• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.269-275
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• 2003

Chitosan-degrading activity induced by chitosan was founf in culture filtrate of Aspergillus flavus IAM2044. Aspergillus flavus IAM2044 had a higher level of chitosanolytic activity when chitosan was used as a carbon source, and yeast extract and peptone were supplemented as nitrogen sources. One of the chitosan-degrading enzymes was purified to homogeneity by ammonium sulfate precipitation followed by cation-exchange and gel filtration chromatographies. The enzyme was monomeric, and its molecular mass was 45 kDa. The optimum pH and temperature of the enzyme were 5.0 and $50^{\circ}C$, respectively. The activity was stable in the pH range of 3.5 to 7.0 and at a temperature below $50^{\circ}C$. Reaction products analyzed by the viscosimetric assay and thin layer chromatography clearly indicated that the enzyme was an exe-type chitosanase, $exo-{\beta}-D-glucosaminidase$, that released GlcN from the nonreducing ends of the oligosaccharide chains.

• BMB Reports
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• 제44권1호
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• pp.64-69
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• 2011

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and $50.0^{\circ}C$, respectively and seasonal variation. The Michaelis-Menten constant ($K_M$) for $H_2O_2$ was 5.27 mM, and the velocity maximum ($V_{max}$) $1.31\;nmol\;min^{-1}$, while the enzyme turnover was $0.148\;s^{-1}$. Finally, the presence of $Ca^{2+}$ and $Mg^{2+}$ enhanced the PD-cP activity, with $Mg^{2+}$ 1.4-fold more effective than $Ca^{2+}$.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.663-667
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• 2007

Pediococcus acidilactici produces bacteriocin, which kills Listeria monocytogenes. The bactericidal mode of action of the bacteriocin against L. monocytogenes V7 was investigated by transmission electron microscopy. The bacteriocin was purified partially from the cell-free extract using Micro-Cel and cation-exchange chromatography, and the specific activity was increased 1,791 fold. The bacteriocin (6,400 AU/ml) was inoculated with L. monocytogenes V7 and incubated for 0.5h, 1h, 3h, and 6h. The bacteriocin was found to destroy most of the cell wall and released most of the inclusions in the cells after 6 h of incubation. These results suggest that the bactericidal effect of the bacteriocin was due to bacterial lysis.

• Journal of Pharmaceutical Investigation
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• 제40권6호
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• pp.379-386
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• 2010

Mono PEGylated rhG-CSF (PEG-G-CSF) prepared by utilizing unique PEG was purified and characterized by cation-exchange chromatography. A unique, trimer-structured PEG was chosen for PEGylation of rhG-CSF among various PEG moieties. The in-vitro bioactivity, stability, and pharmacokinetics of mono-PEG-G-CSF were examined and compared to those of native rhG-CSF. Mono PEG-G-CSF exhibited reduced in-vitro bioactivity to native rhG-CSF but showed an excellent in-vivo bioactivity and stability. Furthermore, it showed markedly reduced clearance in rats, thereby increasing the biological half-life by about 4.5-fold compared to that of native rhG-CSF. The results suggest that this unique, trimer-structured 23 kDa PEG can provide advantages to improve the bioactivity of therapeutic proteins in clinical use.

• The Korean Journal of Ecology
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• 제21권1호
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• pp.73-80
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• 1998

Peroxidase has been isolated to apparent homogeneity from earthworm, Lumbricus rubellus, using ammonium sulfate fractionation, Sephacryl S-2000 gel filtration, CM-cellulose cation exchange chromatography and native-PAGE elution. Some of its enzymatic characteristics were examined. The optimum pH for gruaiacol oxidation of earthworm peroxidase was determined to be 6.0, and the $K_{m}$ values against guaiacol and $H_2O_2$ were 1.25 mM and 3.4mM, respectively. When various compounds were tested as the possible substrates of the enzyme, o-dianisidine was used as the substrate. However, earthworm peroxidase could not oxidize esculetin and ferulic acid as substrates, suggesting the different characteristics of the enzyme from plant peroxidases. The optimum pH for veratryl alcohol and $H_2O_2$ oxidation was determined to be 2.5 when lignin peroxidation activity was examined. The $K_{m}$ values for veratryl alcohol and $H_2O_2$ were 0.02 mM and 0.13 mM, respectively. Furthermore, the earthworm peroxidase could oxidize phenoxyherbicides such as 2,4-D, 2,4-DP and MCPA as substrates. The optimum pHs for 2,4-D, 2,4-DP and MCPA were determined to be 4.0, 2.0 and 2.0, respectively. The most available substrate was 2,4-DP, followed by MCPA and 2,4-D when their peroxidation activities were compared.

• Preventive Nutrition and Food Science
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• 제7권1호
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• pp.43-47
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• 2002

Crude caseinomacropeptide (CMP) was prepared from Na-caseinate using a commercial renneting enzyme. Most of the crude CMP was released from the Na-caseinate by hydrolyzing with the enzyme for 40 min. The hydrolysis of the k-casein with carbohydrate was slower than that of the k-casein without carbohydrate, as shown by the analyses of the sialic acid content and the tricine-SDS-polyacrylamide gel electrophoresis. The yield of crude CMP from Na-caseinate was 3.7%. Cation exchange chromatography showed that the crude CMP consisted of 40.5% CMP and 59.5% caseinogylcomacropetide (CGP). The effect of the TCA concentration on the solubility of CMP and CGP was determined by using crude CMP. The amounts of crude CMP and sialic acid decreased in the proportion to the increase of trichloroacetic acid (TCA) concentration from 2 to 12%, suggesting that the CGP containing carbohydrate, as well as the CMP having no carbohydrate, was precipitated in a range of 4 to 12%, depending on the TCA concentration. This result supports the hypothesis that the different non-glycosylated and glycosylated forms of CMP have different sensitivities to TCA precipitation.

• 약학회지
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• 제54권5호
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• pp.377-386
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• 2010

Single-dimensional (1-D) and two-dimensional (2-D) LC methods were utilized to separate peptides from various sources followed by MS/MS analysis. Two-dimensional ultra-high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than 1-D LC. The most popular 2-D LC approach used today for proteomic research combines strong cation exchange and reversed-phase LC. We have evaluated an alternative mode for 2-D LC of peptides using 2-D RP-RP nano UPLC Q-TOF Mass Spectrometry, employing reversed-phase columns in both separation dimensions. As control experiments, we identified 129 proteins in 1-D LC and 322 proteins in 2-D LC from E. coli extract peptides. Furthermore, we applied this method to rat primary hepatocyte and a total of 170 proteins were identified from 1-D LC, and 527 proteins were identified from all 2-D LC system. The in-depth protein profiling established by this 2-D LC MS/MS from rat primary hepatocyte could be a very useful reference for future applications in regards to drug induced liver toxicity.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1047-1053
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• 2005

Cyclosporin A binding protein type-19 kDa peptidyl-prolyl cis/trans isomerase (PPIases, EC 5.2.1.8) of Euglena gracilis was purified and some of its biochemical characters were elucidated. Purification of the PPIase was achieved by employing a series of steps involving ammonium sulfate precipitation, Superdex G-75 gel filtration chromatography, Mono­Q anion and Mono-S cation exchange chromatographies, and Superdex S-200 gel filtration chromatography on FPLC. Purified PPIase had a specific activity of 8,250 units/mg, showing a 27-fold increase compared with that of cell-free extract of Euglena gracilis. The enzyme consisted of a single polypeptide chain with a molecular mass of 19 kDa. It showed high substrate specificity to succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and $k_{car}/K_{m}$, for this substrate was found to be $61.19{\times}10^5/sec$. The isomer distributions were investigated at an equilibrium of seven different peptide substrates, varying Xaa in Suc-Ala-Xaa-Pro-Phe-p-nitroanilide in dimethylsulfoxide. The cis/trans equilibrium constants were estimated to be from 0.14 (Ile) to 0.63 (Gly), which correspond to $12.00\%\;to\;38.52\%$ of the cis population, respectively, under experimental condition. The enzyme was highly sensitive to the immunosuppressive ligand cyclosporin A, but not to other immunosuppressants such as FK506 and rapamycin. Thus, it appears to belong to the class of cyclophilin.

• Journal of Microbiology and Biotechnology
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• 제17권6호
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• pp.952-959
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• 2007

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multimers. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3mg/l and analyzed by matrixassisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.

• BMB Reports
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• 제43권12호
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• pp.801-806
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• 2010

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.