220 research outputs found
이소플라본의 위치특이적 수산화 반응을 위한 Streptomyces avermitilis 의 P450 수산화 효소의 선별 및 전자 전달 단백질 규명
No full textThe ability of the cytochrome P450 heme monooxygenases to catalyze difficult oxidation reaction, often with high specificity and selectivity, makes them attractive for numerous biotechnological applications. However they are generally limited by low turnover rate and low stability, and their minimum requirement for catalysis include a cofactor as source NAD(P)H, partner proteins for electron transfer, and dioxygen. Here, in this thesis we attempted to deal with these issues in detail. Our goal was to identify the P450s which catalyze the regiospecific hydroxylation of isoflavonoids substrates, development of the host and identification of electron transfer system in Streptomyces avermitilis MA4680
Screening of cytochrome P450 monoxygenases responsible for the regiospecific hydroxylation of flavones, isoflavones and chalcones was attempted using a P450 library constructed from Streptomyces avermitilis MA4680 and Nocarida farcinica IFM10152, among the screened P450 library pools, four cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 and CYP105D7 from S. avermitilis showed hydroxylation activities towards flavones and isoflavones. Further analysis identified that CYP107Y1 (sav2377), CYP125A2 (sav5841), CYP107P2 (sav4539) and CYP105D7 (sav7469) showed regiospecific hydroxylation activities towards genistein (7,4',5 -trihydroxyisoflavone), chrysin (7,5 -dihydroxyisoflavone), apigenin(7,4',5-dihydroxyisoflavone) and daidzein (7,4-dihydroxyisoflavone) to produce 7,3',4',5-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydro- xyflavone, 7,3',4',5-tetrahydroxyflavone and 7,3',4'-trihydroxyisoflavone respectively.
Since, in vitro P450 biocatalysis would require continuous regeneration of the expensive cofactor NAD(P)H from NAD(P)+, adding complexity and cost to the reaction system. It was essential to develop the host based whole cell system to overcome these issues. Here, we have reported that S. avermitilis can be used as a host for the biotransformation of the isoflavonoids. Using recombinant S. avermitilis overexpression CYP105D7, it could be possible to produce 37 mg/L of 7,3',4'–trihydroxyisoflavonoids (3'-ODI). However, degradation of daidzein and its hydroxylated metabolites 7,3',4'-trihydroxyisoflavone was observed in S. avermitilis host system. We have identified that tyrosinase from S. avermitilis function as a suicide substrate for the isoflavonoids. Further deletion of tyrosinase from S. avermitilis prevents the degradation of daidzein as well as its hydroxylation metabolites. However the production yield is very low in tyrosinase (ΔmelC2) deletion mutant. Quantitative real time RT-PCR (qRT-PCR) analysis revealed that, ΔmelC2 greatly affects the expression of FDRs, Fdx and CYP105D7.
Among 33 P450s, 9 ferredoxin and 6 ferredoxin reductase, none of these FDR and Fdx was characterized yet regarding their electron transfer efficiency toward the P450s. Sufficient detail about the relevant homologous electron transfer system in Streptomyces P450s is unclear. Several approaches like gene expression analysis of FDR and Fdx, deletion of FDRs from S. avermitilis chromosomes and further the in vitro reconstitution of FDRs and Fdx, we have identified the primary electron transfer pathway for P450 as being NADPH→FdxH→FprD→CYP105D7.사이토크롬P450은 Heme을 갖고 있는 모노옥시다아제로서, 특이적이고 선택적인 산화 반응을 촉매화한다. 그러나, 낮은 회전율과 낮은 안정성, NAD(P)H와 같은 조효소와 전자 전달 단백질을 필요로 하기 때문에 P450의 생물공학적 응용에 한계가 있다. 본 연구는 아이소 플라보노이드의 위치특이적 수산화 반응을 매개하는 P450을 스크리닝하고 S. avermitilis를 이용한 숙주개발 및 전자 전달 시스템을 규명하는 연구를 다루고 있다.
플라보노이드, 아이소플라보노이드, 칼콘에 대하여 위치특이적 수산화 반응을 매개하는 P450을 스크리닝 하기 위하여 S. avermitilis와 N. farcinica 유래의 P450을 클로닝 하여 라이브러리를 구축하였고, 그 중에서 CYP105D7, CYP107Y1, CYP107P2, CYP125A2가 선택되었다. 이들 P450은 제니스테인 (4',5,7-trihydroxyisoflavone), 크리신 (5,7-dihydroxyisoflavone), 아피게닌 (4',5,7-dihydroxyisoflavone), 다이드제인 (4,7-dihydroxyisoflavone) 에 대하여 수산화 활성이 있다는 것이 규명되었다.
P450 반응에 필요한 전자는 NAD(P)H로부터 공급되기 때문에, 효소 공정 시 조효소 공급을 위한 추가적인 비용이 불가피하다. 따라서, 비용 절감을 위해서는 whole cell 시스템의 도입이 필요하고 이를 위한 숙주개발이 필요하다. 본 연구는 S. avermitilis 내에 CYP105D7의 과발현을 유도함으로서 수산화 아이소 플라보노이드 생산성을 37 mg/L 로 증가시킬 수 있었다. 그러나 기질로 사용된 다이드제인과 그것의 수산화물이 S.avermitilis 내에서 분해되는 부반응이 있음을 보았고, 이는 S.avermitilis의 타이로시나아제가 관여됨을 알게 되었다. S.avermitilis내의 타이로시나아제를 제거한 돌연변이 주를 제작함으로써 부반응을 막을 수 있었지만 생산성이 낮아졌다. 이는 타이로시나아제가 제거된 돌연변이 주 내에서 Fdr, Fdx, CYP105D7의 발현양이 감소하기 때문이었고,이를 real time PCR 을 통하여 확인하였다.
Ferredoxin (Fdx) 과 ferredoxin reductase (FDR)가 P450 반응의 전자 전달 단백질로서 그 중요도가 매우 높기 때문에, S. avermitilis 유래의 9개의 Fdx, 6개의 FDR 중 전자 전달에 관여되는 단백질 규명을 위한 연구가 필요하다. Fdr, Fdx의 mRNA 정량, FDR제거 돌연변이 주 제작, FDR, Fdx, P450의 in vitro 실험 등을 통하여 P450 반응에 필요한 전자는 NADPH로부터 FdrD, FdxH를 거쳐 최종적으로 CYP105D7으로 전달되는 것을 규명하였다.Docto
Voting under temptation
No full textWithin the confines of linear tax and complete market, we show that the efficiency force for a negative capital tax may not be strong enough to reverse the politico-economic force for a positive redistributive taxation under temptation and self-control preferences. (C) 2012 Elsevier B.V. All rights reserved.EconomicsSSCI0ARTICLE3419-42311
Particle Density, Particle Size, and Nutrient Distribution of Flushed Dairy Manure
No full textThe handling of large volumes of liquid manure produced by hydraulic flushing systems can be challenging for dairy farmers. The high solid content in flushed dairy manure causes numerous challenges during manure handling, and the high nutrient content limits the amount of manure that can be applied onto cropland. The knowledge of particle density, particle size, and nutrient distribution of flushed dairy manure would allow dairy farmers to select appropriate manure treatment technologies and make better manure nutrient management on their farms. The major goal of this study was to understand the particle density, particle size, and total nitrogen (TN) and total phosphorous (TP) distributions of flushed dairy manure by using four commercial dairies in Southern Idaho as case studies. The study also aimed to examine the statistical significance of using different pore-sized inclined screen separators for solids and nutrients removal from flushed dairy manures of the four dairies. The particle densities of flushed dairy manure solids were determined by the pycnometer method using a methanol medium. A new technique—wet sieving combined with the hydrometer-pipette method—was used to determine the particle size and nutrient distributions of the flushed dairy manures. Nutrient analyses were carried out using the Hach methods: TNT 880 for TN and TNT 845 for TP. The flushed dairy manures of the four dairies differed in the initial total solid as well as nutrient contents with total solids (TS) ranging from 2.23% to 7.69%, TN ranging from 0.08% to 0.19%, and TP ranging from 0.04% to 0.13%. The particle densities of flushed dairy manure solids were found to vary with particle size, and the average particle densities of dried solids in flushed manures of dairies, #1, #2, #3, and #4 were found to be 1.48, 1.39, 1.37, and 1.30 g/cm3, respectively, much lower than the commonly used particle density of soils of 2.65 g/cm3. The distributions of solids and nutrients in flushed dairy manures also varied between the four dairies and were found to be site-specific. However, regardless of the dairy, the majority of TS, TN, and TP in flushed manures were observed at diameters smaller than 0.5 mm. Dairies, #1, #2, #3, and #4 had 63.85%, 58.17%, 57.94%, and 51.50% of TS smaller than 0.5 mm in diameter. Similarly, the percentages of TN and TP observed at diameters smaller than 0.5 mm for dairies, #1, #2, #3, and #4 were 72.00%, 75.14%, 75.76%, and 61.92% and 85.64%, 70.58%, 69.28%, and 61.35%, respectively. The statistical differences between the solid and nutrient removal capacities of different pore-sized inclined screen separators were found to be dairy-specific. From this study, it was estimated that 0.5-mm pore-sized inclined screen separators would remove between 25.41% and 37.40% of TS, 24.24% and 38.08% of TN, and 14.36% and 38.65% of TP from flushed dairy manures with initial TS ranging from 2.23% to 7.69%. This suggests that the inclined screen separators with pore size larger than 0.5 mm would remove only a fraction of total solids, total nitrogen, and total phosphorus from flushed dairy manures, and most of the TS, TN, and TP would remain in the liquid fraction after solid-liquid separation. Therefore, commercial dairies that rely on inclined screen separators with pore sizes larger than 0.5 mm for solid-liquid separation might need to look beyond the 0.5-mm pore-sized screen separators to remove higher quantities of solids and nutrients from their flushed dairy manures.masters, M.S., Water Resources -- University of Idaho - College of Graduate Studies, 2021-0
Effects of Plant Growth Regulators on Plant Phenotypes and Yield Components of Grass Seed Crops
No full textCreeping red fescue (Festuca rubra L.), meadow brome (Bromus riparius Rehmann) and timothy grass (Phleum pratense L.) are the most important grass species grown for seed production in the Peace River region of Alberta and British Columbia. Creeping red fescue is a popular turf grass species, whereas meadow bromegrass and timothy are commonly used for pasture, hay, and silage production for livestock feed. The seed productivity of these grass crops is limited by lodging under abundant rainfall conditions. Lodging can negatively impact seed yield and quality in forage crops, and make harvesting more difficult, time-consuming, and prone to seed loss through shattering. Plant growth regulators (PGRs) are an effective means of controlling lodging and seed loss, especially when grasses are grown under high nitrogen (N) fertility and non-limiting soil moisture conditions. A study was conducted over two years in 2021 and 2022 to quantify the morphological development of these grasses in response to PGRs at Beaverlodge Research Farm, Agriculture and Agri-Food Canada (AAFC) in northwestern Alberta, Canada. This study aimed to identify beneficial PGRs for regulating plant photosynthesis, growth, lodging tolerance, seed yield, and yield components of these grasses. A field experiment with split-plot design included PGR treatments as main plots and top-dressed spring nitrogen (urea) at 40 kg ha-1 as subplots on three and four-year-old stands of these grasses for two consecutive years. Three PGRs, including trinexapac-ethyl (TE), chlormequat chloride (CC) and ethephon (ETH), applied at 0.20 kg, 1.12 kg, and 0.60 kg active ingredient (ai.) ha-1 were used separately on each grass at the two-node stage of crop development (BBCH 31-32) and compared to untreated grasses. The results showed that TE was efficacious in shortening internode and tiller height and reducing lodging in meadow bromegrass and timothy. However, PGRs did not alter grass seed attributes such as panicle length, seed weight, and seed number, as well as the total seed yield in all three grasses. TE demonstrated similar levels of marginal economic returns to the check, indicating comparable profitability for timothy and creeping red fescue. Spring nitrogen was beneficial in increasing biomass and seed yield in timothy and meadow bromegrass without significant lodging. The effects of PGRs were more pronounced in improving grass morphology under normal rainfall conditions. However, under moisture deficit climatic conditions, PGRs had inconsistent and minimal effects on grass plant morphology.
A greenhouse study was also conducted to investigate the effects of PGRs on several variables related to vegetative growth, root-shoot allometry, chlorophyll pigmentation, and photosynthetic efficiency at the juvenile stage of these grass crops. The study found that TE reduced root, shoot, and total biomass up to 40 days in timothy, and up to 55 days in creeping red fescue after application, but no PGRs affected meadow brome biomass at any point of time. The PGRs had no effect on growth variables such as root area, root length, root to shoot ratio, leaf area, tiller number, final biomass, and other growth parameters like relative growth rate, net assimilation rate, leaf weight ratio and specific leaf area. Both TE and CC reduced the plant height and lodging severity of the three grasses. PGRs did not enhance or hinder the quantum yield of photosystem II (Fv/Fm) from 4 h through 12 days after PGR application. However, TE and CC increased chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents (µg g-1 FW) in timothy up to 70 days after application. Despite these limited responses, a principal component analysis revealed that the ETH and check treatments exhibited higher total biomass, shoot biomass, root-to-shoot ratio, and plant height, which were associated with higher lodging severity, while TE and CC exhibited higher contents of chlorophylls and carotenoids along with greater NAR in all tested grasses. Overall, TE was more efficient and efficacious in reducing plant height and lodging severity of timothy and meadow bromegrass under field conditions and did the same for all grasses including creeping red fescue, in the greenhouse. Multiple studies of PGRs under a range of environmental scenarios can improve our understanding of how these compounds influence grass morpho-physiology. Likewise, long-term field studies under different lodging conditions could lead to the development of more efficient PGRs utilization strategies that maximize forage yield and seed harvestability
CaO-based CO 2 sorbents: A review on screening, enhancement, cyclic stability, regeneration and kinetics modelling
No full textInvestigation into overall heat transfer coefficient in indirectly heated rotary torrefier
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