Economy of scale for green hydrogen-derived fuel production in Nepal.

Opportunity for future green hydrogen development in Nepal comes with end-use infrastructural challenges. The heavy reliance of industries on fossil fuels (63.4%) despite the abundance of hydroelectricity poses an additional challenge to the green transition of Nepal. The presented work aims to study the possibility of storing and utilizing spilled hydroelectricity due to runoff rivers as a compatible alternative to imported petroleum fuels. This is achieved by converting green hydrogen from water electrolysis and carbon dioxide from carbon capture of hard-to-abate industries into synthetic methane for heating applications via the Sabatier process. An economy-of-scale study was conducted to identify the optimal scale for the reference case (Industries in Makwanpur District Nepal) for establishing the Synthetic Natural Gas (SNG) production industry. The techno-economic assessment was carried out for pilot scale and reference scale production unit individually. Uncertainty and sensitivity analyses were performed to study the project profitability and the sensitivity of the parameters influencing the feasibility of the production plant. The reference scale for the production of Synthetic Natural Gas was determined to be 40 Tons Per Day (TPD), with a total capital investment of around 72.15 Million USD. Electricity was identified as the most sensitive parameter affecting the levelized cost of production (LCOP). The 40 TPD plant was found to be price competitive to LPG when electricity price is subsidized below 3.55 NPR/unit (2.7 c/unit) from 12 NPR/unit (9.2 c/unit). In the case of the 2 TPD plant, for it to be profitable, the price of electricity must be subsidized to well below 2 NPR/kWh. The study concludes that the possibility of SNG production in Nepal is profitable and price-competitive at large scales and at the same time limited by the low round efficiency due to conversion losses. Additionally, it was observed that highly favorable conditions driven by government policies would be required for the pilot-scale SNG project to be feasible.

Antioxidant and Enzyme Inhibitory Potential of Streptomyces sp. G-18 Grown in Various Media.

Streptomyces are bacteria well known for producing bioactive secondary metabolites which are commonly found in diverse habitats. The biosynthesis of metabolites from Streptomyces is influenced by various factors such as the growth medium, environmental conditions, and gene regulation. This study aimed to investigate the influence of different growth media on biomass production and the antioxidant and enzyme inhibitory potential of a crude extract obtained from Streptomyces sp. G-18 isolated from high altitudinal soil of Nepal. The highest dry weight growth was observed in R2YE medium (184 mg/L), followed by R5 (144 mg/L), YEME (38 mg/L), and R5M media (30 mg/L). The crude extract showed notable antioxidant activities against free radicals. The highest alpha-amylase inhibition was observed in the R2YE medium, and worthy lipase and tyrosinase inhibition was observed in the YEME medium. However, only the R2YE medium exhibited inhibitory potential against elastase and acetylcholinesterase, while crude extracts from R5, YEME, and R5 modified did not show any such activity. Overall, our findings suggest that the production of bioactive secondary metabolites in Streptomyces sp. G-18 was significantly influenced by the growth medium. This strain may be a promising source of enzyme inhibitors with potential applications in the pharmaceutical and cosmetic industries.

Lichens as spatially transferable bioindicators for monitoring nitrogen pollution.

Excess nitrogen is a pollutant and global problem that harms ecosystems and can severely affect human health. Pollutant nitrogen is becoming more widespread and intensifying in the tropics. There is thus a requirement to develop nitrogen biomonitoring for spatial mapping and trend analysis of tropical biodiversity and ecosystems. In temperate and boreal zones, multiple bioindicators for nitrogen pollution have been developed, with lichen epiphytes among the most sensitive and widely applied. However, the state of our current knowledge on bioindicators is geographically biased, with extensive research effort focused on bioindicators in the temperate and boreal zones. The development of lichen bioindicators in the tropics is further weakened by incomplete taxonomic and ecological knowledge. In this study we performed a literature review and meta-analysis, attempting to identify characteristics of lichens that offer transferability of bioindication into tropical regions. This transferability must overcome the different species pools between source information - drawing on extensive research effort in the temperate and boreal zone - and tropical ecosystems. Focussing on ammonia concentration as the nitrogen pollutant, we identify a set of morphological traits and taxonomic relationships that cause lichen epiphytes to be more sensitive, or more resistant to this excess nitrogen. We perform an independent test of our bioindicator scheme and offer recommendations for its application and future research in the tropics.

Biotransformation of Daidzein, Genistein, and Naringenin by Streptomyces Species Isolated from High-Altitude Soil of Nepal.

Flavonoids have achieved widespread importance in pharmaceutical, food, and cosmetics industries. Furthermore, modification of these naturally occurring flavonoids to structurally diverse compounds through whole cell biotransformation with enhanced biological activities has numerous biotechnological applications. The present study investigated the biotransformation potential of Streptomyces species isolated from a high-altitude-soil sample towards selected flavonoid molecules. The biotransformed metabolites were confirmed by comparing the HPLC chromatogram with authentic compounds and LC-MS/MS analysis. Of these isolates, Streptomyces species G-18 (Accession number: MW663767.1) catalyzed isoflavone molecules daidzein and genistein to produce hydroxylated products at 24 h of reaction condition in a whole cell system. The hydroxylation of daidzein (4',7-dihydroxyisoflavone) was confirmed at 3'-position of the B ring to produce 3',4',7-trihydroxyisoflavone. In addition, Streptomyces species G-14 (Accession number: MW663770.1) and Streptomyces species S4L (Accession number: MW663769.1) also revealed the transformation of daidzein (4',7-dihydroxyisoflavone) to hydroxy daidzein at a distinct position than that of G-18 isolates, whereas thee Streptomyces species S4L reaction mixture with naringenin as a substrate also revealed the hydroxylated product. Our results demonstrated that microorganisms isolated from different ecological niches have broad application.

Technologies to recover nitrogen from livestock manure - A review.

Today, the livestock industry is considered to be one of the biggest emitters of ammonia in the world. The nitrogen present in livestock manure has been linked to the contamination of water bodies. Livestock manures contain a significant quantity of recoverable nitrogen. Recovering nitrogen from livestock manure can minimize negative environmental consequences. This also presents an opportunity to generate some revenue by converting the captured nitrogen to marketable nitrogenous fertilizers. Substantial research efforts have been made toward recovering nitrogen from raw as well as digested livestock manures over the last decade. Many novel technologies as well as ones that have already been implemented to recover nitrogen from municipal wastewaters have been studied for their use in the livestock sector. This paper reviews the common manure nitrogen-recovery technologies reported in the literature, summarizes their efficiencies, discusses their pros and cons, and identifies the areas for future research. Owing to their higher ammonia recovery efficiencies, relatively fewer drawbacks, lower costs, and ability to produce ammonium fertilizers, air stripping by direct aeration, thermal vacuum stripping, and gas-permeable membrane stripping appear to be the most viable choices for livestock farmers. Further studies should focus on the economic feasibility, long-term performance on the manure of varying strengths, and the quality of recovered nitrogenous products.

Bergenia pacumbis from Nepal, an astonishing enzymes inhibitor.

BACKGROUND: The Bergenia species are perennial herbs native to central Asia, and one of the most promising medicinal plants of the family Saxifragaceae which are popularly known as 'Pashanbheda'. The aim of this study was to evaluate antioxidant and α-amylase, α-glucosidase, lipase, tyrosinase, elastase, and cholinesterases inhibition potential of Bergenia pacumbis of Nepali origin collected from the Karnali region of Nepal. METHODS: The sequential crude extracts were made in hexane, ethyl acetate, methanol, and water. Antioxidant activities were analyzed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The α-amylase, α-glucosidase, lipase, tyrosinase, elastase, acetylcholinesterase, and butyrylcholinesterase inhibition were analyzed by the 3,5-Dinitrosalicylic acid (DNSA), p-Nitrophenyl-α-D-glucopyranoside (p-NPG), 4-nitrophenyl butyrate (p-NPB), l-3,4-dihydroxyphenylalanine (L-DOPA), N-Succinyl-Ala-Ala-p-nitroanilide (AAAPVN), acetylthiocholine, and butyrylcholine as a respective substrate. The major metabolites were identified by high performance liquid chromatography with electron spray ionization- quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) profiling. RESULTS: Our results revealed the great antioxidant ability of crude extract of B. pacumbis in ethyl acetate extract against both DPPH (IC50 = 30.14 ± 0.14 µg/mL) and ABTS (IC50 = 17.38 ± 1.12 µg/mL). However, the crude methanol extract of B. pacumbis showed the comparable enzymes inhibitions with standard drugs; α-amylase (IC50 = 14.03 ± 0.04 µg/mL), α-glucosidase (IC50 = 0.29 ± 0.00 µg/mL), lipase (IC50 = 67.26 ± 0.17 µg/mL), tyrosinase (IC50 = 58.25 ± 1.63 µg/mL), elastase (IC50 = 74.00 ± 3.03 µg/mL), acetylcholinesterase (IC50 = 31.52 ± 0.58 µg/mL), and butyrylcholinesterase (IC50 = 11.69 ± 0.14 µg/mL). On the basis of HPLC-ESI-QTOF-MS profiling of metabolites, we identified major compounds such as Bergenin, Catechin, Arbutin, Gallic acid, Protocatechuic acid, Syringic acid, Hyperoside, Afzelechin, Methyl gallate, Paashaanolactone, Astilbin, Quercetin, Kaempferol-7-O-glucoside, Diosmetin, Phloretin, and Morin in methanol extract which has reported beneficial bioactivities. CONCLUSION: Our study provides a plethora of scientific evidence that the crude extracts of B. pacumbis from Nepalese origin in different extracting solvents have shown significant potential on inhibiting free radicals as well as enzymes involved in digestion, skin related problems, and neurological disorders compared with the commercially available drugs.

LC-ESI-QTOF-MS for the Profiling of the Metabolites and in Vitro Enzymes Inhibition Activity of Bryophyllum pinnatum and Oxalis corniculata Collected from Ramechhap District of Nepal.

The objective of this study was to profile the chemical components and biological activity analysis of crude extract of Bryophyllum pinnatum and Oxalis corniculata. Results revealed that the analyzed plant materials encompass the high amount of total phenolic and flavonoids content and have significant antioxidant activities. Furthermore, methanol extracts are the potential source of α-amylase, α-glucosidase, lipase, tyrosinase and elastase inhibitors. High resolution mass spectrometry revealed the presence of diverse metabolites such as quercetin 3-O-α-L-rhamnopyranoside, myricetin 3-rhamnoside, bersaldegenin 1,3,5-orthoacetate, bryophyllin C, syringic acid, caffeic acid, p-coumaric acid, and quercetin in B. pinnatum and isoorientin, swertisin, apigenin 7,4'-diglucoside, vitexin, 4-hydroxybenzoic acid, vanillic acid, ethyl gallate, 3,3',4'-trihydroxy-5,7-dimethoxyflavone, and diosmetin-7-O-ß-D-glucopyranoside in O. corniculata. Our finding suggested that these two plant species have high medicinal importance and are potential source of inhibitors for modern pharmaceuticals, nutraceuticals and cosmetics industries.

Evaluation of α-amylase, lipase inhibition and in-vivo pharmacological activities of Eucalyptus camaladulensis Dehnh leaf extract.

In this present study, phytochemical screening, anti-ulcer assay, anti-diarrhea assay, anti-inflammatory assay, analgesic assay, lipase activity assay, amylase activity assay and the anti-bacterial activity of Eucalyptus camaladulensis Dehnh leaf extracted with methanol and 50% ethanol was analyzed for biological significance. Physical characterization of the non-volatile component revealed the higher yield of 16.92% in 50% ethanol expediting the use of 50% ethanol as a better alternative. Further use of crude extract revealed 33.89% (IC50 = 1.44 mg/ml) of α-amylase inhibition by methanol extract and 33.87% (IC50 = 3.21 mg/ml) lipase inhibition by 50% ethanol extract. Furthermore, 44.44% protective ratio towards ulcer was observed with the methanol extract, whereas 54.58% anti-inflammatory activity was shown by the 50% ethanol extract. The effectiveness of the extract was further enhanced by the presence of 62.54% motility and best analgesic property at 180 min of the exposure of the extract orally. The antioxidant activity of crude methanol extract revealed an IC50 value 601.8 µg/ml whereas, ethanol extract showed 1279.58 µg/ml in DPPH assay. Result revealed several health benefits of E. camaldulensis Dehnh leaf.

Effect of Extracellular Tyrosinase on the Expression Level of P450, Fpr, and Fdx and Ortho-hydroxylation of Daidzein in Streptomyces avermitilis.

We have reported that the expression of CYP105D7 in Streptomyces avermitilis produces 112.5 mg L-1 of 7,3',4'-trihydroxyisoflavone (3'ODI) in 15 h of the reaction time, when 7,4'-dihydroxyisoflavone (daidzein) is used as a substrate. Although production is significant, rapid degradation of 3'ODI after 15 h was observed in a whole-cell biotransformation system, suggesting the further modification of 3'ODI by endogenous enzymes. In this present study, the effect of deletion of extracellular tyrosinase (melC2) in S. avermitilis for 3'ODI production as well as the expressions of CYP105D7, ferredoxin (Fdx), and ferredoxin reductase (Fpr) were investigated. The result revealed that daidzein hydroxylation activity in the ∆melC2 mutant decreased by 40% compared with wild-type S. avermitilis. Further, melC2 deletion significantly affects the messenger RNA (mRNA) expression profile of CYP105D7 and its electron transfer counterparts. Real-time PCR analysis of 9 Fdx, 6 Fpr, and CYP105D7 revealed a significant decrease in mRNA expression level compared to wild-type S. avermitilis. The result clearly shows that the decrease in daidzein hydroxylation activity is due to the lower expression level of CYP105D7 and its electron transfer counterpart in the ∆melC2 mutant. Furthermore, melC2 deletion prevents the degradation of 3'ODI.

Chemical composition, antioxidant and antibacterial activities of essential oil and methanol extract of Artemisia vulgaris and Gaultheria fragrantissima collected from Nepal.

OBJECTIVE: To identify the chemical constituents and biological activities of essential oil and crude methanol extract of Artemisia vulgaris (A. vulgaris) and Gaultheria fragrantissima (G. fragrantissima). METHODS: Phytochemical screening, total phenolic and flavonoid content, antibacterial activities, anti-oxidant assay of the crude extract were carried out to identify the biological activities and phytonutrients present in the extract. Furthermore, the chemical constituents present in the essential oil and crude methanol extract were analyzed using gas chromatography mass spectroscopy and high performance liquid chromatography (HPLC) analysis. RESULTS: Gas chromatography mass spectroscopy analysis of essential oil from the aerial part of A. vulgaris revealed 24 different compounds in it. Sabinene (11.29%), ß-thujone (19.19%), chrysanthenone (4.48%), camphor (11.89%), borneol (4.44%) and germacrene D (8.42%) were the major compounds. Similarly, leaves of G. fragrantissima contained methyl salicylate (95%) and asarone (4.64%). Furthermore, methanol extract of leaves of A. vulgaris and G. fragrantissima were found rich in the total flavonoids and phenolic content. HPLC analysis of the methanol extract of leaves A. vulgaris revealed the presence of morin and luteolin, whereas rutin was found as a major flavonoids compound in the leaves of G. fragrantissima. Further, methanol extract of the A. vulgaris and G. fragrantissima showed the highest antioxidant and antibacterial properties compared to the essential oil. CONCLUSIONS: The HPLC analysis of the methanol extract of A. vulgaris shows the presence of luteolin and morin, whereas G. fragrantissima reveals the presence of rutin and a glycosylated flavonoids. Results reveal that A. vulgaris oil is the rich source of monoterpene and sesquiterpene compounds. Furthermore, A. vulgaris and G. fragrantissima are the rich source of the phenolic and flavonoids compounds and show good antioxidant and antibacterial activity.

Diet and Activity of Macaca assamensis in Wild and Semi-Provisioned Groups in Shivapuri Nagarjun National Park, Nepal.

Studying the behavioural flexibility and adaptability of macaques to different habitats is one approach to designing a conservation plan. To determine the activity budget and feeding behaviour and evaluate the effects of seasonality in wild and human- altered habitats of Assamese macaques (Macaca assamensis), we conducted this study in the Nagarjun forest of Shivapuri-Nagarjun National Park (SNNP) in central Nepal. We also updated the list of plant food items of Assamese macaques in the SNNP. Using scan and all-occurrence sampling, we recorded the diets and activities of Assamese macaques in 2 social groups, a wild-feeding group (WG) and a semi-provisioned group (SPG), throughout the year from August 2013 to July 2014. Both groups spent most of their time in feeding activities and were quite arboreal, but there were significant differences in the activity budgets and diets between the groups. Human food was the main component of the diet for the SPG, whereas it was fruit for the WG, indicating a normally frugivorous diet. Furthermore, the activity budget and diet composition varied in response to the season. These results indicate that provisioning alters the activity and feeding behaviour of macaques, and can also increase human-macaque conflict and disease transmission.

P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System.

(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 µM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

Identification of the specific electron transfer proteins, ferredoxin, and ferredoxin reductase, for CYP105D7 in Streptomyces avermitilis MA4680.

It was previously proposed that regiospecific hydroxylation of daidzein at 3'-position is mediated by cytochrome P450 hydroxylase (CYP105D7) in the presence of putidaredoxin (CamB) and putidaredoxin reductase (CamA) as electron transfer proteins from Pseudomonas putida. The genome sequence of Streptomyces avermitilis MA4680 revealed 33 P450 (CYPs) with 6 ferredoxin reductases (Fprs) and 9 ferredoxins (Fdxs) as their putative electron transfer partner proteins. To identify right endogenous electron transfer proteins for CYP105D7 activity, in vitro reconstitution, gene disruption, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mRNA expression profile analysis were examined. The most effective electron transfer proteins for CYP105D7 appear to be FdxH (SAV7470), which is located downstream to CYP105D7 as a cluster, and FprD (SAV5675). Throughout our overall analysis, we proposed that the primary electron transfer pathway for CYP105D7 follows as such NAD(P)H→FdxH→FprD→CYP105D7.

Engineering of daidzein 3'-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450.

A cytochrome P450 (CYP) enzyme, 3'-daidzein hydroxylase, CYP105D7 (3'-DH), responsible for daidzein hydroxylation at the 3'-position, was recently reported. CYP105D7 (3'-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3'-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a k(cat)/K(m) value of 16.8 µM(-1) min(-1), which was about 24-fold higher than that of the 3'-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3'-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.

Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis.

Among 33 cytochrome P450s (CYPs) of Streptomyces avermitilis, CYP102D1 encoded by the sav575 gene is naturally a unique and self-sufficient CYP. Since the native cyp102D1 gene could not be expressed well in Escherichia coli, its expression was attempted using codon-optimized synthetic DNA. The gene was successfully overexpressed and the recombinant CYP102D1 was functionally active, showing a Soret peak at 450 nm in the reduced CO difference spectrum. FMN/FAD isolated from the reductase domain showed the same fluorescence in thin layer chromatography separation as the authentic standards. Characterization of the substrate specificity of CYP102D1 based on NADPH oxidation rate revealed that it catalysed the oxidation of saturated and unsaturated fatty acids with very good regioselectivity, similar to other CYP102A families depending on NADPH supply. In particular, CYP102D1 catalysed the rapid oxidation of myristoleic acid with a k(cat)/K(m) value of 453.4 ± 181.5 µM(-1)·min(-1). Homology models of CYP102D1 based on other members of the CYP102A family allowed us to alter substrate specificity to aromatic compounds such as daidzein. Interestingly, replacement of F96V/M246I in the active site increased catalytic activity for daidzein with a k(cat)/K(m) value of 100.9 ± 10.4 µM(-1)·min(-1) (15-fold).

Novel iron-sulfur containing NADPH-reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase.

CYP51, a sterol 14α-demethylase, is one of the key enzymes involved in sterol biosynthesis and requires electrons transferred from its redox partners. A unique CYP51 from Nocardia farcinica IFM10152 forms a distinct cluster with iron-sulfur containing NADPH-P450 reductase (FprD) downstream of CYP51. Previously, sequence alignment of nine reductases from N. farcinica revealed that FprC, FprD, and FprH have an additional sequence at their N-termini that has very high identity with iron-sulfur clustered ferredoxin G (FdxG). To construct an artificial self-sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron-sulfur containing FprD were fused together with designed linker sequences. CYP51-FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP. CYP51-FprD F1 and F2 in recombinant Escherichia coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with k(cat) /K(m) values of 96.91 and 105.79 nmol/min/nmol, respectively, which are about 35-fold higher compared to those of CYP51 and FprD alone.

Screening of bacterial cytochrome P450s responsible for regiospecific hydroxylation of (iso)flavonoids.

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, Bacillus and Nocardia farcinica IFM10152 strains. As electron transfer redox partners with the P450s in Escherichia coli system, putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida were used. Among the 50 soluble P450s in the library screened, three cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 from S. avermitilis MA4680 showed good hydroxylation activities towards flavones and isoflavones. However, low product yields prevented us from identifying complete structure of the products. By using S. avermitilis MA4680 as their expression host, further analysis identified that CYP107Y1(SAV2377), CYP125A2(SAV5841) and CYP107P2(SAV4539) showed good regiospecific hydroxylation activities towards genistein (4',5,7-trihydroxyisoflavone), chrysin (5,7-dihydroxyisoflavone) and apigenin (4',5,7-dihydroxyisoflavone) to produce 3',4',5,7,-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydroxyflavone and 3',4',5,7,-tetrahydroxyflavone, respectively. Analyses of the reaction products were performed using HPLC, ESI-MS-MS and GC-MS and 1H NMR.

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680.

Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h.

A-ring ortho-specific monohydroxylation of daidzein by cytochrome P450s of Nocardia farcinica IFM10152.

The bioconversion of the isoflavonoid daidzein using whole cell Nocardia farcinica IFM10152 showed two kinds of major metabolic modifications, i.e. mono-hydroxylation and subsequent O-methylation. The major hydroxylated products of daidzein prior to the O-methylation reaction were 3',4',7-trihydroxyisoflavone (3'-ODI), 4',6,7-trihydroxyisoflavone (6-ODI) and 4',7,8-trihydroxyisoflavone (8-ODI), which are mono-hydroxylated at the ortho position of each hydroxyl group of daidzein. To identify monooxygenases playing a key role in the monohydroxylation of the A-ring of daidzein, all genes of 27 cytochrome P450s from N. farcinica IFM10152 were cloned and transformed into a E. coli BL21 (DE3) host system. By this enzymatic reaction using the mutants and the genome sequence analysis of N. farcinica IFM10152, it was revealed that nfa12130 and nfa33880 P450 genes clustered with their own ferredoxins and ferredoxin reductases (nfa12140+nfa12150 and nfa338870+nfa33860, respectively) are responsible for the hydroxylation of the A-ring of daidzein, and their major reaction products were 6-ODI and 8-ODI, respectively.

Regioselective hydroxylation of isoflavones by Streptomyces avermitilis MA-4680.

Screening of bacterial whole cells was performed for regioselective hydroxylation of daidzein and genistein. Among the strains examined, Streptomyces avermitilis MA-4680 showed high ortho-dihydroxylation activity to produce 3',4',7-trihydroxyisoflavone and 3',4',5,7-tetrahydroxyisoflavone from daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone), respectively. Using 100 mg cells (wet wt.) and 1% (v/v) Triton X100 in 1 ml of total reaction volume, where 100 microl of the substrate solution (0.5 mM in 10% (v/v) mixed solvent of DMSO:MeOH = 3:7) was added to 900 microl of potassium phosphate buffer (100 mM, pH 7.2), a 16% molar conversion yield of 3',4',7-trihydroxyisoflavone was obtained from 0.5 mM daidzein after 24 h of reaction time at 28 degrees C and 200 rpm. Ketoconazole significantly (ca. 90%) inhibited the ortho-hydroxylation activity of daidzein, suggesting that cytochrome P450 enzymes putatively play roles in regiospecific daidzein hydroxylation. The analysis of the reaction products was determined by gas chromatography/mass spectrometry (GC/MS) and (1)H NMR.