• Journal of Industrial Technology
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• v.29 no.B
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• pp.115-119
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• 2009

Green fluorescent protein (GFPuv) was displayed on the surface of ethanol-producing bacteria Zymomonas mobilis using N-terminal domain of ice nucleation protein (INP) as an anchoring motif. To evaluate the ice nucleation protein as plausible anchor motif in Z. mobilis, GFPuv gene was subcloned into Zymomonas expression vector yielding pBBR1MCS-3/pPDC/INPN/GFPuv plasmid., INP-GFPuv fusion protein was expressed in Z. mobilis and its fluorescence was verified by confocal microscopy. The successful display of GFPuv on Zymomonas mobilis suggest that INP anchor motif could be used for future fusion partner in Z. mobilis strain improvement.

• Food Science of Animal Resources
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• v.39 no.6
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• pp.1008-1014
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• 2019

This study investigated the effects of pressure-mediated protein changes on the ice nucleation temperature of pork loins. To variate chemical state of meat proteins, pork loin was pressurized at varying pressure levels (100-500 MPa) for 3 min, and moisture content, expressible moisture (EM) and differential scanning calorimetry (DSC) were analyzed. Although, all treatments showed similar moisture content, EM and degree of protein unfolding of pork loin showed different features as of 300 MPa. At moderate pressure treatments (100-200 MPa), all protein fractions were detected in DSC experiments, and pork loin had lower EM than control (p<0.05). Meanwhile, myosin and actin of pork loin treated at greater than 300 MPa were completely unfolded, and the treatments showed high EM compared to control (p<0.05). Unfolding of meat proteins was a factor suppressing ice nucleation, and the ice nucleation temperature tended to decrease with increasing applied pressure level. The ice nucleation characteristics of pressurized pork loin exhibited a potential application in freezing storage of pressurized meat with less tissue damage comparing to freeze fresh meat, and further exploration regarding the quality change after freezing of fresh and pressurized meat was warranted.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.234-238
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• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.

• Plant Disease and Agriculture
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• v.5 no.1
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• pp.27-33
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• 1999

Frost injury of crops is closely related to the epiphytic population dynamics of ice nucleation-active (INA) bacteria, and the injury can be reduced by decreasing the INA bacterial population. In order to predict the epiphytic population of INA bacteria on crops, a rapid and accurate detection method has to be developed. In the previous report, we produced some antibodies against INA proteins purified from the outer membrane of INA bacteria. However it was difficult to produce the antibodies because the purification procedures of the INA proteins were complicated, and the final yield was too low. We designed a specific peptide from the N-terminal region of INA protein by computer analysis and synthesized the peptide in vitro in this experiment. The peptide sequence was Asp-Ser-Por-Leu-Ser-Leu-His-Ala-Asp, that is corresponding to the highly conserved region in several INA proteins, with predicted beta turn, coiling, and hydrophilic region. A polyclonal anti-INA peptide antiserum produced specifically recognized INA bacteria as few as 10 colony-forming units (CFU) in the ELISA reactions and did not respond to other non-INA bacteria. Serological specificity of the anti-INA peptide antiserum will facilitate the forecasting of the INA bacterial population dynamics on crops.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.379-385
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• 2014

The purpose of this study was to compare the ability of an engineered Escherichia coli to degrade chlorpyrifos (Cp) using an organophosphorus hydrolase enzyme, encoded in both Flavobacterium sp. ATCC 27551 or Pseudomonas diminuta, by employing the Lpp-OmpA chimera and the N-terminal domain of the ice nucleation protein as anchoring motifs. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by both anchors on the outer membrane. This is the first report on the presentation of OPH on the cell surface by Lpp-OmpA under the control of the T7 promoter. The results showed cell growth in the presence of Cp as the sole source of energy, without growth inhibition, and with higher whole-cell activity for both cells harboring plasmids pENVO and pELMO, at approximately 10,342.85 and 10,857.14 U/mg, respectively. Noticeably, the protein displayed by pELMO was lower than the protein displayed by pENVO. It can be concluded that Lpp-OmpA can display less protein, but more functional OPH protein. These results highlight the high potential, of both engineered bacteria, for use in the bioremediation of pesticide-contaminated sources in the environment.

• Journal of Biosystems Engineering
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• v.40 no.3
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• pp.261-270
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• 2015

Purpose: Cold storage is the most popular method used to preserve highly perishable foods such as beef and fish. However, at refrigeration temperatures, the shelf life of these foods is limited, and spoilage leads to massive food waste. Moreover, freezing significantly affects the food's properties. Ice crystallization and growth during freezing can cause irreversible textural damage to foods through volumetric expansion, moisture migration induced by osmotic pressure gradients, and concentration of solutes,which can lead to protein denaturation. Methods: Although freezing can preserve perishable foods for months, these disruptive changes decrease the consumer's perception of the food's quality. Therefore, the development and testing of new and improved cold storage technologies is a worthwhile pursuit. Results: The process of maintaining a food product in an unfrozen state below its equilibrium freezing temperature is known as supercooling. As supercooling has been shown to offer a considerable improvement over refrigeration for extending a perishable product's shelf life, implementation of supercooling in households and commercial refrigeration units would help diminish food waste. Conclusions: A commercially viable supercooling unit for all perishable food items is currently being developed and fabricated. Buildup of this technology will provide a meaningful improvement in the cold storage of perishable foods, and will have a significant impact on the refrigeration market as a whole.

• 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.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1330-1335
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• 2011

N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.712-717
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• 2010

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole-cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a hemeand diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. The surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of a heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins into practically useful whole-cell biocatalysts for extensive biotechnological applications, including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and biochip development.