disulfide bonds between peptides containing CGG residues added to the N-terminus of AFP15 and AFP21 (designated AFP15N and 21N). Their helical contents and antifreeze activities were assessed using circular dichroism (CD) spectroscopy and a nanoliter osmometer, respectively. The helical content of AFP15 AFP21, AFP15N, AFP21N, dAFP15N and dAFP21N was 47, 48, 23.8, 28, 49.1, and 52%, respectively compared to that of wild type AFP37; the antifreeze activity was 8.4, 9.3, 0.05, 5.6, 12.1, 11.2% respectively, compared to that of wild type AFP37. Contrary to our anticipation, the dimerized peptides showed almost the same antifreeze activity as their monomeric counterparts. These results indicate that the dimerized peptides behave as monomeric peptides due to the high rotational freedom of disulfide bonds connecting two monomeric peptides. The star-shaped ice crystals generated by the peptides also demonstrated weak interaction between ice and peptides.

• Bulletin of the Korean Chemical Society
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• v.35 no.1
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• pp.286-288
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• 2014

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.673-679
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• 2004

A microbial cryoprotectant formulation using a W/O/W multiple emulsion system was developed. The psychrotolerant microorganism, B4, isolated from soil in South Korea, was observed by the drop freezing method, in which the microorganism sample inhibited ice nucleation activity. The antifreeze activity was eliminated when the microorganism sample was treated with protease, indicating that the antifreeze activity was due to the presence of antifreeze protein. The result of the l6S rDNA sequencing indicated the B4 strain was most closely related to a species of the genus Bacillus. Culture broth of B4 strain (Bacillus sp.) and rapeseed oil containing 1 % polyglycerine polyricinolate (PGPR) were used as core and wall material, respectively. The most stable W/O emulsion was prepared at a core/oil ratio of 1:2. The highest W/O/W emulsion stability was achieved when the primary emulsion to external aqueous phase containing 0.5% caster oil polyoxyethylene ether $(COG25^{TM})$ ratio was 1:1. Microcrystalline cellulose showed better W/O/W emulsion stability than other polymer types. The viability of cells in a W/O/W emulsion was higher than free cells during storage at $37^\circ{C}$. An acidic pH and UV exposure decreased the viability of free cells, but cells in W/O/W emulsion were more stable under these conditions.

• KSBB Journal
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• v.28 no.5
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• pp.310-314
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• 2013

Antifreeze peptide from Myoxocephalus octodecemspinosus was overexpressed and purified in Escherichia coli. Green fluorescence protein-AFP chimera was constructed by integrating gfp and afp genes. Produced GFP-AFP chimera protein was purified using polyhistidine tag which was inserted at C-terminus. By addition of GFP-AFP chimera protein, freezing point of elution buffer was decreased from $-13^{\circ}C$ to $-20^{\circ}C$. This result suggested that GFP-AFP chimera can be considered as a potential candidate of novel inhibitor for gas hydrates.

• Ocean and Polar Research
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• v.25 no.4
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• pp.607-615
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• 2003

Many organisms are known to survive in icy environments. These include both over wintering terrestrial insects and plants as well the marine fish inhabiting high latitudes. The adaptation of these organisms is both a fascinating and important topic in biology. Marine teleosts in particular, can encounter ice-laden seawater that is approximately $1^{\circ}C$ colder than the colligative freezing point of their body fluids. These animals produce a unique group of proteins, the antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that absorb the ice nuclei and prevent ice crystal growth. Presently, there are at least four different AFP types and one AFGP type that are isolated from a wide variety of fish. Despite their functional similarity, there is no apparent common protein homology or ice-binding motifs among these proteins, except that the surface-surface complementarity between the protein and ice are important for binding. The remarkable diversity of these proteins and their odd phylogenetic distribution would suggest that these proteins might have evolved recently in response to sea level glaciations just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Winter flounder, Pleuronectes americanus, has been used as a popular model to study the regulation of AFP gene expression. It has a built-in annual cycle of AFP expression controlled negatively by the growth hormone. The signal transduction pathways, transcription factors and promoter elements involved in this process have been studied in our laboratory and these studies will be presented.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.43 no.3
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• pp.172-178
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• 1998

Freezing-resistant plants can survive subzero temperatures by withstanding extracellular ice formation. During cold acclimation, their leaves accumulate antifreeze proteins (AFPs) that are secreted into the apoplast and have the ability to modify the normal growth of ice crystals. Three barley, two wheat and two rye cultivars were grown under two different temperature regimes (20/16$^{\circ}C$ and 5/2$^{\circ}C$, day/night). Apoplastic proteins from winter cereals were separated by SDS-PAGE and detected with antisera to AFPs from winter rye. Apoplastic proteins accumulated to much higher levels in cold-acclimated (CA) leaves compared with nonacclimated (NA) ones in winter cereals. After cold acclimation, the protein concentration of apoplastic extracts increased significantly from 0.088 $mgmL^{-1}$ to 0.448 $mgmL^{-1}$, with about 5-fold increment. Also, the apoplastic protein content per gram leaf fresh weight in CA leaves ranged from 31 $\mu\textrm{g}$ $(gFW)^{-1}$ to 120 $\mu\textrm{g}$ $(gFW)^{-1}$ with an averaged value of 77 $\mu\textrm{g}$ $(gFW)^{-1}$, and coefficients of variation of 54.9%. The CA leaves in Musketeer (a Canadian winter rye cultivar) showed the greatest AFPs and antifreeze activity followed by 'Geurumil' (a Korean winter wheat cultivar), and 'Dongbori l' (Korean facultative barley cultivar). The proteins secreted into the wheat leaf apoplast at CA condition were more numerous than those observed in winter rye, where two $\beta$-1,3-glucanase-like proteins (GLPs), two chitinase-like proteins (CLPs) and two thaumatin-like proteins (TLPs) accumulated during cold acclimation. The proteins in barley leaf apoplast at CA conditions were a little different from those in wheat leaves. The AFPs were various among and within species. More freezing-resistant cultivars had more clear and numerous bands than less freezing-resistant ones. The high determination coefficient ($R^2$ =91 %) between freezing resistance and AFPs per gram leaf fresh weight indicated that the amount of AFPs was highly related to freezing resistance in winter cereal crops.

• Bulletin of Food Technology
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• v.23 no.3
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• pp.376-384
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• 2010

• KSBB Journal
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• v.29 no.4
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• pp.303-306
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• 2014

Antifreeze proteins (AFP) inhibit ice growth to permit the survival of polar organisms in the cold environments. The recombinant AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris ice-binding protein), was produced using Pichia pastoris expression system. The optimum fermentation temperature ($30^{\circ}C$) and pH (5) for FfIBP production were determined using a fed-batch culture system. The maximal cell density and purified FfIBP were 112 g/L and 70 mg/L, respectively. The thermal hysteresis (TH) activity (0.85) of FfIBP obtained using a glycerol-methanol fed-batch culture system was 2-fold higher than that of the LeIBP (Leucosporidium ice-binding protein). This work allows for large-scale production of FfIBP, which could be extended to further application studies using recombinant AFPs.

• BMB Reports
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• v.41 no.1
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• pp.72-78
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• 2008

Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.

• Journal of the Korean Magnetic Resonance Society
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• v.26 no.4
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• pp.46-50
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• 2022

Antifreeze proteins (AFPs) bind to the ice crystals and then are able to inhibit the freezing of body fluid at subzero temperatures. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We prepared the QAE2 (AFP11) and SP (AFP6) isoforms of the notched-fin eelpout AFP and their mutant constructs and determined their temperature gradients of amide proton chemical shifts (𝚫δ/𝚫T) using NMR. The nfeAFP11 (QAE2) has the distinct 𝚫δ/𝚫T pattern of the first 3₁₀ helix compared to the QAE1 isoforms. The nfeAFP6 (SP) has the deviated 𝚫δ/𝚫T values of many residues, indicating its backbone conformational distortion. The study suggests the distortion in the H-bonding interactions and backbone conformation that is important for TH activities.