Korean Society for Biotechnology and Bioengineering Journal Korean Society for Biotechnology and Bioengineering Journal

pISSN 1225-7117 eISSN 2288-8268

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  • Review Paper 2022-06-30

    Characteristics of Prussian Blue Nanoparticles and Trends in Their Biotechnological Application Research

    Jeong Un Kim, Kang Jin Lee, and Moon Il Kim

    Abstract : Prussian blue nanoparticles (PBNPs) have gathered an intense attention in diverse fields, based on their unique characteristics such as excellent electrochemical and optical properties, high adsorbing capacity due to the porous nature, enzyme-like multiple catalytic activities, and favorable biocompatibility and biosafety. Particularly, their biotechnological applications for developing potent methods in therapeutics, diagnostics, and remediation hold significant promise. In this review, recent researches on PBNPs, including the synthetic strategies and relevant physicochemical properties, mechanisms of catalytic activity and phototherapy, potentials as drug carrier and scavenger of reactive oxygen species, remediation capability to adsorb/separate/degrade environmentally-harmful molecules, and representative studies are reviewed and discussed. Current challenges and prospects for the utilization of PBNPs in biotechnology are also discussed.

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  • Research Paper 2022-09-30

    Secretional Expression of Glutamate Decarboxylase Gene derived from Streptococcus thermophilus in Bacillus Subtilis: Its Biological Activities by GABA Production

    Minsoo Kim and Jang Won Choi

    Abstract : Gamma-aminobutyric acid (GABA) can be converted from monosodium glutamate (MSG) by glutamate decarboxylase (GAD). GABA is known to be a four-carbon non-protein amino acid existing in the human brain and eye. Recent studies have shown that GABA is effective in accelerating hypotension and anxiety relief, along with acting as a diuretic, antidiabetic, and antidepressant in humans and other mammals. Since the low activity of cell preparations has been a major limitation to commercial enzyme synthesis, it is worthwhile to look for methods to obtain a affordable recombinant enzyme with high GABA synthesizing activity. In order to maximize production of GABA using the culture broth without purification in Bacillus subtilis, the gene (gadB) first was obtained from chromosome of Streptococcus thermophilus (S. thermophilus) expressing the GAD with appropriate amount. The primer set with restriction enzyme sites and His-tag was designed by referring to the DNA sequence of glutamate decarboxylase enrolled in GenBank (DQ871217.2), amplified and cloned into pGemTeasy vector. The right clone was screened and characterized by DNA sequencung analysis. As a result, the DNA sequence was different from that of GenBank in codons encoding 4 amino acids (Tyr189, Gly282, Ser301, and Met425) by point mutations, which were changedto Cys189, Asp282, Gly301, and Thr425, respectively. To secret the GAD in culture medium, the recombinant vector (pRBASGadB, 7.83 kb) containing the gadB gene was constructed using pRBAS secretion vector with alkaline protease promoter (aprE-P, 0.45 kb) and signal sequence (aprS, 87 bp) and then transferred into Bacillus subtilis LKS87. The secretion levels of GAD were analyzed by SDS-PAGE and western-blot and the level in B. subtilis LKS87 harboring the pRBAS-GadB vector was increased to approximately 10-12% of total secreted proteins. The conversion of MSG (monosodiumglutamate) to GABA was measured by paper chromatography using the culture broth and PBS-eluted cell pellet. As a result, the culture broth shows 16% GABA conversion rate from MSG, whereas the pellet shows higher conversion (~43%), indicating that the expressed GAD was not sufficiently secreting into the culture broth caused by the inefficient processing of aprS signal sequence or presence of self signal sequence of the GAD gene. By measuring the ABTS+ scavenging ability, it was confirmed that the GABA converted by GAD showed higher antioxidant activity (55%) than that (50%) of ascorbic acid used as positive control. The survival recovery rate of STZ-treated RINm5F cells was increased to 28.2% by the GABA treatment and also, production of nitric oxide (NO) was drastically reduced with 78.7% in STZ-treated RINm5F cells, suggesting that the GABA possess antidiabetic and antioxidant activities. Finally, although GABA is showing various biological activities, it might be needed to optimize secretion efficiency for industrial applications.

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  • Review Paper 2023-06-30

    Current Status and Development Prospect of Expression Vector Based on Systematic Approach

    Gi-Hye Park, Su-Kyoung Yoo, and Geun-Joong Kim

    Abstract : Plasmid-based expression vectors, the most useful genetic tool in recombinant DNA technology, carry the foreign gene into the cell, and keep it stable, thereby enabling its expression within cells. These vectors typically have a replication origin, selection marker, and promoter as main genetic parts. Each of them enables chromosome-independent replication within cells, exerts pressure for the selection of recombinant cells, and controls the expression of carried foreign genes. Many studies have reported to design and improve vectors by using the targeted replacement and/or mutagenesis to the aforementioned major parts and spanning sequence. Despite the consistent reports on expression vectors with new functions, such approach has some constraints of using unstandardized genetic parts limited in functional sequence space. As an alternative, synthetic biology provides a foundation for tailor-made expression systems by rational design and modification via assembling various modular parts. Nevertheless, due to the limited number of modular parts and the lack of diversity of vector scaffold that offer the basis for assembly of these components, it falls short of constructing the expression systems feasible to graft into getting more useful expression system. This review will give a brief overview of the three major components and current status of vector design for expression of foreign genes, and then discuss the prospect of developing expression systems based on the systematic approaches, consequently expecting to understood the new platform for construction and improvement on target-oriented expression vectors via assembly of functional modules and scaffolds.

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  • Review Paper 2023-03-31

    Current Status of Bacillus subtills Spore Surface Display System and Its Biotechnological Applications for Two Decades

    Junehyung Kim

    Abstract : For the last two decades, Bacillus subtilis spore display system provided new platform for the various biotechnological applications, which overcame many obstacles of traditional bacterial surface display technology. Spore surface display needs Bacillus subtilis spore coat protein such as cotB, cotC and cotG for the anchoring of target protein. When the spore formation is completely done, mother-cell lyse, and spore is liberated to the environment. Because spore display system does not need secretory mechanism through bacterial cell wall or membrane, spore-based display technology has enhanced capability for the display of multimeric protein, large size protein, and co-factor associated protein which could disrupt cell membrane and cell wall of traditional bacterial surface display system. With spore’s nascent robustness, spore surface display technology provides enhanced thermal stability, wide pH stability, organic solvent stability and repeated usage performance in whole cell enzyme reaction. Bacillus subtilis has been Generally Regarded as Safe (GRAS) for the traditional usage in soybean fermented food in Asia. This GRAS characteristics of Bacillus subtilis and its spore has been used for the development of spore based oral vaccine against human, feedstock cattle, fish, and worms. Spore display technology is widening its application in the fields of biosensor, environment, animal nutrition supplementation. In this paper, we describe recent development and application of spore surface technology and present new perspective for the near future.

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  • Research Paper 2022-12-30

    Detection of Alzheimer’s Disease Biomarker using Anti-NFL Antibody Conjugated Gold Nanoparticles

    Hee-Won An, Chi-Yeon Song, Yu-Rim Ahn, Ji-Yeon Park, Miso Jeong, Hyun-Hee Ryu, Sukchan Lee, Hyun-Ouk Kim

    Abstract : Neurofilament light chain (NFL), one of the subunits of neurofilaments present inside neurons, is known as a biomarker of nerve axonal damage in early stage of Alzheimer's disease. Measuring the level of NFL is being actively studied worldwide because it can determine the extent of damage to nerve axons, especially in the early stages of Alzheimer's disease. However, ELISA based NFL detection techniques have several limitations because of expensive equipment and high cost. Herein, we perform the application of antibody conjugated Gold nanoparticles (ACNP) to detect NFL conveniently and efficiently. First, to conjugate Gold nanoparticles and antibody, carboxyl groups are modified on the particle surface to react the amine group of the antibody using EDC/NHS. Then, the prepared ACNP react with the NFL protein to detect NFL. ACNP were characterized by DLS, ELS, Microplate Reader and TEM. Detected NFL by ACNP was confirmed by Western blotting, and the detection efficiency and limit of detection were confirmed through the absorbance change. This platform for detecting NFL using ACNP, which provides a cheap, selective, and convenient response, is a promising tool for diagnosis of neuronal damage through NFL detection in the physiological-pathological range.

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  • Research Paper 2022-06-30

    Bioconversion of Brown Algae Sargassum horneri into Ethanol by Simultaneous Saccharification and Fermentation by Mannitol-fermenting Saccharomyces cerevisiae

    Jueun Jang, In Jung Kim, Suhyeung Kim, Jamin Shin, Sejin Geum, and Soo Rin Kim

    Abstract : Sargassum horneri is a sea-drifting brown macroalga often found along the coast of East Asian countries. It was recently found to be drifting from China toward Jeju Island in South Korea, causing damage to fisheries and vessels. Being considered as a marine waste, a huge amount of S. horneri was collected in the past 5 years, but an efficient and proper way to treat it has still not been found. Therefore, it is required to develop technologies to tackle this issue. Here, we conducted bioconversion of S. horneri by the yeast Saccharomyces cerevisiae in order to utilize it as a biomass source for producing ethanol. First, S. cerevisiae was engineered to extend its substrate range to mannitol, which is one of the major components of brown algae. Activation of the native HXT17 and MAN2 genes enabled the yeast to metabolize mannitol as the sole carbon source. Impact of pretreatment conditions, the type of hydrolytic enzymes, and biomass solid loadings on the ethanol production by the yeast were evaluated. The highest ethanol productivity was obtained when the biomass was pretreated at 121oC and ethanol concentration was the highest when the biomass loading was 24% (w/v), giving the maximum concentrations of monosaccharides and ethanol of 47.29 g/ L and 22.94 g/L, respectively. The results obtained from this study suggest possible utilization of S. horneri as a raw material for cellulosic bioethanol production.

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  • Research Paper 2022-06-30

    Expression of Green Fluorescence Proteins with Non-canonical Amino Acids in Different Escherichia coli Strains

    Jiae Kim and Jong-il Choi

    Abstract : 10 different Escherichia coli strains were evaluated for the host of recombinant proteins with non-canonical amino acids. For the incorporation of non-canonical amino acids, genetic code expansion was performed using a green fluorescent protein, which was mutated to have an amber codon at 204th amino acid. The expression level of green fluorescence proteins and the amber suppression of amino-acyl tRNA synthetase were compared among 10 different E. coli strains. As a result of comparison, E. coli W, DH10B, and EPI300 were shown to be efficient hosts for the expression of recombinant fluorescent protein incorporated with noncanonical amino acids. The results of this study can be utilized not only for selecting a host for the production of recombinant therapeutic proteins with non-canoncial amino acids, but also for the development and improvement of aminoacyltRNA synthetase capable of incorporating new non-canonical amino acids.

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  • Research Paper 2022-06-30

    Evaluation of Bioactivity of Castanopsis cuspidata var. sieboldii Leaves Extract and Isolation of Polyphenolic Compounds

    Nain Kim, Moon-Hee Choi, Seung-Hwa Yang, Deuk-Sil Oh, and Hyun-Jae Shin

    Abstract : Castanopsis cuspidata var. sieboldii (CCS) is a warm-tempered tree that lives on the korean peninsula and has been traditionally used in medicine. Bioactive ingredients from aerial parts of CCS have been identified by several researchers along with anti-inflammatory, antioxidant and whitening effects. However, isolation study of compounds centered on antioxidant activity in CCS leaves (CSL) has not been investigated. In this study, CSL were extracted using 70% ethanol and used as a crude extract. CSL was divided into five fractions using n-hexane, chloroform, ethyl acetate, n-butanol and water. Also, the antioxidant activity of subfraction was determined with DPPH free radical scavenging activity assay and ABTS cation radical scavenging activity assay. The antioxidant contents was confirmed by total polyphenol contents (TPC) and total flavonoid contents (TFC). Through the HPLC qualitative analysis, it was confirmed that CSL and CSL-EA contained various polyphenols. Based on these results, we suggest that extracts, fractions and compounds isolated from CSL provide promising antioxidant abilities and natural cosmetic materials.

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  • Research Paper 2022-06-30

    Development of an Antibody Probe for Specific Detection of Ammonia-Oxidizing Archaea

    Yeon-Jin Chu, Dae-Eun Cheong, and Geun-Joong Kim

    Abstract : Ammonia oxidation is the first step of nitrification and plays an important role in global nitrogen cycle. Over the past 20 years, it has been founded that archaeal amoA gene is more abundant than bacterial amoA gene in diverse habitats. However, most of ammonia-oxidizing archaea (AOA) discovered by molecular techniques so far have not been cultivated yet in pure state. Thus, alternative methods of probing AOA are urgently needed. In this study, we attempted to produce antibody probes that specifically bound to a marine AOA strain, by generating polyclonal antibody using a subunit of specific ammonia monooxygenase (AMO) for ammonia oxidation. The resulting antibody could be used for detection of the AOA strain as illustrated by immunofluorescence staining in this work. Our results thus indicate that the typically generated polyclonal antibody would have broad in-vitro and in-situ applications in environmental microbiology and biotechnology.

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  • Review Paper 2022-09-30

    Recent Advances in Pathogenic Bacteria Detection with Antimicrobial Peptide as Recognition Element

    Jung Eun Kim, and Jong Pil Park

    Abstract : Detection of pathogenic bacteria using antimicrobial peptides can be an alternative way over traditional detection method. Due to its high stability and low cost, it has been studied as a bio-recognition of biosensors. Antimicrobial peptides can be produced naturally to protect host cells from pathogenic bacteria or chemically synthesized in the laboratory with well-known methods. Antibacterial peptides can interact electrostatically with the membrane of pathogenic bacteria, an anionic molecule, and become a source of bacterial detection using electrochemical and optical biosensors. In this review, overall characteristics of antibacterial peptides and its use in biosensor development were summarized.

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December 2023 Volume 38, No.4

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Korean Society for Biotechnology and Bioengineering Journal
pISSN 1225-7117 eISSN 2288-8268