• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.

• 한국축산식품학회지
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• 제27권1호
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• pp.110-116
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• 2007

Lactobacillus salivarius subsp. salivarius Nam27 with a high ${\beta}$-galactosidase activity was selected for enzymatic characterization. For purification, cell pellet was disrupted by Bead Beater, by DEAE-Sepharose and Mono-Q chromatography. The specific activity of the purified enzyme was 5,312 units/mg. The molecular weight of native monomeric ${\beta}$-galactosidase was estimated to be 30,000 dalton (monomer) by the SDS-PAGE. The optimum temperature and optimum pH were $50^{\circ}C$ and 5.0, respectively. This enzyme was stable between 35 and $55^{\circ}C$. ${\beta}$-galactosidase activity was lost rapidly above pH 7.0. But ${\beta}$-galactosidase was more stable at pH 4.0 (acidic conditions). And ${\beta}$-galactosidase activity was lost rapidly above $65^{\circ}C$ after 10 min incubation. $Ca^{2+}$ and $Zn^{2+}$ metal ions enhanced ${\beta}$-galactosidase activity by 164.09% and 127.37% while $Cu^{2+}$, $Fe^{3+}$ and $Mn^{2+}$ lowered ${\beta}$-galactosidase activity by 58.29%,85.10% and 77.66% respectively. Other metal ions didn't affect ${\beta}$-galactosidase activity significantly.

• Journal of Applied Biological Chemistry
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• 제43권3호
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• pp.141-146
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• 2000

Glycosidase activities were tested from the grape berries, Vitis labruscana B. Takasumi. Among various glycosidases, $\alpha$-D-galactosidase was found to be the most active in the flesh and other glycosidases were considerably active in the order of the following: $\alpha$-D-mannosidase>$\alpha$-D-glucosidase>$\beta$-D-glucosidase>$\beta$-D-galactosidase. In the seeds, $\alpha$-D-glucosidase activity was the highest and other glycosidases such as $\alpha$-D-galactosidase, $\beta$-D-glucosidase, and $\beta$-D-galactosidase were still significantly active. The $\alpha$-D-galactosidase in the grape flesh was purified over 83-folds through salting-out with $(NH_4)_2SO_4$ and a series of chromatographies employing Sephadex G-50, Octyl-Sepharose, Q-Sepha- rose, and Biogel P-100. The enzyme was a monomer of 45 kDs as determined through SDS-PAGE and Sephacryl S-200 chromatography. The purified enzyme showed a preference of $\alpha$-D-galactose to $\beta$-D-galactose as a substrate about 5.4 times. Sulfhydryl specific reagents such as N-ethylmaleimide and iodoacetamide significantly inhibited the enzyme activity to the extents of 48 and 52% of its initial activity, respectively. The optimumpH range of $\alpha$-D-galactosidase was around 6.5-7.0. The enzyme activity increased by 46% in the presence of 1mM $Fe^{2+}$.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1415-1423
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• 2010

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular mass of the F. pinicola xylanase was determined to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size-exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of $70^{\circ}C$. The enzyme showed a $t_{1/2}$ value of 33 h at $70^{\circ}C$ and catalytic efficiency ($k_{cat}=77.4\;s^{-1}$, $k_{cat}/K_m$=22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.489-498
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• 2013

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using $1.38{\times}10^9$ CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at $37^{\circ}C$, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and $70^{\circ}C$, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

• BMB Reports
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• 제40권1호
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• pp.107-112
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• 2007

To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by $Ni^{2+}$ affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, $H_6NtHSP70$-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90$^{\circ}C$. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50$^{\circ}C$) for recombinant $H_6NtHSP70$-2, E. coli cells overexpressing $H_6NtHSP70$-2 survived about seven times longer than those lacking $H_6NtHSP70$-2. After 2 h at 50$^{\circ}C$, only the E. coli overexpressing $H_6NtHSP70$-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.

• 약학회지
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• 제41권6호
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• pp.773-781
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• 1997

The activity of $Ca^{2+}$calmodulin (CaM)-dependent protein kinase Ia (CaM kinase Ia) is shown to be regulated through direct phosphorylation by CaM kinase I kinase (CaMK IK). In the present study, three distinct CaMKIK peaks were separated from Q-Sepharose colunm chromatography of pig brain homogenate using a Waters 650 Protein Purification System. The purified CaMKIK from the major peak potently and rapidly enhanced CaM kinase Ia activity, reaching a maximal stimulation within 2min at the concentrations of 12-15nM. The activated state of CaM kinase Ia is characterized by a markedly enhanced $V_{max}4 as well as significantly decreased $K_m\;and\;K_a$ values toward peptide substrate and CaM, respectively. These observations suggest the activation process of CaM kinase Ia. The phosphorylation of CaM kinase Ia by CaMKIK may induce its conformational change responsible for the alterations in the kinetic properties, which ultimately leads to the rapid enzyme activation.

• 대한약침학회지
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• 제14권3호
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• pp.71-79
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• 2011

Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially A${\alpha}$-chain and slowly B${\beta}$-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

• 한국식품영양과학회지
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• 제24권4호
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• pp.563-569
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• 1995

Myrosinase from Korean cabbage(Bogdoli) was purified and its enzymatic properties were investigated. Myrosinase from the Korean cabbage was purified by DEAE Bio-Gel Sepharose, Concanavalin-A, and Mono-Q column chromatography and exhibited a 55KD molecular weight with a single band on the gel of SDS-PAGE. The enzyme was purified about 21-fold compared to its crude enzyme and a specific activity of purified enzyme was 15, 120units/mg. Optimum pH of the myrosinase was 7.0 in both phosphate and Tris-HCl buffer solutions, the enzyme was stable at pH 6.5~7.0. Optimum temperature of enzyme was 37~38$^{\circ}C$. The enzyme activity was significantly inhibited by Cu2+ and Hg2+, but enhanced by ascorbic acid, resulting in a maximum activity at 1mM ascorbic acid. Among the ascorbic acid analogues, dehydro-ascorbic acid did not affect, whereas others showed a little effect on the enzyme activity, but less than ascorbic acid itself. Reducing agents such as 2-mercaptoethanol and dithiothreitol had no effect on the enzyme activity, but the enzyme activity was enhanced when 2-mercaptoethanol was mixed with ascorbic acid.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1087-1092
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• 2001

The effect of Sodium Butyrate (NaBu) on the N-linked oligosaccharide structure of Erythropoietin (EPO) was investigated. Recombinant human EPO was produced by CHO cells grown in an $MEM{\alpha}$ medium with or without 5 mM NaBu, and purified from the culture supernatants using a heparin-sepharose affinity column and immunoaffinity column. The N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were then labeled with a fluorescent dye, 2-aminobenzamide, and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of a GU (glucose unit) vague. A glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction in the proportion of glycans with Sri-and tetrasialylated oligodaccharides from $21.30\%$ (tri-) and $14.86\%$ (tetra-) in the control cultures (without NaBu) to $8.72\%$ (tri-) and $1.25\%$ (tetra-) in the NaBu-treated cultures, respectively. It was also found that the proportion of asialo-glycan increased from $12.54\%\;to\;23.6\%$ when treated with NaBu.