• Journal of Applied Biological Chemistry
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• v.65 no.4
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• pp.383-386
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• 2022

A gene encoding thermostable cellulase B (TmCelB) was isolated from Thermotoga maritima. The open reading frame (ORF) of TmCelB gene was 825bp long which predicted to encode 274 amino acid residues with a molecular weight of 31,732 Da. The 17 amino acid residues from N-terminal of the TmCelB was known as signal peptides. To analyze the enzymatic activity and biochemical properties, the ORF of TmCelB gene excluding a putative signal sequence encoding 17 amino acids were introduced into the E. coli expression vector, pRSET-B, and overexpressed in E. coli BL21. The optimum temperature of recombinant TmCelB was around 95 ℃, and the optimum pH of recombinant TmCelB was around pH 4.5. The recombinant TmCelB was stable at temperature below 100 ℃.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.82-88
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• 2023

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni²⁺-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type I spectral changes, with K_d values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k_cat value of 0.038 ± 0.002 min^-1 and a K_m value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k_cat value of 0.135 ± 0.007 min^-1 and a K_m value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

• Journal of the Korean Society of Marine Environment & Safety
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• v.29 no.1
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• pp.36-43
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• 2023

To establish a culture system with enhanced cellular nutrition, we investigated the effects of light quality (blue, 450 nm; yellow, 590 nm; and red, 630 nm) of a light-emitting diode (LED) on the biochemical composition of Tetraselmis suecica and T. tetrathele. The protein content of both species was higher (42-69%) than the content of other biochemical substances under all wavelengths. Carbohydrate, protein, and lipid contents were higher under the yellow wavelength, which showed a low growth rate, than those under other wavelengths. The contents of all biochemical substances were low under the red wavelength, which showed a high growth rate. These results indicated that protein synthesis occurs in response to decreased cell division rate, while lipid and carbohydrate synthesis occurs owing to altered chemical composition and enzymatic activity. Therefore, we suggested a two-phase LED culture system, which emitted red LED during the early-middle exponential phase and yellow LED during the late exponential and stationary phases, to increase the yield of useful biochemical substances of T. suecica and T. tetrathele.

• Applied Chemistry for Engineering
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• v.35 no.4
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• pp.273-283
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• 2024

As the adverse environmental impacts due to plastic waste become more severe, there is an increasing demand for developing a sustainable ecosystem using biodegradable polymers. Biodegradable polymers are those that can be biochemically decomposed through the enzymatic activity of microorganisms. Currently, a variety of biodegradable polymers with varying properties is being investigated. In particular, polymer blends with an aim to control the biodegradation rate and mechanical properties are under active research. The biodegradable polymer industry, which has not yet reached economies of scale, does not have a cost advantage compared to petroleum-derived polymers. To overcome this challenge, there is an urgent need to expand its application fields to various high-value industries (separators, electronic materials, and medical fields). This review summarizes the current state-of-the-art biodegradable polymers, polymer blends, and recent research trends in new niche applications.

• Journal of Pharmacopuncture
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• v.27 no.3
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• pp.245-252
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• 2024

Objectives: Methotrexate (MTX), an immunosuppressant and anti-cancer medication, can harm the heart. The goal of the current investigation was to assess the cardiotoxicity caused by MTX and the potential cardioprotective properties of silymarin, citral, and thymoquinone as antioxidants. Methods: Forty-eight rats were divided into six groups, which included control, MTX, cosolvent, citral, thymoquinone, and silymarin groups. At the end of the study, the rats were anesthetized (ketamine and xylazine) and killed using CO2. Their blood samples were collected to measure the enzymatic activities of creatine kinase-myoglobin binding (CK-MB), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH). Also, the heart tissue was sampled to determine the antioxidant capacity and examine the histopathology. Results: The findings revealed that the activity of CPK, CK-MB, and LDH enzymes significantly reduced in the thymoquinone treatment group compared to the MTX group (p < 0.05). On the other hand, total antioxidant capacity was significantly increased in the thymoquinone group compared to the MTX group (p < 0.05). The pathological modifications (i.e. severe congestion, edema fluid, the presence of inflammatory cells around the blood vessels, mild to moderate hemorrhaging between cardiac muscle fibers) were seen in the MTX group. The treatment groups, particularly thymoquinone, did not experience any appreciable pathological changes. Conclusion: The thymoquinone was found to have the strongest protective effect against the heart damage caused by MTX.

• Tuberculosis and Respiratory Diseases
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• v.60 no.3
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• pp.304-313
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• 2006

Background : Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. The current evidence has indicated a critical role of HO-1 in cytoprotection and also in other, more diverse biological functions. It is known that the high expression of HO-1 occurs in various tumors, and that HO-1 has an important role in rapid tumor growth because of its antioxidative and antiapoptotic effects. Therefore, the role of HO-1 was analyzed in human lung cancer cell lines, and especially in the A549 cell line. Material and Methods : Human lung cancer cell lines, i.e., A549, NCI-H23, NCI-H157 and NCI-H460, were used for this study. The expression of HO-1 in the untreated state was defined by Western blotting. ZnPP, which is the specific HO inhibitor we used, and the viability of cells were tested for by conducting MTT assaysy. The HO enzymatic activity, as determined via the bilirubin level, was also indirectly measured. Moreover, the generation of intracellular hydrogen peroxide (H2O2) was monitored fluorimetrically with using a scopoletin-horse radish peroxidase (HRP) assay and 2',7'-dichlorofluorescein diacetate (DCFH-DA). We have also transfected small HO-1 interfering RNA (siRNA) into A549 cells, and the apoptotic effects were evaluated by flow cytometric analysis and Western blotting. Results : The A549 cells had a greater expression of HO-1 than the other cell lines, whereas ZnPP significantly decreased the viability of the A549 cells more than the viability of the other lung cancer cells in a dose-dependant fashion. Consistent with the viability, the HO enzymatic activity also was decreased. Moreover, intracellular H2O2 generation via ZnPP was induced in a dose-dependent manner. Apoptotic events were, then induced in the HO-1 siRNA transfected A549 cells. Conclusion : HO-1 provides new important insights into the possible molecular mechanism of the antitumor therapy in lung cancer.

• Applied Biological Chemistry
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• v.43 no.2
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• pp.86-94
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• 2000

In order to develop the enzymatic hydrolysis system concerned with taste and flavor, strains having the high hydrolyzing activity on the soy protein were selected from some traditional Mejus. Two molds and one bacterium producing enzymes which were different in character of hydrolysis were isolated and identified. Leucine and azodye enzyme activities of both M4 and M5 were relatively high among in the isolated molds. And, leucine enzyme activity of B16 was the lowest in the isolated bacteria. These strains were isolated as microorganisms having a dissimilar hydrolysis pattern on the soy protein by enzymatic reactions. Mold M4 on the culture solid media was mycelium colors of white and its sclerotia colors were changed from white to black. According to the result of slide culture, radial conidial head, subclavate vesicle, conidia of subglobose, stipes of uncolored with smooth walls and metula and phialides were existed. Because M4 was taxonomically similar to the characteristics of Aspergillus oryzae (ahlburg) species, M4 was identified and named as Aspergillus oryzae M4.Mold M5 showed white and black mycelium on the MEA medium. Mold M5 colony exhibited grayish-green color and have long(7 mm) sporangiophores at slide culture. Sporangia became brownish-gray and the wall of larger sporangia was broken to form small collars, and smaller sporangia were fomed continually from large basal membrane. Columella is globose and hyaline, and sporangiospores are ellipsoidal of small diameter$(80\;{\mu}m)$. Because M5 was taxonomically similar to the Mucor circinelloides of zygomycetes, M5 was was identified and named as Mucor circinelloides M5. Bacteria B16 colony was opaque white, circular and lobate, and had rod shaped endospore. B16 was found positive in stain, catalase, ${\beta}-glucosidse$ and V-P tests. B16 was found to utilize D-fructose, ${\alpha}-D-glucose$, maltose, D-mannose, D-raffinose, stachyose and sucrose. By the morphological and physiological results, the characteristics of B16 was thought to correspond to that of Bacillus megaterium. However, fatty acid composition was similar to Paenibacillus marcerans, requiring further study for the definite identification. Accordingly, Bacteria B16 was provisionally classified and named as Bacillus megaterium B16.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.3
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• pp.307-314
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• 2006

This study was conducted to screen in vitro angiotensin converting enzyme (ACE) inhibitory activities of methanol (MeOH) and aqueous extracts which were prepared by four different extractions-80% methanol extracts(ME) at $20^{\circ}C\;and\;70^{\circ}C$, respectively and aqueous extracts (AE) at both temperatures with the residue of the MEs-of ten marine green algae and nineteen brown algae collected along Jeju coast of Korea. Most marine brown algae extracts showed higher capacities than those of marine green algae in ACE inhibitory activity. Particularly, $70^{\circ}C$ MeOH extract (70ME) of Hizikia fusiforme showed the strongest inhibition activity (about 87%) among all the extracts. Also, 70 MEs of Enteromorpha linza, Ishige sinicola, Laminaria ochotensis, Petrospongium rugosum, Sagrassum horneri, Undaria pinnatifida and $20^{\circ}C$ MeOH extracts (20ME) of Myagropsis myagroides, Petrospongium rugosum, $20^{\circ}C$ aqueous extracts (20AE) of Codium contractum, Enteromorpha compressa, and $70^{\circ}C$ aqueous extracts (70AE) of Ecklonia cava, Petrospongium rugosum showed moderate ACE inhibitory activities more than 50% and the other extracts exhibited weak activities. On tile other hand, E. cava had the best ACE inhibitory activity among 70AEs. This indicates that 70AE of E. cava contains potential anti-ACE macromolecular. We tried to proteolytic digest 70AE of E. cava to induce production of anti-ACE peptides from E. cava 70AE. The enzymes used are five pretenses including Kojizyme, Flavourzyme, Neutrase, Alcalase, and Protamex, which are food grade-commercial enzymes from Novo Co. Flavourzyme-digest of E. cava 70AE showed the highest inhibitory activity about 90%. And the five different enzymatic digests of the E. cava 70AE ranged from 2.33 to 3.56 ${\mu}g/mL$, respectively in $IC_{50}$ values of anti-ACE activity.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.192-201
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• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.961-967
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• 2005

Macrophage migration inhibitory fartor (MIF), known as a cytokine, is a multifunctional protein that is ubiquitously expressed in a variety of cells and tissues; however, enzymatic function of MIF still remains elusive in cells. In this study, we assessed details of the tautomerase activity of MIF. We established rapid purification condition for MIF by using pGEX system and compared the L-dopachrome tautomerase activity of GST-MIF, tMIF, and MIF. The results show that GST (glutathione S-transferase)-epitope tag or N-terminal amino acids flanking the essential $P^{2}$ almost completely abrogated L-dopachrome tautomerase activity of MIF. Subsequently, to determine whether the N-terminal tags have effects on oligomerization of MIF, protein cross-linking products were analyzed on $15\%$ SDS-PACE. The result demonstrates that N-terminal tags are dispensable for the formation of MIF's homooligomers. Thus, the results imply that exposure of If containing hydrophobic pocket in the active site is critical for L-dopachrome tautomerase activity of MIF. In addition, our study suggest that the MIF's tautomerase activity might be influenced by interacting with cellular partners.