• Journal of Life Science
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• v.34 no.8
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• pp.576-587
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• 2024

In this study, diverse bacterial strains were isolated from fermented foods to screen those with antibacterial activity. Among them, one strain, identified as Bacillus vallismortis MCBL 1012 through 16S rRNA gene sequence analysis, was selected for its bacteriocin production. The culture supernatant of B. vallismortis MCBL 1012 showed antibacterial activity, mainly against Gram-positive bacteria. Scanning electron microscopy (SEM) revealed that bacteriocin treatment led to cellular content leakages in Listeria monocytogenes KCCM 40307, Enterococcus faecium KCCM 12118, and Streptococcus mutans KCTC 3065. PCR analysis confirmed B. vallismortis MCBL 1012 harbored subtilosin A gene (sbo A). Antibacterial activity was decreased by proteolytic enzymes like proteinase K, subtilisin A, and α-chymotrypsin. The bacteriocin demonstrated stability at 40℃ and 60℃ for 120 min, and up to 80℃ for 60 min, with rapid activity loss at 100℃. It retained full antibacterial activity within a pH range of 4.0 to 8.0 and was not affected by up to 100% organic solvents like ethanol, methanol, acetonitrile, and tetrahydrofuran. Nevertheless, activity decreased with more than 40% isopropanol and 80% acetone. Most tested inorganic salts and detergents had no effect on antibacterial activity except, CuSO₄ and NiSO₄ at specified concentrations. The bacteriocin exerted its antibacterial effect through bactericidal action against L. monocytogenes KCCM 40307. The bacteriocin was purified by ammonium sulfate precipitation, DEAE anion exchange chromatography, and RP-HPLC. The purification resulted in a final yield of 0.03% and a 283.7-fold increase in specific activity. MALDI-TOF MS analysis determined the exact molecular weight of purified bacteriocin to be 3,326.1 Da.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.489-494
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• 2008

In order to develop a potent antidementia $\beta$-secretase inhibitor from phytochemicals, $\beta$-secretase inhibitory activities of extracts from many medicinal plants and herbs were determined. Water extracts from Rubus coreanus showed the highest $\beta$-secretase inhibitory activity of 84.5%. After purification of the $\beta$-secretase inhibitor from R. coreanus using systematic solvent extraction, ultrafiltration, Sephadex G-10 column chromatography, and reverse-phase high performance liquid chromatography (HPLC), a purified $\beta$-secretase inhibitor with $IC_{50}$ inhibitory activity of $6.3{\times}10^3\;ng/mL$ ($1.56{\times}10^{-6}\;M)$ was obtained with a 0.08% solid yield. The molecular mass of the purified $\beta$-secretase inhibitor was estimated to be 576 Da by liquid chromatography-mass spectrometry (LC-MS) and $\beta$-secretase inhibitor also is a new tetrapeptide with the sequence Gly-Trp-Trp-Glu. The purified $\beta$-secretase inhibitory peptide inhibited $\beta$-secretase non-competitively and also show less inhibition on trypsin, however no inhibition on other proteases such as $\alpha$-secretase, chymotrypsin, and elastase.

• Animal cells and systems
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• v.14 no.1
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• pp.25-30
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• 2010

We employed a new and improved differential display reverse transcription-polymerase chain reaction (DDRT-PCR) method, which involves annealing control primers (ACPs), to identify the genes that are specifically or prominently expressed in olive flounder (Paralichthys olivaceus) juveniles (35 days post-hatch; dph) compared to larval-stage (dph 21) flounder. Using 60 ACPs, we identified eight differentially expressed genes (DEGs) and basic local alignment search tool (BLAST) searches revealed eight known genes. Gene expression levels were confirmed by RT-PCR. Phosphoglucose isomerase (PGI) was highly expressed at 21 dph, while nephrosin, myosin light chain (MLC), myosin heavy chain (MHC), carboxypeptidase A, chymotrypsin B, fish-egg protein, and matrix protein were expressed at 35 dph. PGI, MLC, and MHC expression was further analyzed by RT-PCR. The differentially expressed genes identified in this study may provide insights into the molecular basis of development in olive flounder.

• Journal of Marine Bioscience and Biotechnology
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• v.7 no.1
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• pp.19-27
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• 2015

The objective of the current study was to evaluate the inhibitory and antioxidant activities of powdered katsuobushi (dried bonito) protein hydrolysates and their corresponding fractions. The powdered katsuobushi (dried bonito) hydrolysates were obtained by enzymatic hydrolysis using Alcalase, ${\alpha}$-chymotrypsin, Neutrase, pepsin, papain, and trypsin. The antioxidant efficacy of the respective hydrolysates were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, superoxide, and alkyl radical-scavenging activities. Among the hydrolysates, the peptic-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, the peptic-derived hydrolysate was further analyzed, and was found to contain an active peptide with an amino acid sequence identified as Pro-Met-Pro-Leu-Asn-Ser-Cys (756 Da). The purified peptides from powdered katsuobushi (dried bonito) had an $EC_{50}$ value of $105.82{\mu}M$, and exhibited an inhibitory effect against DNA oxidation induced by hydroxyl radicals. Taken together, these results suggests that powdered katsuobushi (dried bonito) could be used as a natural antioxidant in functional foods and prevent oxidation reactions in food processing.

• Fibers and Polymers
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• v.6 no.3
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• pp.181-185
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• 2005

Silk fibroin (SF) nanofibers were prepared by electrospinning and their application as an enzyme immobilization support was attempted. By varying the concentration of SF dope solution the diameter of SF nanofiber was controlled. The SF nanofiber web had high capacity of enzyme loading, which reached to $5.6\;wt\%$. The activity of immobilized a-chymotrypsin (CT) on SF nanofiber was 8 times higher than that on silk fiber and it increased as the fiber diameter decreased. Sample SF8 (ca. 205 nm fiber diameter) has excellent stability at $25^{\circ}C$ by retaining more than $90\%$ of initial activity after 24 hours, while sample SF11 (ca. 320 nm fiber diameter) shows higher stability in ethanol, retaining more than $45\%$ of initial activity. The formation of multipoint attachment between enzyme and support might increase the stability of enzyme. From these results, it is expected that the electrospun SF nanofibers can be used as an excellent support for enzyme immobilization.

• Preventive Nutrition and Food Science
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• v.2 no.4
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• pp.321-327
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• 1997

High Temperature-cooking conditions of cultured fishes(loach, crucian carp, bastard halibut, and jacopever) were optimized by response surface methodology(RSM), and plastein products were prepared using enzymatic hydrolysis. Four models were proposed with regard to effects of time(t), temperature(T), and water/fish meat (w/f) ratio on the amount of 0.3M TCA soluble fractions. The model coefficients were ranged from p<0.0001 for jacopever to p<0.0433 for bastared halibut. Cooking conditions for 60% hydrolysis were optimized at 1) 14$0^{\circ}C$ except for crucian carp(136$^{\circ}C$); 2) 10.08 hours(loach), 7.25 hours(crucian carp), 9.85 hours(ba-stard harlibut), and 9.37 hours(iacopever); 3) 1:1(w/f) ratio except for the crucian carp(1.1:1). When protein hydrolyzates were employed for the plastein synthesis, optimum plastein-reaction conditions were determined to be pH 9.0 with chymotrypsin for the loach and crucian carp hydrolyzates, pH 9.0 with papain for the bastard halibut hydrolyzate, and pH 11.0 with trypsin for the jacopever hydrolyzate. Plastein reaction could be performed in water at concentration up to 20%(w/f).

• Proceedings of the Korean Society of Food Hygiene and Safety Conference
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• 2002.05a
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• pp.215-215
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• 2002

Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major funational domains such as dihydrplipoamide adetyltransferase(E2)-binding domain, regulatory subunit of PDP(PDP)r-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase(rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPc binds to the inner lipoyl domain (L2) of E2 of ppyruvate dehydrogenase complex (PDC) in the presence of Ca+2, not under EGTA. PDPc was limited-proteolysed by typsin, chymotypsin, Arg-C, and elastase at pH 7.0 and 30C and N-terminal analysis of the fragments was done. Chymotrypsin, trypsin, and elastase made two major fragments: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx.10 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• Food Science of Animal Resources
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• v.38 no.5
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• pp.1008-1018
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• 2018

Although bacteriocins with anti-listerial activity have been isolated from a wide variety of lactic acid bacteria, little is known about those from Leuconostoc lactis, a heterofermentative bacterium that produces diacetyl and exopolysaccharides in dairy foods. In this study, an anti-listerial bacteriocin was isolated from Leuc. lactis SD501 and characterized. It was particularly potent against Listeria monocytogenes and also inhibited Enterococcus faecalis. Anti-listerial activity reached a maximum during the early stationary phase and then decreased gradually. The anti-listerial substance was sensitive to proteinase K and ${\alpha}$-chymotrypsin, confirming its proteinaceous nature. Its activity remained stable at pH values ranging from 1 to 10. In addition, it was strongly resistant to high temperatures, retaining its activity even after incubation for 15 min at $121^{\circ}C$. The apparent molecular mass of the partially purified anti-listerial bacteriocin was approximately 7 kDa. The characteristics of the SD501 bacteriocin, including its small molecular size (<10 kDa), strong anti-listerial activity, wide pH stability and good thermostability, indicate its classification as a Class IIa bacteriocin.

• Biomedical Science Letters
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• v.10 no.3
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• pp.179-186
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• 2004

We examined a role of proteasome, the non-lysosomal multicatalytic protease complex,on the differentiation of chick embryonic myoblasts in culture. The peptide hydrolyzing activities of proteasome were found to change; the hydrolyzing activity against N-succinyl-Leu-Leu- Val- Tyr-7 -amido-4-methy1coumarin (SLLVY-AMC) was prominent and increased with myogenic differentiation. Proteasome inhibitors, N-carbobenzoxy-Leu-Leu-norvalinal (MG115) and N-carbobenzoxy-Ile-Glu (O-t-butyl)-Ala-Leucinal (PSI), blocked membrane fusion of myoblasts as well as the SLLVY-AMC hydrolyzing activity. Those inhibitory activities of the agents occurred in parallel, but were reversible and both cell fusion and the peptidase activity were restored when the agents were withdrawn from the culture medium. On the other hand, the agents caused accumulation of the ubiquitinylated proteins in the cytoskeletal proteins. These results suggest that each of the peptide hydrolyzing activities of proteasome is independently regulated during the myogenic differentiation and the chymotrypsin-like activity may play an important role in that process.

• BMB Reports
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• v.30 no.2
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• pp.125-131
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• 1997

This work reports studies of the catalytic and structural properties of pyridoxal kinase (ATP: pyridoxal 5' -phosphotransferase, EC. 2.7.1.35), Pyridoxal kinase catalyzes the phosphorylation of vitamin $B_6$ (pyridoxal, pyridoxamine, pyridoxine) using ATP-Zn as a phosphoryl donor. The enzyme purified from brain tissues is made up of two identical subunits of 40 kDa each. Native enzyme was inhibited by a substrate analogue, pyridoxal-oxime. Limited chymotrypsin digestion of pyridoxal kinase yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were isolated by DEAE ion exchange chromatography and used for binding studies with fluorescent ATP and pyridoxal analogues. The spectroscopic properties of both fluorescent pyridoxal analogue and Anthraniloyl ATP (Ant-ATP) bound to the 24 kDa fragment are indistinguishable from those of both pyridoxal analogue and Ant-ATP bound to the native pyridoxal kinase, respectively. The small 16 kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the substrate analogues. Binding characteristics of Ant-ATP were extensively studied by measuring the changes in fluorescence spectra at various conditions. From the results presented herein, it is postulated that the structural domain associated with catalytic activity comprises approximately one-half of the molecular mass of pyridoxal kinase (24 kDa). whereas the remaining portion (16 kDa) of the enzyme contains a regulatory binding domain.