• Animal Bioscience
• /
• 제36권10호
• /
• pp.1604-1611
• /
• 2023

Objective: The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Methods: Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Results: Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dose-dependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Conclusion: Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

• Preventive Nutrition and Food Science
• /
• 제2권4호
• /
• pp.354-359
• /
• 1997

Optimal cultivation conditions for the production of phenylalanine ammonia-lyase(PAL) from Rhodotorula aurantiaca K-505 were selected, and the kinetic parameters of the produced PAL were determined. The most suitable carbon and nitrogen sources were glucose and tryptone, respectively. The strain expressed PAL constituttively when using the optimized semi-complex media. High cell density culture could be critical for maximal production of PAl since the PAL ynthesis was growth associated. maximum PAL activity was observed at initial pH 6.0. although the ll growth was not markedly affected by temperature between 22 and 28$^{\circ}C$, the cells yielded the maximum PAL activity when cultivated at 22$^{\circ}C$. The maximum activity for deamination of L-phenylalnine to trans-cinnamic acid was observed around pH 8.8. The PAL activity gave the maximum at 45$^{\circ}C$, and greatly decreased at higher than 5$0^{\circ}C$. Activation energy({TEX}$E_{a}${/TEX}) calculated from Arrhenius equation was 6.28 kcal/mol in the range of 22$^{\circ}C$ to 4$0^{\circ}C$. A oolf plot showed that the enzyme reaction follows Michaelis-Menten equation, whose {TEX}$K_{M}${/TEX} and {TEX}$V_{max}${/TEX} values were 4.65$\times${TEX}$10^{-3}${/TEX} M and 0.89$\mu$ mol/mg-min respectively.

• Food Science and Biotechnology
• /
• 제18권4호
• /
• pp.954-958
• /
• 2009

Effects of controlled- and uncontrolled-pH operations on phenylalanine ammonia lyase (PAL) production by a recombinant Escherichia coli strain were investigated at uncontrolled-pH ($pH_{UC}$) and controlled-pH ($pH_C$) of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 in bioreactor systems. The results showed that the recombinant PAL activity was improved significantly by controlled pH strategy. Among the $pH_C$ operations, the highest PAL activities were obtained under $pH_C$ 7.5 strategy where cell mass ($OD_{600\;nm}$) and PAL activity was 1.3 and 1.8 fold higher than those of $pH_{UC}$, respectively. The maximum PAL activity reached 123 U/g. The $pH_C$ 7.5 strategy made recombinant plasmid more stable and therefore allowed easier expression of PAL recombinant plasmid, which increased PAL production. It was indicated that the new approach (controlled-pH strategy) obtained in this work possessed a high potential for the industrial production of PAL, especially in the biosynthesis of L-phenylalanine.

• BMB Reports
• /
• 제31권1호
• /
• pp.31-36
• /
• 1998

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme in the phenylpropanoid biosynthesis, catalyzes the elimination reaction of ammonium ion from L-phenylalanine. PAL was purified from the cytosolic fraction of Chinese cabbage (Brassica campestris ssp. napus var. pekinensis) through ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-200 chromatography, and Q-Sepharose chromatography. It consists of four identical subunits, the molecular mass of which was estimated to be about 38,000 daltons on SDS-PAGE. The optimal pH and temperature of the purified enzyme are 8~9 and $45^{\circ}C$, respectively. Its activity is greatly inhibited by $Zn^{2+}$ ion, and strongly activated by caffeic acid. The purified PAL has some different characteristics compared to those obtained with other PALs.

• Journal of Pharmaceutical Investigation
• /
• 제41권2호
• /
• pp.95-102
• /
• 2011

An amphiphilic cationic branched methoxy poly (ethylene glycol)-branched polyethylenimine - poly(L-phenylalanine) (mPEG-bPEI-pPhe) block copolymer was successfully synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of L-phenylalanine (Phe-NCA) with mPEG-bPEI for the preparation of more stable polyelectrolyte complex (PEC) included a hydrophobic interaction. mPEG-bPEI was firstly prepared by the coupling of mPEG and bPEI using hexamethylene diisocyanate (HMDI). The structural properties of mPEG-bPEI-pPhe copolymers were confirmed by $^1H$ NMR. The copolymers exhibited a self-assemble behavior in water above critical aggregate concentration (CAC) in the range of 0.01-0.14 g/L. The CAC of copolymers obviously depended on the hydrophobic block content in the copolymers (the value decreased with the increase of the pPhe block content). The cationic copolymers have the ability to form multi-interaction complex (MIC) with bovine serum albumin (BSA) and plasmid DNA through multi-interaction (electrostatic and hydrophobic interaction). The physicochemical characterization of the complex was carried out by the measurement of zeta potential and particle size. Their zeta-potentials were positive (approximately +10 mV) and their sizes decreased with increasing pPhe contents in the copolymers (PPF/BSA wt% ratio = 2). The complex showed good stability at high ionic strength. Therefore, mPEG-bPEI-pPhe block copolymer was considered as a potential material to enhance the stability of complex including biotherapuetic drugs.

• 한국동물학회지
• /
• 제33권1호
• /
• pp.53-62
• /
• 1990

양서류의 여포난자를 생체외에서 배양하면서 호르몬을 처리하면 난자의 성숙(핵붕괴)을 일으킨다. 본 연구는 북방산개구리의 여포난자를 배양하면서 난자내 단백질 분해효소들의 활성변화를 유도하여 이것이 난자의 성숙에 어떠한 영향을 미치는가를 조사하였다. chymotrypsin의 저해제로 알려진 N$\alpha$ -tosyl-L-phenylalanine-chloromethyl-ketone(TPCK)을 배양액에 처리하면 비교적 낮은 농도(0.001-1 $\mu$M)에서는 호르몬의 도움없이도 난자의 성숙을 유도하나 높은 농도 (100 $\mu$M)에서는 호르몬에 의한 난자의 성숙까지도 억제하는 이중적인 효과를 나타내었다. Trypsin의 저해제인 N$\alpha$ -tosyl-L-phenylalanine-chloromethyl-ketone(TPCK)은 성숙유도능력이 없을 뿐 아니라 progesterone에 의한 난자의 성숙을 억제하였다. Trypsin을 직접 처리했을 때에는 농도에 의존하여(0.001-1$\mu$g/2ml) 호르몬의 도움없이도 난자의 성숙을 유도함을 발견하였다. TLCK나 TPCK의 억제효과는 성숙 초기에만 나타났다. 본 결과는 양서류 난자의 성숙조절 과정에 몇종의 단백질 분해효소들이 참여한다는 것을 시사해주고 있다.

• 한국식품영양과학회지
• /
• 제24권6호
• /
• pp.998-1004
• /
• 1995

23종의 amino acid와 fish oil과의 갈변 반응에서 Aw 및 온도의 영향을 조사한 결과 Aw 0.33에서 Aw 0.85에서 3가지 형태를 보였다. Type I은 Aw 0.33과 Aw 0.95에서의 갈변도가 Aw 0.52와 0.75 보다 높은 값을 나타낸 것이며(phenylalanine, trans-4-hydroxy-L-proline, me0thionine, valine), Type II는 수분활성도가 증가할수록 갈변도가 감소한 것이며(poline, leucine, isoleucine, arginine), Type III은 Aw 0,33과 0.95 보다는 Aw 0.52와 0.75에서 높은 갈변도를 나타냈다(tryptophan, cystein, threonine, lysine). 온도에 대한 갈변도의 정도는 높은 온도 의존도를 보였는데 특히 phenylalanine, valine, trans-4-hydroxy-proline 및 methionin이었다. Activation energy는 8~40kcal/mole이었고, $Q_{10}$ 값은 2~10이었다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제9권3호
• /
• pp.255-259
• /
• 1996

A study was carried out to investigate the action of central L-pheylalanine (Phe) and L-tyrosine (Tyr) on food intake of the chicken. In the first trial, Phe ($200{\mu}g/10{\mu}l$) or saline was acutely administered into the right lateral ventricle (i.c.v.) of chickens (5 birds per each group). Birds (4 birds per each group) were administered with the i.c.v. Tyr ($200{\mu}g/10{\mu}l$) or saline in the second trial. The brains of the birds were removed for catecholamine assy 30 min postadministration. Catecholamine concentrations were measured at specific sites of the brain (LH: lateral hypothalamus, PVN: paraventricular nucleus, and VMH: ventromedial hypothalamus). No significant effect of amino acids on the concentration of norepinephrine of brain sites investigated was detected. Food intake and rectal body temperature were also monitored for 6 h after central administrations of Phe, Tyr or saline (5 birds per each group). Both Phe and Tyr, up to $1mg/10{\mu}l$, failed to modulate food intake or rectal body temperature.

• 한국식품영양과학회지
• /
• 제16권1호
• /
• pp.10-17
• /
• 1987

고추의 배양세포(培養細胞)에 있어서 고추의 신미성분(辛味成分)인 capsaicinoids 생성(生成)에 미치는 growth regulator의 영향을 조사하기 위하며 growth regulator들을 각각 함유한 Linsmaier-Skoog 기본배지에서 배양(培養)한 callus의 capsaicinoids을 GLC에 의하여 조사하고 또한 capsaicinoids의 중간대사 산물인 phenylpropanoids와 phenylalanine ammonia-lyase activity을 측정하여 다음과 같은 결과를 얻었다. 1. Growth regulators가 배양세포(培養細胞)의 capsaicinoids 생성(生成)에 미치는 영향을 조사한 결과 growth regulators을 달리한 배지(培地)에서 생성(生成)된 capsaicinoids의 함량(含量)은 $2,4-D\;10^{-6}M$에서 가장 capsaicinoids의 생성량(生成量)이 많았으며 $IAA\;10^{-6}M,\;NAA\;10^{-6}M,\;kinetin\;10^{-6}M$의 배지(培地)에서는 2,4-D에 비(比)하여 각각 65%, 38%, 68%이었다. 2. Growth regulators을 달리한 기본배지에서 생성(生成)된 phenylpropanoids는 cinnamic acid, coumaric acid의 생성(生成)은 5, 15, 25일(日)에 점진적으로 증가하였으며 NAA 함유 배지에서는 2,4-D에 비해 약 30%, kinetin, IAA 함유배지에서는 약 70% 생성(生成)되었다. 3. $2,4-B\;10^{-6}M$을 함유한 배양세포(培養細胞)에서 phenylalanine ammonia-lyase activity을 측정한 결과 배양후(培養後) 5, 15, 25일(日) 배양일수에 따라 phenylalanine ammonia-lyase activity는 신선중량 g당 생성된 cinnamate는 각각 52, 81 및 209 % nmole로 증가하였다.

• 대한화학회지
• /
• 제14권2호
• /
• pp.161-168
• /
• 1970

In order to obtain the more effective evidence, supporting the hypothesis which have been previously described by former report that pepsin (EC 3.4. 4.1) forms a hydrophobic bond with the nonpolar side chain of its substrate, the inhibitory effect of carboxylic acids(from formic acid to iso-butyric acid) on the activity of pepsin to the synthetic dipeptide, N-Carbobenzoxy-L-glutamyl-L-tyrosine, was discussed. The kinetic study showed that the inhibition by carboxylic acids was competitive. The Kidecreased with increasing size of the inhibitor molecule. The $-{\Delta}F^{\circ}$increased linearly with increasing number of carbon atoms in the hydrocarbon chain of the inhibitor. It was confirmed that the hydrophobic bond between more than one side chain of amino acid residues(phenylalanine) in the binding region of the active center of pepsin and the side chain of amino acid residues in the substrate was formed as the first step of its enzymic mechanism. The inhibitory effect of carboxylic acids was due to the competition of the hydrocarbon group of the carboxylic acids with the side chain of the substrate for the hydrophobic binding site(the side chain of phenylalanine) of the pepsin.