• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.317-325
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• 2001

For heuristic purposes, the relative ratio of urease contents inside and outside cells was surveyed using nine ureB+ strains of Helicobacter pylori. the ratio of the enzyme specific activity appeared to vary greatly between the various H. pylori strains, ranging from 0.5 to 2.5. Besides the above compartment, urease was also richly found in the membrane fraction, especially in either peripheral or integral form. The urease distribution across the H. pylori membrane was significantly influenced by the ambient pH; the specific activity of external urease was highest at pH 5.5 with a narrow plateau, whereas the internal specific activity was highest within a pH range of 4.5 to 6.5 with a broad plateau. These finding strongly suggest that H. pylori urease is secretory and responded to the external pH. However, at pH 4.0 or below, no urease activity was detected in either the internal or external compartment, although an increase in the color development with 2,4,6-trinitrobenzene sulfonate (TNBS) was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these phenomena may be related to a specific proteolysis in certain proteins, including urease or ${\gamma}$-glutamyl transpeptidase. Interestingly, the effect of ammonium ions n alleviating the enzyme inactivation inside the H. pylori cells was remarkably similar to that of D-glucose. In addition, it would appear that the cation acted as a surrogate of not only $Na^+$ but also $K^+$ thereby increasing the H. pylori P-type ATPase activity. This is of great interest, as it implies that the urease action in H. pylori is indispensible at any locus.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.379-386
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• 1998

Deterioration of food is in general caused by the presence of microorganisms and chemical compounds of food itself. There exists antimicrobial compound in the food, however, addition of food antiseptics, additives, or physico-chemical processing is a common practice. The safety of artificial chemical antiseptics became a serious public concern, therefore, new natural antiseptic compounds are in need to be developed. We have isolated a new natural antifungal protein (KBS-B2) from Astragal seed through ammonium sulfate precipitation and column chromatography using FPLC Mono-S and Superose 12HR. The purified protein inhibited growth of Candida albicans, and spore germination of food spoiling fungi such as Aspergillus ochraceus, Penicillium expensum, P. digitatum and Botrytis cineria. Antifungal effect of the KBS-B2 protein could be directly assayed by bioautography overlaying the fungal spores on the electrophoresed acrylamide gel. The comparison of N-terminal amino acid sequences of the KBS-B2 with known antifungal protein revealed that had 50% homology to thaumatin and zeamatin like proteins.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.483-493
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• 2011

An acid phosphatase (HppA) activated by $NH_4Cl$ was purified 192- and 34-fold from the periplasmic and membrane fractions of Helicobacter pylori, respectively. SDS-polyacrylamide gel electrophoresis revealed that HppA from the latter appears to be several kilodaltons larger in molecular mass than from the former by about 24 kDa. Under acidic conditions (pH${\leq}$4.5), the enzyme activity was entirely dependent on the presence of certain mono- and/or divalent metal cations (e.g., $K^+$,$ NH_4{^+}$, and/or $Ni^{2+}$). In particular, $Ni^{2+}$ appeared to lower the enzyme's $K_m$ for the substrates, without changing $V_{max}$. The purified enzyme showed differential specificity against nucleotide substrates with pH; for example, the enzyme hydrolyzed adenosine nucleotides more rapidly at pH 5.5 than at pH 6.0, and vice versa for CTP or TTP. Analyses of the enzyme's N-terminal sequence and of an $HppA^-$ H. pylori mutant revealed that the purified enzyme is identical to rHppA, a cloned H. pylori class C acid phosphatase, and shown to be the sole bacterial 5'-nucleotidase uniquely activated by $NH_4Cl$. In contrast to wild type, $HppA^-$ H. pylori cells grew more slowly. Strikingly, they imported $Mg^{2+}$ at a markedly lowered rate, but assimilated urea rapidly, with a subsequent increase in extracellular pH. Moreover, mutant cells were much more sensitive to extracellular potassium ions, as well as to metronidazole, omeprazole, or thiophenol, with considerably lowered MIC values, than wild-type cells. From these data, we suggest that the role of the acid phosphatase HppA in H. pylori may extend beyond 5'-nucleotidase function to include cation-flux as well as pH regulation on the cell envelope.

• Korean Journal of Food Science and Technology
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• v.35 no.2
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• pp.260-265
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• 2003

Transglutaminase (TG) was prepared from Streptomyces mobaraensis to improve texture and self-life of food. In preliminary experiments, texture of the dough was not improved due to the interference in microbial TG reaction by proteases present in the crude enzyme. Among the cation exchange resins tested for the removal of proteases, MonoPlus S 100 was the most efficient. Further purification steps with a quaternary ammonia salt resin and gel permeation chromatography effectively removed proteases from crude enzyme. Molecular weight of purified enzyme was about 38,000 on SDS-polyacrylamide gel electrophoresis. Farinograph data showed the addition of purified enzyme to wheat flour gave higher stability and lower weakness values those that of crude enzyme.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.296-300
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• 2002

Bacterial cell surface components are the major factors responsible for pathogenesis and bioremediation. In particular, the surface of a Gram-negative bacterium cell has a variety of components compared to that of a Gram-positive cell. In our previous study, we isolated an isogenic mutant of Bradyrhizobium japonicum, which exhibited altered cell surface characteristics, including an increased hydrophobicity. Polyacrylamide gel electrophoretic analysis of the lipopolysaccharide (LPS) in the mutant demonstrated that the O-polysaccharide part was completely absent. Meanwhile, a gel permeation chromatographic analysis of the exopolysaccharide (EPS) in the mutant demonstrated that it was unaltered. Since LPSs are known to have several anion groups that interact with various cation groups and metal ions, the mutant provided an opportunity to examine the direct role of LPS in metal binding by B. japonicum. Using atomic absorption spectrophotometry, it was clearly demonstrated that LPS was involved in metal binding. The binding capacity of the LPS mutant to various metal ions $(Cd^{2+},\;Cu^{2+},\;Pb^{2+},\;and\;Zn^{2+})$ was 50-70% lower than that of the wild-type strain. Also, through an EPS analysis and desorption experiment, it was found that EPS and centrifugal force had no effect on the metal binding. Accordingly, it would appear that LPS molecules on B. japonicum effect the properties, which precipitate more distinctly metal-rich mineral phase.

• KSBB Journal
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• v.14 no.1
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• pp.9-16
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• 1999

Transforming growth factor $\beta$1(TGF-$\beta$1) has potentials to be used as a new therapeutic agent. However, studies with TGF-$\beta$ were hindered by its high cost. In this study, we developed an improved method to purify TGF-$\beta$1 from human platelets, for which four purification steps were used: platelet extraction, gel filtration, cation exchange chromatography, and reverse phase high performance liquid chromatography. After a final step of purification, a pure protein with a molecular weight of 25,000 corresponding to the commercially available TGF-$\beta$1 was obtained, which were confirmed by silver staining and Western blotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It was confirmed by the inhibitory effects of TGF-$\beta$1 on a mink lung epithelial cell line that the purified TGF-$\beta$1 had its biological activity, whose activity is slightly higher than that of the commercially available TGF-$\beta$1. About 3.7$\mug of the purified TGF-$\beta$1 was obtained from 10 units of concentrated human platelets, the final yield of which is about 21%.

• Food Engineering Progress
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• v.21 no.2
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• pp.103-109
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• 2017

Alginate lyase from Streptomyces violaceoruber was purified by DEAE sephacel chromatography and SP sepharose chromatography. The specific activity of the purified enzyme was 14.6 units/mg protein, representing a 40.6-fold purification of the crude extract. The final preparation thus obtained showed a single band on Tricine-SDS polyacrylamide gel electrophoresis whose molecular weight was determined to be 23.3 kDa. The polyMG block of sodium alginate was hydrolyzed by the purified alginate lyase and then separated by activated carbon column chromatography and bio gel P-2 gel filtration. The main hydrolysates were composed of hetero type M/G-oligosaccharides with the degrees of polymerization (D.P.) being 6 and 8. To investigate the effects of hetero type M/G-oligosaccharides from the sodium alginate on the growth of some intestinal bacteria, cells were cultivated individually on the modified-MRS medium containing D.P. 6 and 8 M/G-oligosaccharides. B. longumgrew 4.25-fold and 6.44-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides compared with those of standard MRS medium. In addition, B. bifidumgrew 3.3-fold and 5.4-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides. In conclusion, D.P. 8 was more effective than D.P. 6 hetero M/G-oligosaccharides as regards the growth of Bifidobacteriumspp. and Lactobacillus spp.