• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.166-172
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• 1997

Aspergillus niger was selected as a strain producing the potent raw starch hydorlyzing enzyme. These experiments were conducted to investigate the conditions of the glucoa- mylase production, the purification of the enzyme, some characteristics of the purified enzyme and hydrolysis rate on various raw starches such as com, rice, potato, glutinous rice, sweet potato, wheat and barley. The optimum cultural temperature and time for the enzyme production on wheat bran medium were $30^{\circ}C$ and 96hrs, respectively. The respective addition of yeast extract and nutrient broth on wheat bran medium increased slightly the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. The specific activity of the purified enzyme was 30.7u/mg-protein and the yield of enzyme activity was 25.8%. The purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and its molecular weight was estimated to be 56,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelectric point for the purified enzyme was pH3.7. The optimum temperature and pH were $65^{\circ}C$ and pH 4.0, respectively. The purified enzyme was stable in the pH range of pH 3.0-9.5 and below $45^{\circ}C$, and its thermal stability was slightly increased by the addition of $Ca^{2+}$. The purified enzyme was activated by $Co^{2+},\;Sr^{2+},\;Mn^{2+},\;Fe^{2+},\;Cu^{2+}$. Raw rice starch, raw corn starch, raw glutinous rice starch, raw sweet potato starch, raw wheat starch and raw barley starch showed more than 90% hydrolysis rate in 48hrs incubation. Even raw potato starch, most difficult to be hydrolyzed, showed 80% hydrolysis rate. The purified enzyme was identified as glucoamylase.

• Korean Journal of Food Science and Technology
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• v.17 no.5
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• pp.383-388
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• 1985

Soy protein isolate was acylated with succinic anhydride and acetic anhydride. The functional properties are markedly improved by acylation of the $\varepsilon$-amino groups. Acylation of the available amino groups shifted the isoelectric point from 4.5 to 4.0 and enhanced the solubility between pH 4.0-6.0. In the 0.03M-$CaCl_2$ solution the solubility of the modified soy protein is much larger than that of the unmodified protein above the isoelectric point. The emulsion properties and foaming properties also improved by the modification and the effects of pH on the properties paralleled its effect on protein solubility. The changes of reduced viscosity with concentration followed Huggin's equation and by modification the intrinsic viscosity of the soy protein increased and the interaction coefficient decreased.

• Archives of Pharmacal Research
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• v.24 no.6
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• pp.590-596
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• 2001

A new extended-spectrum ${\beta}-lactamase$ with an isoelectric point (pl) of 6.2 was detected in Klebsiella pneumoniae Fl 61 that was isolated from a patient with infection. This strain was highly resistant to the third or fourth generation cephalosporins such as cceftazidime ceftriaxone, cefoperzaone, and cefpirome. Analysis of this strain by the double disk diffusion test showed synergies between amoxicillin-clavulanate (AMX-CA) and cefotaxime, and AMX-CA and aztreonam, which suggested that this strain produced a extended-spectrum ${\beta}-lactamase$ (ESBL). Cenetic analysis revealed that the resistance was due to the presence of a 9.4-kb plasmic, designated as pkpl 61, encoding for new ${\beta}-lactamase$ gene (bla). Sequence analysis showed that a new bla gene of pkpl 61 differed from $bla_{TEM-1}$ by three mutations leading to the following amino acid substitutions: $Val_{84}{\rightarrow}lie,{\;}Ala_{184}{\rightarrow}Val,{\;}and{\;}Gly_{238}{\rightarrow}Ser$. These mutations have not been reported previously in the TIM type ${\beta}-lactamases$ produced by clinical strains. The novel ${\beta}-lactamase$ was overexpressed in E. coli and purified by ion exchange chromatography on Q-Sepharose and CM-Sepharose, and then further purified by gel filtration on Sehadex G-200. The catalytic activity of th8 purified ${\beta}-lactamase$ was confirmed by the nitrocefin disk.

• Korean Journal of Plant Resources
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• v.11 no.3
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• pp.272-278
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• 1998

This study was carried out to investigate the functional properties such as nitrogen solubility, emulsifying property , foaming capapcity , water and oil absorption of Camellia (Camellia japonica .) seed protein isolate in condition of distilled water and 0.5M NaCl solution at pH 2.0∼10.0. Nitrogen solubility of Camellia protein isolate in distilled water showed the minimum value at pH 4.0 and increased at pH lower or higher than the isoelectric point(pH 4.0). It was 90.0 %at pH 10.0 Nitrogen solubility of 0.5M NaCl solution showed a similar pattern with that of distrille dwater but was higher than that of distilled water except pH 2.0 and pH 10.0. Emulsifying activity of Camellia seed protein islate showed the minimum value at pH 4.0, but was higher at ether value of pH. Emulsifying stability of protein isolate was stable by heat treatment for 30min, at 80℃ and increased in 0.5M NaCl solution more than that of distille dwater. Foaming capacity of Camellia seed protein isolate in distill3ed water showed the minimum value near the isoelectric point, While it changed little at other values of pH. Foaming stability slowly decreased as, but didn't make a significant difference as time was delayed . Oil absorption was 1.4ml per a sample of 1g and water absorption was 0.9ml per a sample of 1g. The former was higher than the latter . The content of total amino acid of Camellia protein isolate was 43.67% and the major total amino acid of Camellia protein isolate was 43.67% and the major total amino acid was in the order of glutamic acid , arginine, aspartic acid, and leucine.

• Mass Spectrometry Letters
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• v.3 no.1
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• pp.10-14
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• 2012

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.251-262
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• 1986

High-molecular-weight ${\beta}-glucosidase$ (EC 3.2.1.21) was purified from the culture filtrate of Trichoderma koningii through a four-step procedure including chromatography on Bio-Gel P-150, DEAE-Sephadex A-50 and SP-Sephadex C-50; and chromatofocusing on Polybuffer exchanger PBE 94. The molecular weight of the enzyme was determined to be about 101,000 by SDS-polyacrylamide gel electrophoreses, and the isoelectric point was estimated to be 4.96 by analytical isoelectric focusing. The temperature optimum for activity was about $55^{\circ}C$, and the pH optimumwas 3.5. The enzyme was considerably thermostable, for no loss of activity was observed when the enzyme was preincubated at $60^{\circ}C$ for 5h. Km values for cellobiose, gentiobiose, sophorose, salicin and $p-nitrophenyl-{\betha}-D-glucoside$ were 99.2, 14.7, 7.09, 3.15 and 0.70 mM, respectively, which indicates that the enzyme has much higher affinity towards $p-nitrophenyl-{\betha}-D-glucoside$ than towards the other substrates, especially cellobiose. Substrate inhibition by $p-nitrophenyl-{\betha}-D-glucoside$ and salicin was observed at the conecntrations exceeding 5mM. Gluconolactone was a powerful inhibitor against the action of the enzyme on $p-nitrophenyl-{\betha}-D-glucoside\;(K_i\;37.9\;{\mu}M)$, wherease glucose was much less effective ($K_i$ 1.95 mM). Inhibition was of the competitive type in each case. Transglucosylation activity was detected shen the readtion products formed from $p-nitrophenyl-{\betha}-D-glucoside$ by the enzyme were analysed using high-performance liquid chromatography.

• International Journal of Industrial Entomology and Biomaterials
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• v.16 no.2
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• pp.37-48
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• 2008

The diapause and non diapause associated proteins of multivoltine silkworm eggs were analysed by two dimensional (2D) gel electrophoresis. The study was made at 0 hr, 24 hrs and 48 hrs after oviposition. A total of four protein spots in diapause eggs at 24 hrs of oviposition and two protein spots in non diapause eggs at 0 hrs of oviposition were observed. All the six protein spots were considered to have association with diapause and non diapause characters. The molecular weight (MW) and isoelectric point (PI) of these 6 protein spots were calculated. The protein spots 1 and 2 observed in 0 hr of non diapause eggs were found to have the MW of 67 and 75 KDa and PI of 8.6 and 8.4 respectively. Similarly the four protein spots observed in diapause egg at 24 hrs of oviposition exhibited MW viz., 15, 17,20 and 25 KDa and PI of 5.3, 5.8, 6.5 and 6.0 respectively. All these 6 identified protein spots were subjected to in-gel digestion and resulted tryptic peptides were analyzed by Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI TOF-MS). Databases searched based on experimentally determined molecular weights of peptides for the determination of the identities of proteins. The identified proteins indicated homology of 34% to 95%. The results indicate that the proteins may playa role in development of diapause and non diapause eggs.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.476-482
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• 2002

Methanol dehydrogenase (MDH) and c-type cytochromes from marine methanol-oxidizing bacterium, Methylophaga sp. MP, were purified and characterized. The native MDH had a molecular mass of 148 kDa and its isoelectric point was 5.5. Two c-type cytochromes, $c_L\;and\;c_H$, were found, and their isoelectric points were 3.4 and 8.0, respectively. The purified MDH had higher thermal stability than that of the other soil methylotrophic bacteria. The electron flow rate from MDH to cytochrome $c_L$was higher than that from MDH to cytochrome $c_H$, indicating that the physiological primary electron acceptor for MDH is cytochrome $c_L$. The electron transfer from MDH to phenazine ethosulfate (PES, artificial electron acceptor) in the two dye (PES/DCPIP)-linked assay system was not inhibited by NaCl, whereas the electron flow from MDH to cytochrome $c_L$ in the cytochrome/DCPIP-linked assay system was suppressed significantly by NaCl. Metal chelating agents such as EDTA showed the same effects on the MDH activity.

• Archives of Pharmacal Research
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• v.29 no.5
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.477-486
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• 1991

To elucidate enzymatic properties of a-glactosidase (EC 3.2.1.22) from Asp. niger, a-galactosidase from wheat bran culture was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. And then its enzymatic propeties were investigated. The highest level of $\alpha$-galactosidase activity was obtained when Asp. niger was grown on wheat bran medium at $30^{\circ}C$ for 96 hours. The $\alpha$-galactosidase was purified by 23.7 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Celluose and Sephadex A-50, and gel filtration on Sephadex G-150 and its specific activity was 1,229 Unitslmg protein and the yield was 14% of the total activity of wheat bran culture. The purified $\alpha$-galactosidase was found to be homogeneous by polyacrylamide gel electrophoresis and HPLC. The $\alpha$-galactosidase was a tetrameric glycoprotein which consisted of identical subunits with molelcular weight of 28,000 each by SDS-PAGE and isoelectric point was determined analytical isoelectric focusing to be pH 4.6. The optimal temperature and pH for the $\alpha$-galactosidase activity were $40^{\circ}C$ and pH 6.5, respectively, and 54% of its activity was lost by heating at $60^{\circ}C$ for 10 mins, It was appeared to have higher affinty to raffinose than to stachyose. The K, value and activation energy of $\alpha$-galactosidase were 5.0 mM and 8.515 Kcal per mole for p-nitrophenyl- $\alpha$--D-galactopyranoside, respectively.