• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1705-1713
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• 2012

A gene encoding ${\beta}$-glucosidase was cloned from Weissella cibaria 37, an isolate from human feces. Sequence analysis showed that the gene could encode a protein of 415 amino acids in length, and the translated amino acid sequence showed homology (34-31%) with glycosyl hydrolase family 1 ${\beta}$-glucosidases. The gene was overexpressed in E. coli BL21(DE3) using pET26b(+) and a 50 kDa protein was overproduced, which matched well with the calculated size of the enzyme, 49,950.87 Da. Recombinant ${\beta}$-glucosidase was purified by using a his-tag affinity column. The purified ${\beta}$-glucosidase had an optimum pH and a temperature of 5.5 and $45^{\circ}C$, respectively. Among the metal ions (5mM concentration), $Ca^{2+}$ slightly increased the activity (108.2%) whereas $Cu^{2+}$ (46.1%) and $Zn^{2+}$ (56.7%) reduced the activity. Among the enzyme inhibitors (1 mM concentration), SDS was the strongest inhibitor (16.9%), followed by pepstatin A (45.2%). The $K_m$ and $V_{max}$ values of purified enzyme were 4.04 mM and 0.92 ${\mu}mol/min$, respectively, when assayed using pNPG (p-nitrophenyl-${\beta}$-D-glucopyranoside) as the substrate. The enzyme liberated reducing sugars from carboxymethyl cellulose (CMC).

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.935-940
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• 2005

Fluorescence change of coumarin labeled phosphate binding protein (PBP-MDCC) was monitored to measure the amount of released inorganic phosphate ($P_{i}$) during nucleoside triphosphate (NTP) hydrolysis reaction. After purification of PBP-MDCC, fluorescence emission spectra showed that fluorescence responded linearly to $P_{i}$ up to about 0.7 molar ratio of $P_{i}$ to protein. The correlation of fluorescence signal and $P_{i}$ standard was measured to obtain [$P_{i}$] - fluorescence intensity standard curve on the stopped-flow instrument. When T7 bacteriophage helicase, double-stranded DNA unwinding enzyme using dTTP hydrolysis as an energy source, reacted with dTTP, the change of fluorescence was able to be converted to the amount of released $P_{i}$ by the $P_{i}$ standard curve. $P_{i}$ release results showed that single-stranded Ml3 DNA stimulated dTTP hydrolysis reaction several folds by T7 helicase. Instead of end point assay in NTP hydrolysis reaction, real time $P_{i}$-release assay by PBP-MDCC was proven to be very easy and convenient to measure released $P_{i}$.

• International Journal of Stem Cells
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• v.10 no.1
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• pp.1-11
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• 2017

Human cardiomyocytes (CMs) cease to proliferate and remain terminally differentiated thereafter, when humans reach the mid-20s. Thus, any damages sustained by myocardium tissue are irreversible, and they require medical interventions to regain functionality. To date, new surgical procedures and drugs have been developed, albeit with limited success, to treat various heart diseases including myocardial infarction. Hence, there is a pressing need to develop more effective treatment methods to address the increasing mortality rate of the heart diseases. Functional CMs are not only an important in vitro cellular tool to model various types of heart diseases for drug development, but they are also a promising therapeutic agent for cell therapy. However, the limited proliferative capacity entails difficulties in acquiring functional CMs in the scale that is required for pathological studies and cell therapy development. Stem cells, human pluripotent stem cells (hPSCs) in particular, have been considered as an unlimited cellular source for providing functional CMs for various applications. Notable progress has already been made: the first clinical trials of hPSCs derived CMs (hPSC-CMs) for treating myocardial infarction was approved in 2015, and their potential use in disease modeling and drug discovery is being fully explored. This concise review gives an account of current development of differentiation, purification and maturation techniques for hPSC-CMs, and their application in cell therapy development and pharmaceutical industries will be discussed with the latest experimental evidence.

• Food Engineering Progress
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• v.13 no.4
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• pp.326-334
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• 2009

Blood coagulation and aggregation of platelet are crucial events in the pathogenesis of various ischemic diseases. The substance which can prevent blood coagulation and platelet aggregation was extracted from wood ear mushroom (Auricularia auricular-judae) and its anticoagulation activity was investigated. The dried A. auricular-judae was extracted with 0.1 N NaOH and its supernatant was further extracted with methanol and ethanol followed by $H_{2}O$. The resulting methanol soluble fraction showed significant antithrombotic activity in activated partial thromboplastin time, thrombin time, and prothrombin time assays with values of 100, 124, and 54 sec, respectively. This active substance was purified with DEAE-Sepharose CL6B and Sephacryl 400-HR and was found to be polysaccharide with the average molecular weight of over 150 kDa. This polysaccharide was xyloglucomannan of which the main component was mannose, and its anticoagulant activity was mostly mediated by inhibition of thrombin activity.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1470-1477
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• 2019

${\beta}_2$-adrenergic receptor (${\beta}_2-AR$) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of ${\beta}$-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and $59.57{\mu}g/l$, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was $3.2{\mu}g/l$ and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified ${\beta}_2-AR$ would have great application potential in detection of ${\beta}$-agonist residues.

• Membrane Journal
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• v.31 no.5
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• pp.293-303
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• 2021

Separation membranes used in water filtration, protein purification or biomedical filtration device frequently undergo membrane fouling for several reasons. The formation of biofilm on the membrane surface by bacteria causes a severe problem for durability of the membrane. For the protein separation, the membrane pores get blocked due to surface hydrophobicity of the membrane. There are several approaches controlling the membrane fouling and one of them is the incorporation of silver nanoparticles. Antibacterial properties of silver nanoparticles are well known and thus widely used in several applications. In this review, we have focused on the membranes where silver nanoparticles or its derivatives are either incorporated in the active layer of thin film composite membranes or uniformly distributed throughout the whole membranes.

• Korean Journal of Agricultural Science
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• v.48 no.4
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• pp.1003-1013
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• 2021

Thioredoxin (TRX) protein is an antioxidant responsible for reducing other proteins by exchanging cysteine thiol-disulfide and is also known for its anti-allergic and anti-aging properties. On the other hand, epidermal growth factor (EGF) is an important material used in the cosmetics industry and an essential protein necessary for dermal wound healing facilitated by the proliferation and migration of keratinocytes. EGF also assists in the formation of granulation tissues and stimulates the motility of fibroblasts. Hence, genetically modified soybeans were developed to overexpress these industrially important proteins for mass production. A single-dose oral-toxicity-based study was conducted to evaluate the potential toxic effects of TRX and EGF proteins, as safety assessments are necessary for the commercial use of seed-specific protein-expressing transgenic soybeans. To achieve this rationale, TRX and EGF proteins were mass purified from recombinant E. coli. The single-dose oral-toxicity tests of the TRX and EGF proteins were carried out in six-week old male and female Institute of Cancer Research (ICR) mice. The initial evaluation of the single-dose TRF and EGF treatments was based on monitoring the toxicity signatures and mortality rates among the mice, and the resultant mortality rates did not show any specific clinical symptoms related to the proteins. Furthermore, no significant differences were observed in the weights between the treatment and control groups of male and female ICR mice. After 14 days of treatment, no differences were observed in the autopsy reports between the various treatment and control groups. These results suggest that the minimum lethal dose of TRX and EGF proteins is higher than the allowed 2,000 mg·kg^-1 limit.

• Korean Journal of Agricultural Science
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• v.48 no.4
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• pp.935-944
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• 2021

It has been suggested that bisphenol A (BPA), a known endocrine disruptor, interferes with the endocrine system, causing reproductive dysfunction. Recently, BPA has been found in waste water due to incomplete sewage purification, possibly threatening health through its ingestion via tap water. In this study, young male mice (6 - 7 weeks old) were administered water containing BPA (50 mg·kg^-1) for four weeks, while control mice consumed water without BPA. Serum, epididymal spermatozoa and testicular sections were assessed after sacrificing the mice on day 28. No significant differences were obtained between the groups in the body, testis and seminal vesicle weights. However, the epididymal sperm motility and count levels were significantly reduced in BPA-fed mice. Significantly higher hepatotoxicity levels were also observed in mice ingesting BPA as compared to the control mice. The level of serum testosterone was reduced, and testicular sections revealed incomplete and irregular spermatogenesis in BPA-ingested mice. The sperm proteasomal-proteolytic activity level has been implicated in sperm function and is measured in motile spermatozoa using fluorometric substrates. High ubiquitin C-terminal hydrolase activity levels were observed in the control mice without BPA. During a mating trial, a low pregnancy rate (71.4%) was observed in females mated with males who had consumed BPA (100% in the control mice). Overall, BPA adversely affected spermatogenesis and quality, as indicated by decreased sperm motility, concentration and serum testosterone levels, resulting in reduced fertility competence.

• Journal of Mushroom
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• v.21 no.4
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• pp.209-214
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• 2023

In this study, Pleurotus ostreatus No.42 was cultured in glucose-peptone-yeast-wheat bran medium using a previously reported novel rotary draft tube bioreactor. Versatile peroxidase (VP), a lignin-degrading enzyme, was isolated from a pellet-type mycelium culture grown in the medium for seven days. The VP was purified by sequentially applying ultra-filtration, DEAE-Sepharose CL-6B column, and Mono Q column. SDS-PAGE analysis revealed the molecular weight of VP to be 36.4 KDa with an isoelectric point of 3.65. The amino acid sequence was confirmed as VTCATGQTT. The purified VP was observed to possess the property of not only oxidizing Mn ions but also decomposing veratryl alcohol, a non-phenolic compound. The catalytic ability of VP is a subject for future research.