• Journal of Applied Biological Chemistry
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• 제63권4호
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• pp.443-449
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• 2020

본 연구에서는 섬괴불나무 잎으로부터 phenol성 화합물의 생리활성 탐색을 통하여 기능성 소재로 활용 가능성을 살펴보았다. 인체에 무해한 용매인 열수와 ethanol로 추출 했을 때 열수는 3.53 mg/g, 40% ethanol은 2.82 mg/g의 TPC를 나타내었으며, 추출물을 이용하여 항산화 활성, xanthine oxidase (XOase), angiotensin converting enzyme (ACE), α-glucosidase 억제효과 및 항균 활성을 검정하였다. 그 결과 섬괴불나무 잎의 열수와 ethanol 추출물 50-200 ㎍/mL TPC에서 농도의존적으로 DPPH 유리라디칼 소거활성 및 PF가 증가하는 경향을 보였고, 50 ㎍/mL의 저농도에서도 모두 매우 높은 활성을 나타내었다. XOase 저해효과에서는 200 ㎍/mL TPC에서 열수와 ethanol 추출물이 각각 76.35, 99.83%의 높은 저해효과 나타냈으며, ACE 저해효과 또한 200 ㎍/mL TPC에서 열수와 ethanol 추출물 각각 79.06, 87.14%의 저해효과를 확인하였다. α-Glucosidase 저해효과를 측정한 결과 200 ㎍/mL TPC에서 열수와 ethanol 추출물 각각 80.45, 63.58%의 저해효과를 보였다. 생육저해환으로 항균활성을 측정 결과 열수 추출물 200 ㎍/100 μL TPC에서 H. pylori, P. acne균에 대해 각각 11.5, 18.5 mm의 clear zone을 형성 하였고, ethanol 추출물 200 ㎍/100 μL 농도에서는 P. acne균에 대해 10 mm의 clear zone을 형성하였다. 따라서 본 연구를 통하여 섬괴불나무 잎 추출물의 항산화 활성, xanthine oxidase (XOase), angiotensin converting enzyme (ACE), α-glucosidase 억제효과 및 항균 활성이 있다고 판단되었다.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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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.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.272-279
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• 2006

The gene encoding Thermus sp. T351 alkaline phosphatase (T351 APase) was cloned and sequenced. The gene consisted of 1,503 bp coding for a protein with 500 amino acid residues including a signal peptide. The deduced amino acid sequence of T351 APase showed relatively low similarity to other Thermus APases. The T351 APase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21 (DE3). The expressed enzyme was purified by heat treatment, and $UNO^{TM}$ Q and $HiTrap^{TM}$ Heparin HP column chromatographies. The purified enzyme exhibited high activity at extremely alkaline pHs, reaching a maximum at pH 12.0. The optimum temperature of the enzyme was $80^{\circ}C$, and the half-life at $85^{\circ}C$ was approximately 103 min. The enzyme activity was found to be dependent on metal ions: the addition of $Mg^{2+}$ and $CO^{2+}$ increased the activity, whereas EDTA inhibited it. With p-nitrophenyl phosphate as the substrate, T351 APase had a Michaelis constant ($K_{m}$) of $3.9{\times}10^{-5}M$. The enzyme catalyzed the hydrolysis of a wide variety of phosphorylated compounds.

• Journal of Pharmaceutical Investigation
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• 제29권1호
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• pp.13-19
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• 1999

Canalicular liver plasma membrane vesicles (cLPM) were prepared according to two different methods (Inoue method and Meier method), and were evaluated for their protein yield, enzyme activity and transport characteristics. No difference was found between the methods in the protein yield (i.e., $0.14{\pm}0.031$ and $0.15{\pm}0.050$ mglg liver for Inoue method and Meier method, respectively). The activity of alkaline phosphatase, a marker enzyme of canalicular membrane, was significantly (P<0.05) higher in the vesicles of Meier method $(3.52{\pm}0.91\;mmol/mg/hr)$than in the vesicles of Inoue method ($2.28{\pm}0.94$ mmol/mg/hr) indicating that more purified cLPM were obtained from Meier method compared with Inoue method. ATP-dependent vesicular uptake of taurocholate and tributylmethylammonium (TBuMA) was observed for vesicles of both methods, and the kinetic parameters responsible for the transport were similar between the vesicles of both methods (for example, $V_{max}:$ 9.72 nmol/mg protein/30sec and $K_m:$ 0.63 mM for Inoue method; $V_{max}:$ 10.1 nmol/mg protein/30sec and $K_m:$ 0.70 mM for Meier method). A pH gradient dependent counter transport of TBuMA was also observed for both vesicles with similar kinetic characteristics. Either the uptake of taurocholate in the absence of ATP or that of TBuMA in the absence of pH gradient, which may represent passive diffusion of respective compound into the vesicles, was more rapid for the vesicles of Meier method than for the vesicles of Inoue method. For example, passive diffusion rate constants $(K_d)$ for TBuMA uptake into the vesicles were 0.00030 and 0.00052\;{\mu}l/mg$ protein/min for the vesicles of Inoue method and Meier method, respectively. It may indicate that more leaky vesicles are obtained form the Meier method compared with the Inoue method. These aspects together with the time necessary to prepare the vesicles (i.e., 8 hr for Inoue method and 23 hr for Meier method) should be considered before selecting an appropriate method for the preparation of cLPM.

• Journal of Microbiology and Biotechnology
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• 제19권6호
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• pp.604-609
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• 2009

To investigate the potential use of microbial heme as an iron source, recombinant Escherichia coli coexpressing ALA synthase (HemA) as well as the NADP-dependent malic enzyme (MaeB) and dicarboxylic acid transporter (DctA) were cultured. The typical red pigment extracted from the recombinant E. coli after 38 h showed highest absorbance at 407 nm, and the amount of iron in 38.4 mg of microbial heme extract derived from 6-1 fermentation broth was 4.1 mg. To determine the commercial potential of the recombinant E.coli-synthesized iron-associated heme as an iron source, mice were fed the iron-free provender with the microbial heme extract. The average body weight reduction of mice fed non-iron provender was 2.3%, whereas no detectable weight loss was evident in mice fed microbial heme addition after 15 days. The heme content of the blood from microbial heme fed mice was 4.2 mg/ml whereas that of controls was 2.4 mg/ml, which implies that the microbial heme could be available for use as an animal iron source.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.764-769
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• 2000

The lipase gene(lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution: S113($AGC{\rightarrow}AGT$), L252 ($TTG{\rightarrow}TTA$), and G275E ($GGA{\rightarrow}GAA$). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity ($V_{max}$) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was $68^{\circ}C$ and it showed a typical $Ca^{2+}$-dependent thermostability, properties fo which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.

• Journal of Microbiology and Biotechnology
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• 제26권8호
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• pp.1392-1397
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• 2016

Curcumin is a polyphenol derived from the plant Curcuma longa, which is used for the treatment of diseases associated with oxidative stress and inflammation. The present study was undertaken to determine the protective effect of curcumin against naproxen-induced gastric antral ulcerations in rats. Different doses (10, 50, and 100 mg/kg) of curcumin or vehicle (curcumin, 0 mg/kg) were pretreated for 3 days by oral gavage, and then gastric mucosal lesions were caused by 80 mg/kg naproxen applied for 3 days. Curcumin significantly inhibited the naproxen-induced gastric antral ulcer area and lipid peroxidation in a dose-dependent manner. In addition, curcumin markedly increased activities of radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase in a dose-dependent manner. Specifically, 100 mg/kg curcumin completely protected the gastric mucosa against the loss in the enzyme, resulting in a drastic increase of activities of radical scavenging enzymes up to more than the level of untreated normal rats. Histological examination obviously showed that curcumin prevents naproxen-induced gastric antral ulceration as a result of direct protection of the gastric mucosa. These results suggest that curcumin blocks naproxen-induced gastric antral ulcerations through prevention of lipid peroxidation and activation of radical scavenging enzymes, and it may offer a potential remedy of gastric antral ulcerations.

• Toxicological Research
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• 제12권2호
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• 한국식품영양과학회지
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• 제29권4호
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• pp.737-742
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• 2000

대두발효식품의 기능성 탐색 연구의 일환으로 Bacillus natto 를 접종한 납두의 발효과정 중 고혈압을 유발하는 angiotension converting enzyme의 저해 peptide를 분리하고 저해효과를 검토함으로서 대두발효식품의 우수성을 입증하기 위한 과학 적 접근을 시도하고자 하였다. 납두는 Bacillus natto균을 이용하여 제조하였고, 2$0^{\circ}C$, 3$0^{\circ}C$, 4$0^{\circ}C$, 5$0^{\circ}C$, 6$0^{\circ}C$에서 0~72시간 동안 배양하면서 protein량, protease activity, ACE 저해율 을 측정하고 저해활성을 가지는 peptide를 정제 후 아미노산 조상을 분석하였다. Bacillus natto에 의한 납두의 발효시간이 경과함에 따라 protein 함량이 증가하여 4$0^{\circ}C$, 60시간에서 최대를 나타낸 후 감소하였다. 발효시간에 따른 protease activity는 4$0^{\circ}C$, 60 시간 배양이 최적의 조건이었으며, 최적 발효조건에 따라 납두를 제조한 후 20 mM sodium phosphate buffer(pH 7.0)를 가해 추출한 추출물을 Amicon membrane YM-3 filtration 과 Sephadex G-10, G-25를 이용한 gel filtration으로 부분정제하였다. 또한 정제한 peptide는 첨가함량이 높아질수록 저해활성은 높게 나타났으며, 1 mg 정도의 peptide 함량으로 74.74%의 저해율 을 나타내었다. 정제한 peptide의 아미노산 조성은 alanine(30.84%), phenylalanine(30.03%), histidine(20.24%) 순서로 그 함량이 높게 나타났다.

• Preventive Nutrition and Food Science
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• 제14권4호
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• pp.335-342
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• 2009

Bioactive compounds were produced from carrot pomace by solid-state fermentation using Bacillus subtilis HA and Leuconostoc mesenteroides. The carrot pomace (CP) fermented by B. subtilis HA with 3% monosodium glutamate (MSG) showed higher production of various bioactive compounds, with 1.64 Pa·sn of consistency, 2.31% of mucilage content, 16.95 unit/g of fibrinolytic enzyme activity, 35.3 unit/g of proteolytic activity and 37.5 mg% of tyrosine content. The mucilage production was greatly dependent upon the concentration of MSG added. Most MSG added in CP was converted into mucilage (2.3%) including 0.83% poly-$gamma$-glutamic acid (PGA) with 1,505 kDa of molecular weight. The CP fermented secondly by Leuc. mesenteroides showed acidic pH and lower consistency. However, the fibrinolytic and proteolytic activities were increased. The secondly fermented CP showed the viable cell counts with $2.5{\time}108$ CFU/g of B. subtilis HA and $3.7{\time}109$ CFU/g of Leuc. mesenteroides, respectively. The freeze-dried fermented CP showed 2.88 Pa·sn of consistency, 24% of mucilage content and 104.9 unit/g of fibrinolytic enzyme activity, respectively. Also, the powder of fermented CP indicated viable cell counts of $8.0{\time}107$ CFU/g of B. subtilis and $4.0{\time}108$ CFU/g of Leuc. mesenteroides. Therefore, the fermented CP that was fortified with dietary fibers, fibrinolytic enzyme and probiotics could be utilized as valuable ingredients of functional foods in food or cosmetic industries.