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

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.

Oxygen Transfer Rate (OTR) Prediction Model for CHO Cell Culture in Shake Flasks

Abstract : Shake flasks are widely used as small-scale mammalian culture vessels during cell line development and bioprocess optimization for the production of biopharmaceuticals. However, the lack of information as well as controllability on dissolved oxygen often leads to oxygen limitation that can adversely affect cell growth and productivity during cell culture, thereby challenging cell line development and scale-up processes. In this work, we report an oxygen transfer rate (OTR) prediction model of shake flasks using Chinese hamster ovary (CHO) cells to avoid oxygen limitation issues in shake flasks. First, we varied shaking speed and culture volume to measure maximum OTR (OTR_max) and created an OTR_max regression function. Then, we estimated theoretical maximum viable cell density with the specific oxygen uptake rate (sOUR) of CHO cells and the OTR_max values. Lastly, we tested if decreasing culture volume or increasing shaker speed could mitigate the adverse effect of oxygen limitation. In conclusion, we believe this OTR prediction model would provide information about the oxygen availability in shake flasks, presumably assisting to achieve better culture performances.

Electropolymerized Molecularly Imprinted Polymers (EMIPs)-Based Electrochemical Sensor

Abstract : Biosensors have been widely studied and developed as a tool in various fields, including pharmaceutical, food safety, environmental and medical applications. Antibodybased immunosensors are considered as gold standard method, however, more advanced biosensor without need of antibody as receptor is needed. Molecularly imprinted polymers (MIPs) are synthetic receptors obtained by polymerization of functional monomers in the presence of the target molecule and can form high specific binding sites to the target molecules depending on shape, size, and functionality. One of the most synthesis of MIPs is electropolymerization that use electrochemical method. This method can easily manipulate the desired morphology and thickness of the polymer films. Various EMIPs (electropolymerized molecular imprinted polymers) based biosensors have been developed for detection of small molecule such as chemicals, drugs, and toxins. In this review, we summarize the synthetic methods and characteristics of EMIPs-based biosensor for various targets.

Microbial Production and Industrial Applications of Keratinase

Abstract : Keratin, which is the hard and insoluble structural protein found in many parts of birds, reptiles, and tissues such as feathers, hair, horns, wool. Some keratinaceous materials such as feathers known as industrial wastes, hard to degrade naturally because of their complex structure. To date, several bacteria and fungi have been discovered that have keratinase activity, which breaks down keratin into amino acids. In addition, various studies are being conducted to increase the activity of this enzyme through of recombinant keratinase or to facilitate purification. This potential enzyme can be used not only for the degradation of keratinaceous industrial wastes but also for the leather and healthcare industries. In this review, keratin degradation mechanisms, keratinase production, and their industrial applications will be discussed.

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

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.

Effect of Macroporous Gelatin Cryogel as Biomaterial Scaffold on Osteogenic Differentiation of Mesenchymal Stem Cells and Direct Conversion of Fibroblasts into Osteoblasts

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

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.

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

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.

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

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.

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

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.