• Korean Journal of Food Science and Technology
• /
• v.21 no.4
• /
• pp.569-574
• /
• 1989

These studies were conducted to investigate the purification and characterization of Kiwifruit protease, and the results obtained were as follows The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography and purified enzyme gave a single protein band on polyacrylamide gel electrophoresis The specific activity of purified enzyme was 30,10 units/mg protein and the yield was 7.48. The purified enzyme showed a high affinity for casein and hemoglobin. The optimal pH and temperature for enzyme activity were 7.0 and $45^{\circ}C$, respectively. The enzyme activity was strongly inhibited by $HgCl_2,\;MnSO_4$. However. the enzyme was activated by cysteine and EDTA. The Michaelis constant for casein was calculated to be 50.5mg/ml according to the Line weaver-Burk method, and its molecular weight was determied as 23,500 by polyacrylamide gel electrophoresis.

• BMB Reports
• /
• v.43 no.8
• /
• pp.541-546
• /
• 2010

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

• BMB Reports
• /
• v.32 no.6
• /
• pp.605-609
• /
• 1999

Thioltransferase, also called glutaredoxin, is a general GSH-disulfide reductase of importance for redox regulation. Previously, the protein thioltransferase, now called S-type thioltransferase, was purified and characterized from Arabidopsis thaliana seed. In the present study, a second thioltransferase, called L-type thioltransferase, was purified to homogeneity from Arabidopsis thaliana leaves. The purification procedures included DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme was confirmed to show a unique band on SDS-PAGE and its molecular weight was estimated to be 26.6 kDa, which appeared to be atypical compared with those of most other thioltransferase. It could utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates, and also contained dehydroascorbate reductase activity. Its optimum pH was 8.5 and its activity was greatly activated by L-cysteine. When it was kept for 30 min, it appeared to be very stable up to $70^{\circ}C$. It was activated by $MgCl_2$ and, on the contrary, inhibited by $ZnCl_2$, $MnCl_2$, and $AlCl_3$.

• Archives of Pharmacal Research
• /
• v.19 no.3
• /
• pp.207-212
• /
• 1996

To find pharmacologically active components of Agrocybe cylin(Iracea, its basidiocarps were extracted with water. The extracts were separated by DEAE cellulose column chromatography, Sepharose CL-4B gel filtration, and Concanavalin A-Sepharose 4B affinity chromatography. Among the obtained fractions from A, cylinclracea, fraction IN which was the neutral proteinbound-polysaccharide fraction exhibited a marked antitumor activity and it was tentatively named "Cylindan". It showed about 70% of tumor inhibition against the solid form of sarcoma 180 when a dose of 30 mg/kg/day was intraperitoneally injected into ICR mice. When each fraction was examined by chemical analysis, Cylindan consisted of 85% polysaccharide, 3% protein and 1% hexosamine. Its polysaccharide moiety contained glucose, mannose, fucose and galactose and its protein moiety contained the comparatively large amounts of aspartic acid and glycine, and other 11 amino acids.

• Bulletin of the Korean Chemical Society
• /
• v.15 no.9
• /
• pp.719-724
• /
• 1994

Major cellulase components, such as three endoglucanases (endoglucanases I, II, and III) and one exoglucanase (exoglucanase II), were isolated from a commercial cellulase (Meicelase TP 60) derived from the fungus Trichoderma viride by a series of chromatography procedures. These procedures were the gel filtration on Bio-Gel, the anion exchange on DEAE-Bio-Gel A, the cation exchange on SP-Sephadex C50, and the affinity chromatography on Avicel cellulose. The average molecular weights determined by SDS-polyacrylamide gel electrophoretic analysis were 51,000, 59,000, 41,000 and 62,000 Da for endoglucanases I, II and III and exoglucanase II, respectively. The extinction coefficients, ${\varepsilon}^{1%}$ 280 nm, of these enzymes were 11.7, 3.3, 7.2 and 11.3, respectively. Among them, the endoglucanase II showed the very low value of the coefficient compared with the others. On the other hand, it was found that endoglucanase II and III were of more random hydrolytic mode on carboxymethylcellulose as compared with those of endoglucanase I and exoglucanase II. Especially, endoglucanase I showed less random action than that of exoglucanase II. In the hydrolysis of insoluble cellulose by the enzyme components, cellobiose was the major product, but glucose was the major product by endoglucanase III.

• Korean Chemical Engineering Research
• /
• v.55 no.3
• /
• pp.369-378
• /
• 2017

Vascular endotherial growth factor (VEGF) is a potent mitogen that stimulates vascular permeability and angiogenesis and has a potential in therapeutic applications. An industrial production method that provides high yield as well as purity is needed. Researches for various factors of mild solubilization with combination of ubiquitin fusion protein to increase solubility were carried out as well as by changing pH and denaturant concentration. Usage of pET28-a bacteral expression vector in BL21 (DE3) host cell was capable of producing approximately 14 g/L VEGF fusion protein in 20L fermentor. A purification process consisting of four chromatography steps including refolding and digestion with UBP1 resulted in mild solublization under the conditions of 2M urea and pH 10.0 due to ubiquitin fusion tag protein that increases in solubility of target protein VEGF. High yield of refolding and dimerization could be obtained between two step Ni-affinity chromatography. Multimeric and misfolded proteins and endotoxin were removed by DEAE anion exchange chromatography. Final monomers were removed from dimers by gel filtration chromatography. Characterization analysis of purified dimeric VEGF was performed using SDS-PAGE and RP-HPLC with a purity of 97%.

• Korean Journal of Food Science and Technology
• /
• v.26 no.1
• /
• pp.81-87
• /
• 1994

Two different trypsin inhibitors, TRTI-1 and TRTI-2, were purified to near homogenity from Trichosanthes kirilowii root, by $0{\sim}90%$ saturated ammonium sulfate salting out, DEAE-Sephacel ion exchange chromatography, Sephadex G-50 gel filtration chromatography and trypsin-affinity chromatography. The molecular weight of TRTI-1 and TRTI-2 were estimated to be about 5,000 Da and 24,000 Da, respectively, by gel filtration and must be monomer and homodimer since they contain 4,000 Da and 10,000 Da each on SDS-polyacrylamide gel electrophoresis. TRTI-1 was stable after heating for at least 2 hr at $100^{\circ}C$ but TRTI-2 was completely inactivated after heating for 10 min at $90^{\circ}C$. When Bz-dl-Arg-pNA was used as a substrate of TPCK-treated trypsin, half-maximal inhibitions of TRTI-1 and TRTI-2 were observed at $0.8\;{\mu}M$ and 6\;${\mu}M$, repectively. Both TRTI-1 and TRTI-2 inhibited the hydrolysis of trypsin competitively and Km values were $0.97\;{\mu}M$ and $0.63\;{\mu}M$, respectively. Both TRTI-1 and TRTI-2 specifically inhibited trypsin but they did not inhibit other proteases tested, chymotrypsin, papain, elastase, collagenase, thermolysin, Nagarase, pepsin, and thrombin.

• Journal of Life Science
• /
• v.13 no.1
• /
• pp.90-98
• /
• 2003

An allium rhizobacterium Serratia plymuthica AL-1 was previously selected as a biocontrol agent of allium white rot. The chitinase from S. plymuthica AL-1 produced in medium containing colloidal chitin was purified by ammonium sulfate precipitation (40~70%), affinity adsorption, column chromatography on DEAE-sephadex A-50 and sephadex C-200 gel filtration. The enzyme was purified 10.8-fold with a yield of 7.3% from the starting culture broth. The purified chtinase gave a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it's molecular weight was estimated to be 55 kDa. The optimum pH and temperature of the purified enzyme were pH 5.5 and $55^{\circ}C$, respectively and it is stable up to $50^{\circ}C$ and maintains around 90% of its activity for 60min. The enzyme were activated by $Ca^{2+}$, $Mn^{2+}$ and $Mg^{2+}$ and inhibited by $Cu^{2+}$, SDS, $\rho$-CMB, MIA, respectively. The purified chitinase showed broad spectrum of antifungal activities against plant pathogenic fungi Sclerotium cepivoruin, Alternana alternnta, Colletotrichum glceosporioidrs, Phoma sp., Sclerotinia sclerotiorum, Stemphylium solani, Fusarium oxysporium f. sp. niveum but rarely inhibited Phytophthora capsici and Pythium ultimum.. The purified chitinase from S. plymuthica AL-1 caused swelling, lysis, deceleration and degradation of the hyphal tips of S. sczerotiorum causing allium white rot. It suggest that S. prymuthica AL-1 chitinase play an important part in the bifunctional chitinase / lysozyme activity.

• The Korean Journal of Food And Nutrition
• /
• v.23 no.2
• /
• pp.171-177
• /
• 2010

The catabolic threonine dehydratase from Serratia marcescens ATCC 25419 was purified to homogeniety using Sephadex G-200 gel filtration and AMP-Sepharose 4B affinity chromatography. The molecular weight of the native enzyme was 120,000 by native pore gradient PAGE. The enzyme was composed of four identical subunits with subunit molecular weights of 30,000 by SDS-PAGE. The Km values of the enzyme for L-threonine with and without AMP were 7.3 and 92 mM, respectively. There were 2 moles of pyridoxal phosphate and 16 moles of free -SH groups per 1 mole of enzyme. The enzyme was inhibited by $\alpha$-ketobutyrate, pyruvate, glyoxylate, and phosphoenol pyruvate(PEP) in the presence of AMP, yet stimulated by cAMP and ADP. For enzyme properties in comparison with S. marcescens, E. coli, and S. typhimurium enzyme, such as the PLP content, number of free sulfhydryl groups, and existence of ADP binding site, the S. marcescens enzyme was more similar to the S. typhimurium enzyme than the E. coli enzyme. Of the three enteric bacteria, the E. coli and S. typhimurium enzyme was increased the activity by ADP and cAMP, respectively, but only the S. marcescens enzyme was increased the activity by both ADP and cAMP. Therefore, the subtle differences in the properties between enzymes from the three enteric bacteria may represent minor structural differences among these enzymes and warrants further study.

• Korean Journal of Food Science and Technology
• /
• v.34 no.4
• /
• pp.552-558
• /
• 2002

Polyphenol oxidase from Flammulina velutipes was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Superdex G-200 gel filtration chromatography, Phenyl superose affinity chromatography, Mono-Q anion exchange chromatography and Superdex S-200 gel filtration chromatography on FPLC. After these purification steps specific activity of purified polyphenol oxidase increased to 199.1 units/mg. Polyphenol oxidase from F. velutipes was composed of a single polypeptide with molecular weight of about 40 kDa. Optimum pH and temperature for the enzyme reaction were found to be 6.0 and $25^{\circ}C$, respectively. The activity of the enzyme gradually decreased at acidic pH between 3 and 5, and the enzyme lost its activity at alkaline pH between 8 and 10. This enzyme exhibited high substrate specificity to o-diphenols. Km-values for L-DOPA and caffeic acid were found to be 3.97 mM and 1.78 mM, respectively. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA and $Mg^{2+}$ inhibited the activity of pholyphenol oxidase and $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ increased enzyme activity. The activity of enzyme was well maintained at $-70^{\circ}C$ for over 4 months, and at $-20^{\circ}C$ for 1 months.