Assessment of the stabilization effect of ferrous sulfate for arsenic-contaminated soils based on chemical extraction methods and in vitro methods: Methodological differences and linkages.

Stabilization of arsenic-contaminated soils with ferrous sulfate has been reported in many studies, but there are few stabilization effects assessments simultaneously combined chemical extraction methods and in vitro methods, and further explored the corresponding alternative relationships. In this study, ferrous sulfate was added at FeAs molar ratio of 0, 5, 10 and 20 to stabilize As in 10 As spiked soils. Stabilization effects were assessed by 6 chemical extraction methods (toxicity characteristic leaching procedures (TCLP), HCl, diethylenetriamine pentaacetic acid (DTPA), CaCl2, CH3COONH4, (NH4)2SO4), and 4 in vitro methods (physiologically based extraction test (PBET), in vitro gastrointestinal method (IVG), Solubility Bioaccessibility Research Consortium (SBRC) method, and the Unified Bioaccessibility Research Group of Europe method (UBM)). The results showed that the HCl method provides the most conservative assessment results in non-calcareous soils, and in alkaline calcareous soils, (NH4)2SO4 method provides a more conservative assessment. In vitro methods provided significantly higher As concentrations than chemical extraction methods. The components of the simulated digestion solution as well as the parameters may have contributed to this result. The small intestinal phase of PBET and SBRC method produced the highest and lowest ranges of As concentrations, and in the range of 127-462 mg/kg and 68-222 mg/kg when the FeAs molar ratio was 5. So the small intestinal phase of PBET method may provide the most conservative assessment results, while the same phase of SBRC may underestimate the human health risks of As in stabilized soil by 51 %(at a FeAs molar ratio of 5). Spearman correlation analysis indicated that the small intestinal phase of PBET method correlated best with HCl method (correlation coefficient: 0.71). This study provides ideas for the assessment of stabilization efforts to ensure that stabilization meets ecological needs while also being less harmful to humans.

Curative effect of wrist-ankle acupuncture on postpartum abdominal pain and its influence on serum ß-EP level in puerpera. / è •è¸é’ˆæ²»ç–—äº§åŽè ¹ç—›çš„ä¸´åºŠç–—æ•ˆåŠå¯¹äº§å¦‡è¡€æ¸ ß-EP水平的影响.

OBJECTIVES: To observe the clinical effect of wrist-ankle acupuncture on postpartum abdominal pain and its influence on serum beta-endorphin (ß-EP) level in puerpera. METHODS: Seventy patients with postpartum abdominal pain were randomly divided into an acupuncture + herbal medication group (35 cases, 1 case dropped out) and a herbal medication group (35 cases, 2 cases dropped out). In the herbal medication group, 1 day after delivery, modified shenghua decoction was taken orally, one dose a day. In the acupuncture + herbal medication group, on the basis of herbal medication, wrist-ankle acupuncture was given at the Lower 1 and Lower 2 of the ankles, once daily. The duration of treatment was 3 days in the two groups. Before and after treatment, the score of visual analogue scale (VAS) for pain, serum ß-EP level, uterine fundus height, postpartum conditions of lochia and the uterine recovery at 42 days postpartum were compared in the patients of the two groups. RESULTS: At each time point after treatment (24 h, 48 h and 72 h after delivery), VAS scores and the uterine fundus height were reduced as compared with those before treatment (2 h after delivery) in the two groups (P<0.05); these indexes in the acupuncture + herbal medication group were lower than those in the herbal medication group (P<0.05). After treatment (72 h after delivery), ß-EP levels in the serum were increased when compared with those before treatment in the two groups (P<0.05), and the ß-EP level in the acupuncture + herbal medication group was higher than that in the herbal medication group (P<0.05). The volume of postpartum lochia discharge in the acupuncture + herbal medication group was higher than that in the herbal medication group (P<0.05), while the duration of postpartum lochia discharge and the total time of lochia discharge were shorter (P<0.05). Regarding the recovery of the uterus at 42 days postpartum, there was no statistical significance between the two groups (P>0.05). CONCLUSIONS: Wrist-ankle acupuncture obviously reduces the degree of postpartum abdominal pain and promotes the lochia discharge and the uterine recovery. The effect mechanism may be related to the up-regulation of serum ß-EP level and the increase of pain threshold so that analgesia is obtained.

Effects of foods and food components on the in vitro bioaccessibility of total arsenic and arsenic species from Hizikia fusiforme seaweed.

Seaweed is an important food source, especially in many Asian countries, because of its high nutritional value; however, increasing arsenic (As) accumulation may pose serious hazards to human health. The influence of food components on As bioaccessibility and transformation in the high As-containing seaweed Hizikia fusiforme was determined using an in vitro gastrointestinal digestion method. The results showed that co-digestion with several daily foods (such as celery, broccoli, onion, green chili, tomato) produced a higher As bioaccessibility (approximately 6-11 % increase) compared with that of seaweed alone. Vegetables such as fennel (Foeniculum valgare Mill.), celery (Apium grareolens L.), blanched garlic leaves (Allium sativum L.), scallions (Allium fistulosum L.), ginger (Zingiber officinale Rosc.), and green pepper (Capsicum frutescens L. vat. grussum Bailey) decreased bioaccessible inorganic As (18-35 %) in both the gastric and small intestinal phases. Meanwhile, the process of reducing As(V) to As(III) also occurred during co-digestion with some food matrices. Egg white and other animal proteins were the most effective reducing agents, transforming >70 % As(V) into As(III) in the solution system. These results may have important implications for health risk assessment via co-consumption. The present study provides the first evidence showing that the co-consumption of some vegetables and proteins leads to a higher toxicity of inorganic arsenic-containing food. In addition, the positive and negative effects of co-digestion on the bioaccessibility of essential metals (iron, manganese) compared to single digestion were evaluated in this study.

A dual-response drug delivery system with X-ray and ROS to boost the anti-tumor efficiency of TPZ via enhancement of tumor hypoxia levels.

The selective anti-tumor activity and less toxic nature of hypoxia-activated prodrugs including tirapazamine (TPZ) are harbored by hypoxia levels in tumors, the inadequacy of which leads to failure in clinical trials. Thus, the development of effective clinical applications of TPZ requires advanced strategies to intensify hypoxia levels in tumors effectively and safely. In this study, we designed and fabricated a paclitaxel (PTX)-loaded dual-response delivery system with a low dose (e.g., 2 Gy) of X-ray and reactive oxygen species on the basis of diselenide block copolymers. Upon the external X-ray stimulus, the system accurately released encapsulated PTX at tumor sites and remarkably improved tumor hypoxia levels by causing severe damage to tumor blood vessels. Subsequently, these enhanced tumor hypoxia levels effectively activated the reduction of TPZ into benzotriazinyl free radicals, which significantly improved the antitumor efficacy of our system against 4T1 breast cancer cells with an initial tumor volume of 500 mm3. Moreover, the dual-stimulus coordinated and controlled release of PTX was found to largely avoid the off-target effects of PTX on normal cells while exhibiting very limited side effects in experimental mice. The current novel strategy for regulating tumor hypoxia levels offers an effective and safe way to activate TPZ for the treatment of large solid tumors.

Effect of vitamin C supplement on lead bioaccessibility in contaminated soils using multiple in vitro gastrointestinal assays: Mechanisms and health risks.

Effects of vitamin C supplementation on the oral bioaccessibility of lead (Pb) present in contaminated soils were examined using a number of in vitro assays (PBET, SBRC, UBM and IVG). In the presence of vitamin C, an increase in Pb bioaccessibility was observed in the gastric phase by 1.3-fold (30.5%-85.5%) and in the intestinal phase by 3.1-fold (0.9%-58.9%). Lead mobilization was regulated by reductive dissolution of Fe(III) and sequestration of Pb on secondary Fe minerals. Sequential extraction by the Bureau Community of Reference (BCR) provided more evidence that reducible fraction and residual fraction were major contributor of gastric Pb bioaccessibility, as well as reduced fractions in intestinal Pb bioaccessibility. In addition, higher non-carcinogenic risks may occur based on target hazard quotient (THQ ≥ 1). For people exposed to Pb present in soil, the management of vitamin C supplements is of serious concern.

PrMYB5 activates anthocyanin biosynthetic PrDFR to promote the distinct pigmentation pattern in the petal of Paeonia rockii.

Paeonia rockii is well-known for its distinctive large dark-purple spot at the white petal base and has been considered to be the main genetic source of spotted tree peony cultivars. In this study, the petal base and petal background of Paeonia ostii (pure white petals without any spot), P. rockii, and other three tree peony cultivars were sampled at four blooming stages from the small bell-like bud stage to the initial blooming stage. There is a distinct difference between the pigmentation processes of spots and petal backgrounds; the spot pigmentation was about 10 days earlier than the petal background. Moreover, the cyanin and peonidin type anthocyanin accumulation at the petal base mainly contributed to the petal spot formation. Then, we identified a C1 subgroup R2R3-MYB transcription factor, PrMYB5, predominantly transcribing at the petal base. This is extremely consistent with PrDFR and PrANS expression, the contents of anthocyanins, and spot formation. Furthermore, PrMYB5 could bind to and activate the promoter of PrDFR in yeast one-hybrid and dual-luciferase assays, which was further verified in overexpression of PrMYB5 in tobacco and PrMYB5-silenced petals of P. rockii by comparing the color change, anthocyanin contents, and gene expression. In summary, these results shed light on the mechanism of petal spot formation in P. rockii and speed up the molecular breeding process of tree peony cultivars with novel spot pigmentation patterns.

Role of human gut bacteria in arsenic biosorption and biotransformation.

There is growing evidence that human gut microbiota can metabolize arsenic (As); however, which bacteria play roles in this metabolism is unclear. In this study, we measured the abilities of 21 human gut bacteria strains from diverse clades to adsorb and transform As using in vitro method with the aim of determining which bacteria play a role in As metabolism. Seven strains showed high biosorption of As, ranging from 20.1 to 29.8%, which was attributed to functional groups on the bacterial surfaces, such as hydroxyl, amino, and carboxyl groups. Moreover, six of these seven strains were versatile, as they also had roles in reducing As(V) to As(III), which is mainly regulated by the arsC gene. Escherichia coli had the strongest tolerance to As and the highest reducing ability, with a value of 71.04-73.13 µM As/h. This study reveals that gut bacteria play essential roles in As biosorption and biotransformation, and provides a better understanding of which strains are involved, which has implications for the regulation of As toxicity-based gut bacteria and provides basic data for regulating arsenic to human health.

Predictive capabilities of in vitro colon bioaccessibility for estimating in vivo relative bioavailability of arsenic from contaminated soils: Arsenic speciation and gut microbiota considerations.

Arsenic (As) transformation by human gut microbiota has been evidenced to impact As toxicity and human health. However, little is known about the influence of gut microbiota on As bioavailability from incidental ingestion of soil. In this study, we assessed As relative bioavailability (RBA) using an in vivo mouse model and As bioaccessibility in the colon phase of in vitro assays. Strong in vivo-in vitro correlations (R2 = 0.70-0.92, P < 0.05) were observed between soil As RBA (10.2%-57.7%) and colon bioaccessibility (4.8%-49.0%) in 13 As-contaminated soils. Upon in vitro incubation of human colon microbiota, we found a high degree of As transformation and 65.9% of generated As(III) was observed in soil residues. For in vivo mouse assay, DMA(V) accounted for 79.0% of cumulative urinary As excretion. Except for As(V), dominant As species including As(III), DMA(V) and As sulfides were also detected in mouse feces. Gut bacteria (families Rikenellaceae and Marinifilaceae) could be significantly correlated with As intake and excretion in mice (P < 0.05). Our findings provide evidence that gut microbiota can affect transformation, bioavailability, and fate of the orally ingested soil As in human gastrointestinal tract.

Integrating full-length transcriptomics and metabolomics reveals the regulatory mechanisms underlying yellow pigmentation in tree peony (Paeonia suffruticosa Andr.) flowers.

Tree peony (Paeonia suffruticosa Andr.) is a popular ornamental plant in China due to its showy and colorful flowers. However, yellow-colored flowers are rare in both wild species and domesticated cultivars. The molecular mechanisms underlying yellow pigmentation remain poorly understood. Here, petal tissues of two tree peony cultivars, "High Noon" (yellow flowers) and "Roufurong" (purple-red flowers), were sampled at five developmental stages (S1-S5) from early flower buds to full blooms. Five petal color indices (brightness, redness, yellowness, chroma, and hue angle) and the contents of ten different flavonoids were determined. Compared to "Roufurong," which accumulated abundant anthocyanins at S3-S5, the yellow-colored "High Noon" displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols but no anthocyanin production. The contents of THC, flavones, and flavonols in "High Noon" peaked at S3 and dropped gradually as the flower bloomed, consistent with the color index patterns. Furthermore, RNA-seq analyses at S3 showed that structural genes such as PsC4Hs, PsDFRs, and PsUFGTs in the flavonoid biosynthesis pathway were downregulated in "High Noon," whereas most PsFLSs, PsF3Hs, and PsF3'Hs were upregulated. Five transcription factor (TF) genes related to flavonoid biosynthesis were also upregulated in "High Noon." One of these TFs, PsMYB111, was overexpressed in tobacco, which led to increased flavonols but decreased anthocyanins. Dual-luciferase assays further confirmed that PsMYB111 upregulated PsFLS. These results improve our understanding of yellow pigmentation in tree peony and provide a guide for future molecular-assisted breeding experiments in tree peony with novel flower colors.

Arsenic speciation and bioaccessibility in raw and cooked seafood: Influence of seafood species and gut microbiota.

Seafood is an important source of arsenic (As) exposure for humans. In this study, 34 seafood samples (fishes, shellfishes, and seaweeds) collected from different markets in China were analysed for total and speciated As before and after boiling. Furthermore, the As bioaccessibility was also assessed using a physiologically based extraction test combined with the Simulator of Human Intestinal Microbial Ecosystems. The results showed that the total As (tAs) contents of seaweeds (raw: 44.12; boiled: 31.13, µg·g-1 dw) were higher than those of shellfishes (raw: 8.34; boiled: 5.14, µg·g-1 dw) and fishes (raw: 6.01; boiled: 3.25, µg·g-1 dw). Boiling significantly decreased the As content by 22.24% for seaweeds, 32.27% for shellfishes, and 41.42% in fishes, respectively (p < 0.05). During in vitro digestion, the bioaccessibility of tAs and arsenobetaine (AsB) significantly varied between the investigated species of seafood samples in gastric (G) and small intestinal phases (I) (p < 0.05). Higher tAs bioaccessibility (G: 68.6%, I: 81.9%) were obtained in fishes than shellfishes (G: 40.9%, I: 52.5%) and seaweeds (G: 31%, I: 53.6%). However, there was no significant differences in colonic phase (C) (p > 0.05). With the effect of gut microbiota, arsenate (AsⅤ) was transformed into monomethylarsonic acid (MMA) and arsenite (AsⅢ) in C. Moreover, as for seaweeds, an unknown As compound was produced.

Assessment of arsenic distribution, bioaccessibility and speciation in rice utilizing continuous extraction and in vitro digestion.

Rice, a staple food for half the world's population, easily accumulates arsenic (As). Research on As distribution in rice protein and starch and its relationship with rice As bioaccessibility remains limited. This study investigated As distribution, chemical composition, As bioaccessibility and speciation in rice by continuous extraction and in vitro digestion. Of the total As, 87.5-94.5% was in rice protein and 5.0-9.8% in rice starch. The As amount in different protein fractions decreased as follows: glutelin > globulin > albumin > prolamin. As(V), As(III) and DMA in rice were more bioaccessible in the small intestinal phase than the gastric phase, and almost all As(V) dissolved in the small intestinal phase. Bioaccessible As in gastrointestinal digestive solution and As mass in protein fractions (albumin, globulin, and glutelin) were significantly positively correlated (p < 0.05). These results illuminate the bioaccessibility of As to humans consuming As-contaminated rice and avoid overassessment.

Genome-Wide Identification and Comparative Profiling of MicroRNAs Reveal Flavonoid Biosynthesis in Two Contrasting Flower Color Cultivars of Tree Peony.

MicroRNA (miRNA)-mediated gene regulation is involved in various physiological processes in plants. Flower color is one of the vital ornamental traits of tree peony (Paeonia suffruticosa Andr.). However, the yellow-flowered tree peony cultivars are particularly rare. To elucidate the miRNA-mediated gene regulatory mechanism underlying yellow pigmentation in tree peony, we combined pigment assessment, miRNA identification, expression analysis, and gene functional verification in two contrasting flower color cultivars "High Noon" and "Roufurong." Flavones/flavonols and anthocyanins were found to be the main contributors to the coloration of "High Noon" and "Roufurong" petals, respectively. Subsequently, miRNA analysis based on available genome data identified 9 differentially expressed miRNAs and 12 relevant target genes implicated in flavonoid biosynthesis. Their dynamic expression patterns determined the key role of mdm-miR156b-PsSPL2 module in yellow pigmentation of tree peony flowers. The sequence analysis and subcellular localization validated that PsSPL2 might function as a nuclear-localized transcription factor. Overexpression of PsSPL2 in tobacco resulted in a decrease of anthocyanin content and down-regulation of NtF3'H and NtDFR transcripts. PsSPL2-silenced petals exhibited lighter yellow color, and the contents of THC, Ap, and Ch decreased significantly. Meanwhile, expression levels of PsCHS, PsCHI, and PsF3H were significantly decreased in the petals with PsSPL2 silencing, while those of PsF3'H and PsDFR were remarkably increased. This study offers a novel insight into yellow pigmentation-related miRNA regulation network in tree peony, and further provides the valuable information on physiological changes during yellow coloring process of tree peony.

A peroxidase coordinating to Zn (II) preventing heme bleaching and resistant to the interference of H2 O2.

Dehaloperoxidase (DHP) catalyzes detoxifying halophenols. It is a heme-containing enzyme using H2 O2 as the oxidant. Heme bleaching from the active site is of great concern. In addition, the interference of DHP by H2 O2 leads to the inactivation of the enzyme. To solve these two problems, DHP is coordinated to Zn (II) in PBS buffer to form a biomineralized composite (DHP&Zn-CP). DHP&Zn-CP was characterized by measuring SEM and confocal images, as well as energy dispersive X-ray spectrometry mapping. Fluorescence spectra demonstrated that DHP&Zn-CP can prevent heme bleaching. Two-dimensional FTIR spectra were measured, dynamically providing insight into the structural change of DHP along the coordination process. Raman spectra were performed to analyze the structural change. The optical spectra confirmed that the forming of DHP&Zn-CP had a little effect on the structures of DHP. For the dehalogenation of 2,4,6-trichlorophenol, DHP&Zn-CP can tolerate the presence of H2 O2 and is resistant to the interference by H2 O2 . The catalytic efficiency of DHP&Zn-CP is much higher than that of free DHP.

Assessment of nutrients effect on the bioaccessibility of Cd and Cu in contaminated soil.

Soil is considered as a sink for heavy metals. Human health is severely affected by the continuous intake of toxic heavy metals even in a very low concentration. In the present experiment, we determined the influence of nutritional status including control (fasted condition), glucose (fed state), plant protein (fed state), animal protein (fed state) and calcium (fed state) on soil cadmium (Cd) and copper (Cu) bioaccessibility using physiologically-based extraction test (PBET) method together with simulator of the human intestinal microbial ecosystem (SHIME) model. The bioaccessibility of Cd was 1.06-73.58%, 0.44-54.79% and 0-17.78% and Cu was 3.81-67.32%, 4.98-71.14%, and 0-17.54% in the phase-I, phase-II and Phase-III respectively (in this study gastric phase, small intestinal phase and colon phase were considered as phase-I, phase-II and Phase-III respectively). The outcomes showed that, the average Cd bioaccessibility was higher with animal protein addition compared with other treatments in different phases. So, the effect of animal protein on Cd bioaccessibility was higher than other treatments in the phase-I, phase-II and phase-III. Due to the addition of plant protein, the higher average bioaccessibility of Cu was noticed in phase-I and phase-II in comparison to other treatments. However, in phase-III, the higher average bioaccessibility of Cu was found due to control treatment comparing with other treatments. Therefore, the influence of plant protein was higher than other nutrients on Cu bioaccessibility in the phase-I and phase-II. Moreover, other nutrients showed variable influence on Cd and Cu bioaccessibility. So, nutritional status has a significant effect on bioaccessibility as well as human health risk assessment.

Lead bioaccessibility in farming and mining soils: The influence of soil properties, types and human gut microbiota.

To better understand the risk assessment of Lead (Pb) in contaminated soils, 78 soil samples were collected from different locations in China and Pb bioaccessibility was assessed using the PBET (The Physiologically Based Extraction Test) method combined with SHIME (The Simulator of the Human Intestinal Microbial Ecosystem), and Pb bioaccessibility data from the PBET method on 88 soil samples that found in the literature were also used for the assessment. For all the soils, the mean Pb bioaccessibility was as follows: the gastric phase (31.25%) > colon phase (17.78%) > small intestinal phase (10.13%). The values of Pb bioaccessibility in most soils were lower than 60%, which is the typical default assumption for Pb (RBA, relatively bioavailability) by the US EPA. Mean Pb bioaccessibility (41.10% and 14.00% for gastric and small intestinal phases, respectively) in the present study was slightly higher than the values from the literature (24.80% and 8.68% for gastric and small intestinal phases, respectively) in the gastrointestinal tract. Mean Pb bioaccessibility was lower in acidic soil during the small intestinal phase, while the values for the alkaline soil were higher in the small intestinal and colon phases. In the gastric and small intestinal phases, mean Pb bioaccessibility in farming soils was slightly lower than it was in mining soils. However, the mean Pb bioaccessibility from farming soils was increased compared with mining soils in the colon phase given the action of human gut microbiota. Soil pH and type are important factors for predicting soil Pb bioaccessibility and health risk.

The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi.

Soil-dwelling entomopathogenic nematodes (EPNs) kill arthropod hosts by injecting their symbiotic bacteria into the host hemolymph and feed on the bacteria and the tissue of the dying host for several generations cycles until the arthropod cadaver is completely depleted. The EPN-bacteria-arthropod cadaver complex represents a rich energy source for the surrounding opportunistic soil fungal biota and other competitors. We hypothesized that EPNs need to protect their food source until depletion and that the EPN symbiotic bacteria produce volatile and non-volatile exudations that deter different soil fungal groups in the soil. We isolated the symbiotic bacteria species (Alcaligenes faecalis) from the EPN Oscheius spp. and ran infectivity bioassays against entomopathogenic fungi (EPF) as well as against plant pathogenic fungi (PPF). We found that both volatile and non-volatile symbiotic bacterial exudations had negative effects on both EPF and PPF. Such deterrent function on functionally different fungal strains suggests a common mode of action of A. faecalis bacterial exudates, which has the potential to influence the structure of soil microbial communities, and could be integrated into pest management programs for increasing crop protection against fungal pathogens.

A Comparison of Novel Entomopathogenic Nematode Application Methods for Control of the Chive Gnat, Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga Yang & Zhang (Diptera: Sciaridae) is the most serious pest of Chinese chive, Allium tuberosum Chemical pesticide application has become a necessary step to control B. odoriphaga after each of the four to six harvests during the growing season. We compared the effects of application type (nematode-infected cadaver, aqueous nematode suspension, and mixture of cadaver and aqueous suspension) and nematode species (Heterorhabditis bacteriophora vs. Steinernema carpocapsae) on B. odoriphaga control. Nematode species combinations and the use of the cadaver method has only been tested in a relatively few studies, and has not been tested for this target pest, B. odoriphaga. Furthermore, this is the first report of combining application methods (aqueous and cadaver). Results indicated that the cadaver treatment produced higher mortality of B. odoriphaga than the aqueous treatment, and H. bacteriophora caused higher mortality of B. odoriphaga than S. carpocapsae. The mortality of B. odoriphaga was 96.7% in H. bacteriophora cadaver treatment and only 27.5% in control treatment without nematode addition. Cadaver treatments caused higher biomass of A. tuberosum than that of aqueous treatment. The plant biomass in H. bacteriophora cadaver treatment was 10 folds as much as that in the control treatment. The mixed aqueous suspension of H. bacteriophora and S. carpocapsae did not increase the mortality in pot trial. Our findings indicate that entomopathogenic nematodes applied as cadavers might be a potential alternative biological agent in the integrated management of B. odoriphaga for Chinese chive production.

Genetically-modified R-ω-transaminase: purification and self-assembly facilitating interaction with substrate droplets.

OBJECTIVES: An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase. RESULTS: R-ω-Transaminase (R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP-R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-R-ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %. CONCLUSIONS: The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports.