• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.237-244
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• 1998

A study was carried out to elucidate the relation between the production of flavonol glycosides and the change of phenylalanine ammonia-lyase activity in cell suspension cultures of Ginkgo biloba by the unassisted and synergistic effects of various factors. The quercetin production showed a mixed-growth-associated pattern in cell suspension cultures. Fluorescent light and UV radiation increased phenylalanine ammonia-lyase (PAL) activity, and resulted in the increase of the production of quercetin and kaempferol ten- and four-fold, respectively, as compared to that obtained in the normal culture condition. The cell growth of Ginkgo biloba was enhanced .at higher temperatures whereas the quercetin production was at its maximum at low temperatures. Moreover, the quercetin production was increased by temperature change during the culture period. In particular, the quercetin production was at the highest level when the culture temperature was elevated from $10^{\circ}C\;to\;30^{\circ}C$. The addition of phenylalanine as a precursor in the culture medium stimulated an 8-fold increase in the production of quercetin; the addition of naringenin caused a l0-fold increase. The quercetin production was also greatly increased by feeding enzyme cofactors such as 2-ketoglutarate and ascorbic acid in the culture medium, but specific PAL activity was not increased except with phenylalanine feeding. The synergistic effect of UV radiation and naringenin feeding was observed, resulting in the increase of flavonol glycoside production at a rate higher than in any other case investigated.

• Journal of Life Science
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• v.19 no.9
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• pp.1226-1231
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• 2009

Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

• Korean Journal of Plant Tissue Culture
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• v.26 no.1
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• pp.41-47
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• 1999

Phenylalanine ammonia-lyase (PAL; EC 4, 3, 1, 5) genomic clones were isolated from tomato(Lycopersicon esculentum L.) genomic DNA libraries using tomato PAL5 cDNA sequences as probes. The nucleotide sequences of tPAL1, tPAL4 and tPAL5 were compared. tPAL5 contains an open reading frame encoding a polypeptide of 722 amino acids, interrupted by a 710 bp intron in the codon for the amino acid 139. tPAL1 encodes a polypeptide of 249 amino acids which is much shorter than tPAL5 gene due to a premature stop codon and does not contain an intron. tPAL4 encodes a polypeptide of 357 amino acids, interrupted by a 305 bp intron in the codon for the amino acid 138. Premature stop codons observed in tPAL1 and tPAL4 gene produce a short polypeptide rather than a normal polypeptide (722 aa). tPALl shows 87.2% homology with tPAL4 and 85.3% homology with tPAL5 gene whereas tPAL4 showes 91.4% homology with tPAL5 at nucleotide level. In general, phylogenetic analysis showed that genes isolated from tomato, potato, and sweet potato were belong to the same group and another dicot plants such as parsley, bean, soybean, pea and alfalfa formed another group. PAL genes isolated from rice and yeast showed very low homology with other PAL genes and formed the other group.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.89-93
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• 1998

Tomato phenylalanine ammonia-lyase 5 (tPAL5) was identified that alternate initiation sites were utilized differentially in response to environmental stimuli (Lee et al, 1992b). In this study, we tried to look into tissue -or cell- specific expression pattern of tPAL5 gene by fusing with ${\beta}-glucuronidase$ (GUS) gene in transgenic tobacco plants. In transgenic plants, root and stem extracts contained 8~12 fold higher levels of GUS activity than petiole or leaf tissue while the highest levels of induction was observed from leaf tissue by mechanical wounding (5~11 fold). In trans-sections of stems and petioles, GUS activity was restricted to phloem cells(outer region) of developing vascular bundle and mainly at apical tip region in the root tissues. The levels of GUS activity was drastically reduced (10~12 fold reduction) when the 5'-upstream region of tPAL5 gene (-1151bp from ATG codon) was deleted up to -665. The levels of GUS expression, however, raised up by 6~8 fold when deleted up to -455. Therefore, we conclude that there are positive cis-elements at the region -1151 to -1008 and at -455 to -195 while the negative cis-element is at -1008 to -455.

• Proceeding of EDISON Challenge
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• 2013.04a
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• pp.203-204
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• 2013

Phenylalanine Ammonia Lyase (PAL)는 phenylalanine의 산성도가 더 큰 ammonium hydrogen 과는 반응하지 않고 그것의 nonacidic ${\beta}$ proton을 제거하는 역할을 한다. Phenylalanine은 electrophilic 그룹을 갖고 있으며 지난 30 여년 동안 phenylalanine의 electrophilic 그룹이 PAL의 반응 기작에 중요한 역할을 한다고 믿어왔다. 그러나 최근 X-ray와 UV spectroscopy를 통하여 상당히 electrophilic 한 5-methylene-3,5-dihydroimidazol-4-one (MIO) 그룹이 발견되었다. 이전의 연구들은 실험을 통하여 MIO와 관련된 electrophile에 의한 Friedel-Crafts Attack 메커니즘과 electrophile에 대한 nucleophilic addition 메커니즘을 제시하였으며, 본 연구진은 양자계산을 통하여 두 가지 메커니즘의 에너지 차이를 살펴봄으로써 더욱 합리적인 메커니즘을 제시, 규명하고자 한다. 본 연구에서는 특히 electrophile에 대한 nucleophilic addition 메커니즘에 대하여 양자계산을 이용하여 반응물, 생성물, 전이상태의 분자 구조를 제시하고 반응이 일어나는데 필요한 에너지를 계산하고자 한다.

• Journal of Life Science
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• v.12 no.3
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• pp.274-280
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• 2002

The metabolites related to phenylpropanoid pathway play an important role in the self-defense of plants and induced by environmental stress like wounding, pathogen attack, UV-irradiation and so on. The mRNA level of rite phenylalanine ammonia lyase (PAL) was increased at 12 h to 48 h, however it was gradually decreased 48 h to 60 h after UV irradiation. The PAL enzyme activities in rice were peaked at the time of 24 h after UV irradiation, on the other hand, it was not affected by wounding. The PAL enzyme activities in pepper were raised high at 24 h and 10 h by UV irradiation and wounding respectively. The cinnamic acid 4-hydroxylase (C4H) activities were increased by wounding treatment and were detected from 12 h to end time point of experiment, while UV-irradiation didn't affect the C4H activity in rice and pepper. These results were assumed that the action of isoflavonid has an alternative effect on the defenses which include wounding and UV irradiation and on the diverse roles in rice and hot pepper.

• Journal of the Korean Society of Food Science and Nutrition
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• v.16 no.1
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• pp.10-17
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• 1987

In order to investigate the effects of growth regulators on the formation of capsaicinoids in callus of Capsicum annuum L. tissues were cultured in the Linsmaier and Skoog RM 1964 medium containing various growth regulators. Production of capsaicinoids during culture was monitored by gas chromatography. In the presence of $10^{-6}M$ of 2,4-D and kinetin in the medium, $1182{\mu}g$ of capsaicinoids were formed per 100g dry wt. of tissue, of which was greater than with any of three other growth regulators. IAA, NAA, and kinetin of same concentrations had 65%, 38%, 68% effect of 2.4-D in capsaicinoids formation, respectively. Production of capsaicinoids increased gradually in the presence of 2,4-B as culture period was proceeded. Of phenylpropanoids formed, cinnamic acid and coumaric acid were not significantly different in their levels, although growth regulators were varied. On the other hand, caffeic acid and ferulic acid formation were highest in the presence of 2,4-D. Effects of kinetin and IAA were about 70 percent of that of 2,4-D, whereas NAA had only about 30 percent effect. Phenylalanine ammonia-lyase activity in cultured tissue was increased during the periods; 52, 81, and 209 n moles of cinnamic acid per g fresh wt. were formed after 5, 15, and 25 days of culture, respectively.

• The Plant Pathology Journal
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• v.24 no.2
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• pp.171-177
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• 2008

In this study, we investigated the induced defense response and protective effects against Stemphylium vesicarium by application of benzothiadiazole ($Bion^{(R)}$) and di-potassium phosphate salt $(K_2HPO_4)$ to onion. Onion leaves were sprayed with $Bion^{(R)}$ and $K_2HPO_4$, then inoculated 2 days later with a virulent strain of S. vesicarium under greenhouse conditions. Disease severity and activities of peroxidase (PO), polyphenoloxidase, phenylalanine ammonia-lyase (PAL) and phenol contents were evaluated in the treated leaf tissues. Reduction in the disease severity was observed in plants treated with $Bion^{(R)}$ and $K_2HPO_4$. Onion plants treated with $Bion^{(R)}$ and $K_2HPO_4$ and inoculated with the pathogen showed significantly higher PAL activity, PO activity, and phenol contents than inoculated water-treated plants 2 days after the treatment. In conclusion, the results of this study provide evidence that application of simple non-toxic chemical solutions as di-potassium phosphate and $Bion^{(R)}$ can control Stemphylium leaf blight of onion.

• Journal of The Korean Society of Grassland and Forage Science
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• v.34 no.1
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• pp.15-20
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• 2014

To investigate lignification process and its physiological significance under water-deficit condition, the responses of peroxidases, polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) in relation to leaf water status to the short term of water deficit treatment in the leaves with different maturities in forage rape were measured. The significant decrease in relative water content (RWC) and leaf osmotic potential (${\Psi}{\pi}$) were apparent after 5 d of water-deficit treatment. The activity of guaiacol peroxidase (GPOD), ascorbate peroxidase (APOD), coniferyl alcohol peroxidase (CPOD), and syringaldazine peroxidase (SPOD) was depressed especially in middle and old leaves when compared with that of control leaves. On the other hand, in young leaves, a significant increase in CPOD (+34%) and SPOD (+24%) activity as affected by water-deficit treatment was apparent. The activation of PAL and PPO was observed in middle and old leaves for PAL and in young and middle leaves for PPO. These results suggest that peroxidases in middle and old leaves did not involve in lignification under mild water-deficit stress, whereas CPOD and SPOD in young leaves participate in lignification by a coordination with PAL and PPO to incorporate phenol and lignin into the cell walls.

• Journal of Environmental Science International
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• v.14 no.12
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• pp.1093-1102
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• 2005

Effects of abscisic acid(ABA) and temperature on the anthocyanin accumulation and phenylalanine ammonia Iyase(PAL) activity were investigated in seedlings of Arabidopsis thaliana. In time course study, exogenous application of ABA $(50-1000\;{\mu}M)$ led to a noticeable increase in anthocyanin pigments which persisted over the following 5 days. Anthocyanins increased in concert with the chlorophyll loss. The activity of PAL, a key enzyme in the phenylpropanoid pathway, increased on exposure to ABA and reached maximum on the 4th day, This result shows that anthocyanin synthesis and PAL activity have a close physiological relationships. In the effects of temperatures ($10^{\circ}C,\;17^{\circ}C,\;25^{\circ}C$and $30^{\circ}C$) on anthocyanin accumulation and PAL activity in seedlings, a moderate-low temperatures ($17^{\circ}C$) enhanced both anthocyanin content and PAL activity, whereas elevated temperatures ($30^{\circ}C$) showed low levels of anthocyanin and PAL activity, suggesting a correlation between temperature-induced anthocyanin synthesis and the accumulation of PAL mRNA. Simultaneous application of ABA with temperatures Induced higher anthocyanin synthesis and PAL activity in seedlings than ABA or temperature stress alone. Moderate-low temperature with ABA exposure elicited the maximal induction of anthocyanin synthesis and PAL activity. Therefore, ABA treatment significantly increased thermotolerance in .A. thalinan seedlings. Ethephon and ABA showed similar mode of action in physiological effects on anthocyanin accumulation and PAL activity. Our data support that anthocyanins may be protective in preventing damage caused by environmental stresses and play an important role in the acquisition of freezing tolerance.