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Objective: The present study aimed to investigate whether recombinant human erythropoietin (rHuEPO) plays an immunomodulatory function by regulating the TLR4/NF-κB signaling pathway.Materials and m...

Genes encoding six feruloyl esterases (FAEs; lbff0997, lbff0272, lbff1432, lbff1695, lbff1849, lbff0153) from Lactobacillus fermentum JN248 were cloned, overexpressed and characterised. Maximum enzyme activity was observed at 35 °C for recombinant FAEs LFFae0997, LFFae0272 and LFFae0153, at 30 °C for LFFae1695, and at 40 °C for LFFae1432and LFFae1849. For five of the enzymes, optimal activity was observed at pH 7.0 or pH 8.0, and high thermostability was measured up to 55 °C. By contrast, LFFae1432 lost less than 10.0% activity after incubation at 40 °C for 2 h, and pH stability was highest between pH 7.0 and pH 9.0. In addition, LFFae1432 was the most robust esterase, with a higher affinity and hydrolytic activity against synthetic esters. The enzymes released ferulic acids (FAs) from de-starched wheat bran (DSWB), and 60.7% of the total alkali-extractable FAs were released when LFFae1432 was added alone, compared with less than 10% for the other enzymes. The amount of FAs released by FAEs increased when combined with xylanase. These FAEs could serve as promising biocatalysts for biodegradation, and LFFae1432 may hold promise for potential industrial applications.

Sepsis is common and frequently fatal condition in critically ill patients and is a major cause of acute kidney injury (AKI). In this retrospective study, we sought to develop a comprehensive risk score model of sepsis associated-AKI (SA-AKI). A total of 2617 patients were randomly assigned to a development (1554 patients) and a validation group (777 patients). The risk score model for SA-AKI was developed with multivariate regression analysis in development group and the model was further evaluated on validation group. We identified 16 independent predictors of SA-AKI in development group (age ≥ 60 years, hypertension/coronary heart disease, diabetes, chronic kidney disease, heart failure, chronic obstructive pulmonary disease, acute severe pancreatitis, hypotension, hypoproteinemia, lactic acidosis, the length of stay in intensive care unit(ICU), 60 g/L

D-Danshensu (D-DSS), a traditional Chinese medicine, is used to treat cardiovascular and cerebrovascular diseases. However, current isolation protocols for D-DSS both natural and synthetic are not ideal; therefore, in this study, we have developed a whole-cell biotransformation method to produce D-DSS from L-DOPA. This was done by co-expressing L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD), and glucose dehydrogenase (gdh). To begin to optimize the production of D-DSS, varying copy number plasmids were used to express each of the required genes. The resulting strain, Escherichia coli ALG7, which strongly overexpressed aadL, ldhD, and weakly overexpressed gdh, yielded a 378% increase in D-DSS production compared to E. coli ALG1. Furthermore, the optimal reaction conditions for the production of D-DSS were found to be a pH of 7.5, temperature at 35 °C, and 50 g/L wet cells for 12 h. Under these optimized conditions, the D-DSS amount achieved 119.1 mM with an excellent ee (> 99.9%) and a productivity of 9.9 mM/h.

A novel alanine dehydrogenase (ADH; EC.1.4.1.1) with high pyruvate reduced activity was isolated from Helicobacter aurati and expressed in Escherichia coli BL21 (DE3). The optimum pH of the reduction and oxidation reaction were 8.0 and 9.0, respectively, and the optimum temperature was 55 °C. With pyruvate and alanine as substrates, the specific activity of HAADH1 were 268 U·mg−1 and 26 U·mg−1, respectively. HAADH1 had a prominent substrate specificity for alanine (Km = 2.23 mM, kcat/Km = 8.1 s−1·mM−1). In the reduction reaction, HAADH1 showed the highest substrate affinity for pyruvate (Km = 0.56 mM, kcat/Km = 364 s−1·mM−1). Compared to pyruvate, oxaloacetic acid, 2-ketobutyric acid, 3-fluoropyruvate, α-ketoglutaric acids, glyoxylic acid showed a residual activity of 93.30%, 8.93%, 5.62%, 2.57%, 2.51%, respectively. Phylogenetic tree analysis showed that this is a new type of ADH which have a low sequence similarity to available ADH reported in references. 3-Fluoropyruvate was effectively reduced to 3-fluoro-L-alanine by whole-cell catalysis.

Filamentous fungi comprise an abundance of gene clusters that encode high-value metabolites, whereas affluent gene clusters remain silent during laboratory conditions. Complex cellular metabolism further limits these metabolite yields. Therefore, diverse strategies such as genetic engineering and chemical mutagenesis have been developed to activate these cryptic pathways and improve metabolite productivity. However, lower efficiencies of gene modifications and screen tools delayed the above processes. To address the above issues, this review describes an alternative design-construction evaluation optimization (DCEO) approach. The DCEO tool provides theoretical and practical principles to identify potential pathways, modify endogenous pathways, integrate exogenous pathways, and exploit novel pathways for their diverse metabolites and desirable productivities. This DCEO method also offers different tactics to balance the cellular metabolisms, facilitate the genetic engineering, and exploit the scalable metabolites in filamentous fungi.

AIMS The purpose of this work was to find an efficient enzyme to synthesize d-3,4-dihydroxyphenyllactic acid (d-DSS). METHODS AND RESULTS Nineteen lactic acid bacteria strains were screened for production of d-DSS using 3,4-dihydroxyphenylpyruvic acid (DPA) as a substrate. Lactobacillus reuteri JN516 exhibited the highest d-DSS yield. A nonspecific coenzyme, d-lactate dehydrogenase (d-LDH82319), from L. reuteri JN516 with high DPA reducing activity was identified. This enzyme reduced DPA to form d-DSS with excellent optical purity (enantioselectivity >99%). Its molecular weight was 35 kDa based on SDS-PAGE migration. The Michaelis-Menten constant (Km ), turnover number (kcat ), and catalytic efficiency (kcat /Km ) of d-LDH82319 for DPA were 0·09 mmol l-1 , 2·17 s-1 and 24·07 (mmol l-1 )-1 s-1 , respectively, with NADH as the coenzyme. The (Km ), (kcat ) and (kcat /Km ) of d-LDH82319 for DPA were 0·10 mmol l-1 , 0·13 s-1 and 1·30 (mmol l-1 )-1 s-1 , respectively, with NADPH as the coenzyme. The optimum temperature and pH of d-LDH82319 were 25°C and pH 8 respectively. Additionally, d-LDH82319 had a broad substrate range for alpha-keto acids, among which the activity of reducing pyruvate was the strongest; therefore, it belongs to the group of d-lactate dehydrogenases. d-LDH82319 and glucose dehydrogenase (GDH) were coexpressed to produce d-DSS from DPA. CONCLUSIONS d-LDH82319 from L. reuteri JN516 with high DPA reducing activity has the characteristics of a nonspecific coenzyme. SIGNIFICANCE AND IMPACT OF THE STUDY d-LDH82319 is the first reported coenzyme nonspecific d-lactate dehydrogenase with DPA-reducing activity. The coexpression system provided an effective method to produce d-DSS.

Danshensu (DSS), also known as 3,4-dihydroxyphenyllactate, is an herbal preparation with prominent pharmacological activities. It has lactic acid structure and may be obtained by reducing 3,4-dihydroxyphenylpyruvate with lactate dehydrogenase. We screened a coenzyme-aspecific L-lactate dehydrogenase (LF-L-LDH0845) from lactobacillus fermentum for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee ≥ 99.99%) L-DSS. LF-L-LDH0845 has an approximate molecular weight of 33.65 kDa, exhibits wide substrate scope for 2-keto-carboxylic acids. Values of Km, Kcat, and Kcat/Km for LF-L-LDH0845 with 3,4-dihydroxy-phenylpyruvate substrate were 11.37 mM, 0.2931 s−1, and 0.0258 mM−1 s−1, respectively. LF-L-LDH0845 was most active and stable at pH 6.0, the optimum temperature was 25 °C, stability decreased with increasing temperature, and activity was lost completely at 50 °C. K+ stimulated while Fe2+ and Cu2+ inhibited the enzyme activity significantly. Glucose dehydrogenase gene was coexpressed with lf-l-ldh0845 in E. coli to regenerate cofactors by oxidising glucose, which efficiently reduced 3,4-dihydroxyphenylpyruvate to L-DSS with 95.45% isolation yield.

Bacteria rarely produce natural 2-phenylethanol. We verified a new pathway from Proteus mirabilis JN458 to produce 2-phenylethanol using Escherichia coli to coexpress l-amino acid deaminase, α-keto acid decarboxylase, and alcohol dehydrogenase from P. mirabilis. Based on this pathway, a glucose dehydrogenase coenzyme regeneration system was constructed. The optimal conditions of biotransformation by the recombinant strain E-pAEAKaG were at 40 °C and pH 7.0. Finally, the recombinant strain E-pAEAKaG produced 3.21 ± 0.10 g/L 2-phenylethanol in M9 medium containing 10 g/L l-phenylalanine after a 16 h transformation. Furthermore, when the concentration of l-phenylalanine was 4 g/L (24 mM), the production of 2-phenylethanol reached 2.88 ± 0.18 g/L and displayed a higher conversion rate of 97.38 mol %.

Background Epilepsy is one of chronic neurological disorders that affects 0.5–1.0% of the world’s population during their lifetime. There is a still significant need to develop novel anticonvulsant drugs that possess superior efficacy, broad spectrum of activities and good safety profile. Methods α-Asaronol and two current antiseizure drugs (α-asarone and carbamazepine (CBZ)) were assessed by in vivo anticonvulsant screening with the three most employed standard animal seizure models, including maximal electroshock seizure (MES), subcutaneous injection-pentylenetetrazole (PTZ)-induced seizures and 3-mercaptopropionic acid (3-MP)-induced seizures in mice. Considering drug safety evaluation, acute neurotoxicity was assessed with minimal motor impairment screening determined in the rotarod test, and acute toxicity was also detected in mice. Results In our results, α-asaronol displayed a broad spectrum of anticonvulsant activity (ACA) and showed better protective indexes (PI = 11.11 in MES, PI = 8.68 in PTZ) and lower acute toxicity (LD50 = 2940 mg/kg) than its metabolic parent compound (α-asarone). Additionally, α-asaronol displayed a prominent anticonvulsant profile with ED50 values of 62.02 mg/kg in the MES and 79.45 mg/kg in the sc-PTZ screen as compared with stiripentol of ED50 of 240 mg/kg and 115 mg/kg in the relevant test, respectively. Conclusion The results of the present study revealed α-asaronol can be developed as a novel molecular in the search for safer and efficient anticonvulsants having neuroprotective effects as well as low toxicity. Meanwhile, the results also suggested that α-asaronol has great potential to develop into another new aromatic allylic alcohols type anticonvulsant drug for add-on therapy of Dravet’s syndrome.

Traditional Chinese medicine (TCM) has a long history and been widely used in prevention and treatment of epilepsy in China. This paper is intended to review the advances in the active anticonvulsant compounds isolated from herbs in the prescription of TCM in the treatment of epilepsy. These compounds were introduced with the details including classification, CAS number specific structure and druggability data. Meanwhile, much of the research in these compounds in the last two decades has shown that they exhibited favorable pharmacological properties in treatment of epilepsy both in in vivo and in vitro models. In addition, in this present review, the evaluation of the effects of the anticonvulsant classical TCM prescriptions is discussed. According to these rewarding pharmacological effects and chemical substances, the prescription of TCM herbs could be an effective therapeutic strategy for epilepsy patients, and also could be a promising source for the development of new drugs.

Phenyllactic acid (PLA) is a novel antimicrobial compound. A novel NADH‐dependent d‐lactate dehydrogenase (d‐LDH), named as LF‐d‐LDH0653, with high phenylpyruvate (PPA) reducing activity was isolated from Lactobacillus fermentumJN248. Its optimum pH and temperature were 8.0 and 50 °C, respectively. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for NADH were 1.20 mmol/L, 67.39 s−1, and 56.16 (mmol/L)−1s−1, respectively. The (Km), (kcat), and (kcat/Km) for phenylpyruvate were 1.68 mmol/L, 122.66 s−1, and 73.01 (mmol/L)−1s−1, respectively. This enzyme can catalyze phenylpyruvate and the product presented excellent optical purity (enantioselectivity >99%). The results suggest that LF‐d‐LDH0653 is a promising biocatalyst for the efficient synthesis of optically pure d‐PLA. A novel d‐LDH with phenylpyruvate reducing activity has been isolated and identified. It could be used as a reference for improving the production of optically pure d‐PLA. d‐PLA has a potential for application as antimicrobial an agent in dairy industry and baking industry, pharmaceutical agent in medicine and cosmetics.

Feruloyl esterases (Faes) are a subclass of the carboxylic esterases that hydrolyze the ester bonds between ferulic acid and polysaccharides in plant cell walls. Until now, the biochemical characteristics of FAEs from Bacillus spp. have not been reported. In this study, a strain with high activity of FAEs, Bacillus amyloliquefaciens H47 was screened from 122 Bacillus – type strains. Finally, three FAEs (BaFae04, BaFae06, and BaFae09) were identified. Comparing with other bacterial FAEs, these novel FAEs exhibited low sequence identities (less than 30%). The profiles of 52 esterase substrates showed that the three FAEs had a broad substrate spectrum and could effectively hydrolyze several common FAE substrates, such as methyl ferulate, ethyl caffeate, methyl p-coumarate, methyl sinapate, and chlorogenic acid. Furthermore, the three FAEs also can release ferulic acid from destarched wheat bran. They showed maximal activity with an optimal pH of 8.0 at 30 °C, 35 °C, and 40 °C, respectively. BaFae04 showed high stability in the temperature range of 25–60 °C for 1 h and retained 59% of its activity at 60 °C. The present study displays some useful characteristics of FAEs for potential industrial application and contributes to our understanding of FAEs.

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.

Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, yet lacks effective therapeutic agents. Previously, we discovered one novel synthetic compound, tanshinol borneol ester (DBZ), possesses anti-inflammatory and anti-atherosclerotic activities, whereas little is known about its effects in CNS. Therefore, the present study aims to explore the effects and potential mechanism of DBZ on neuroinflammation and microglial function. Our studies revealed that DBZ significantly inhibited NF-κB activity, suppressed the production of pro-inflammatory mediators meanwhile promoted M2 mediators expression in LPS-stimulated BV2 cells and mouse primary microglia cells. DBZ also exhibited antioxidant activity by enhancing Nrf2 nuclear accumulation and transcriptional activity, increasing HO-1 and NQO1 expression, and inhibiting LPS-induced ROS generation in BV2 cells. Importantly, the anti-neuroinflammatory and antioxidant effects of DBZ above were reversed by Nrf2 knockdown. Additionally, DBZ ameliorated sickness behaviors of neuroinflammatory mice induced by systemic LPS administration, and significantly reduced infract volume, improved sensorimotor and cognitive function in rats subjected to transient middle cerebral artery occlusion (tMCAO); besides, DBZ restored microglia morphological alterations and shifted the M1/M2 polarization in both murine models. Mechanistically, DBZ-induced Nrf2 nuclear accumulation and antioxidant enzymes expression were accompanied by increased level of p-Akt(Ser473) (activation) and p-GSK3β(Ser9) (inactivation), and decreased nuclear level of Fyn both in vitro and in vivo. Pharmacologically inhibiting PI3K or activating GSK3β markedly increased nuclear density of Fyn in microglia cells, which blocked the promoting effect of DBZ on Nrf2 nuclear accumulation and its antioxidant and anti-neuroinflammatory activities. Collectively, these results indicated the effects of DBZ on microglia-mediated neuroinflammation were strongly associated with the nuclear accumulation and stabilization of Nrf2 via the Akt(Ser473)/GSK3β(Ser9)/Fyn pathway. With anti-neuroinflammatory and antioxidant properties, DBZ could be a promising new drug candidate for prevention and/or treatment of cerebral ischemia and other neuroinflammatory disorders.

Putrescine, a biogenic amine, is a highly valued compound in medicine, industry, and agriculture. In this study, we report a whole-cell biocatalytic method in Escherichia coli for the production of putrescine, using L-arginine as the substrate. L-arginine decarboxylase and agmatine ureohydrolase were co-expressed to produce putrescine from L-arginine. Ten plasmids with different copy numbers and ordering of genes were constructed to balance the expression of the two enzymes, and the best strain was pACYCDuet-speB-speA. The optimal concentration of L-arginine was determined to be 20 mM for this strain. The optimum pH of the biotransformation was 9.5, and the optimum temperature was 45 &deg, C, under these conditions, the yield of putrescine was 98%. This whole-cell biocatalytic method appeared to have great potential for the production of putrescine.

In mammals, peroxisome proliferators activated receptors (PPARs), the nuclear hormone receptors, have been reported to be involved in seizure control. Selective agonists and antagonists of PPARs raise seizure thresholds and suppress seizures, respectively. In this study, we evaluated the anticonvulsant effects of α-asaronol, a metabolic product of α-asarone, on pentylenetetrazole (PTZ)-induced seizures in zebrafish and investigated the underlying mechanisms. As a result, α-asaronol ameliorated seizures with increase of seizure latency, as well as decrease of seizure-like behavior, c-fos expression, and abnormal neuronal discharge in a concentration dependent manner. By comparing gene expression profiles of zebrafish undergoing seizures and α-asaronol pretreated zebrafish, we found that α-asaronol attenuate seizures through increase of PPAR γ expression, while PPAR γ antagonist GW9662 inhibit the anti-seizures actions of α-asaronol. Moreover, molecular docking simulation implied the physical interaction between α-asaronol and PPAR γ. The overall results indicated that the anticonvulsant effects of α-asaronol are regulated through PPAR γ-mediated pathway, which shed light on development of α-asaronol as a potential antiepileptic drug. In addition, it is for first time to report that PPAR γ is associated with seizures in zebrafish, supporting previous evidence that zebrafish is a suitable alternative for studying seizures.

We developed an efficient multi-enzyme cascade reaction to produce (R)- or (S)-3,4-Dihydroxyphenyllactic acid [(R)- or (S)-Danshensu, (R)- or (S)-DSS] from 3,4-Dihydroxyphenyl-l-alanine (l-DOPA) in Escherichia coli by introducing tyrosine aminotransferase (tyrB), glutamate dehydrogenase (cdgdh) and d-aromatic lactate dehydrogenase (csldhD) or l-aromatic lactate dehydrogenase (tcldhL). First, the genes in the pathway were overexpressed and fine-tuned for (R)- or (S)-DSS production. The resulting strain, E. coli TGL 2.1 and E. coli TGL 2.2, which overexpressed tyrB with the stronger T7 promoter and cdgdh, cs ldhD or tcldhL with the weaker Trc promoter, E. coli TGL 2.1 yielded 57% increase in (R)-DSS production: 59.8 ± 2.9 mM. Meanwhile, E. coli TGL 2.2 yielded 54% increase in (S)-DSS production: 52.2 ± 2.4 mM. The optimal concentration of L-glutamate was found to be 20 mM for production of (R)- or (S)-DSS. Finally, l-DOPA were transformed into (R)- or (S)-DSS with an excellent enantiopure form (enantiomeric excess > 99.99%) and productivity of 6.61 mM/h and 4.48 mM/h, respectively.

Epilepsy is a common chronic neurological disorder disease, and there is an urgent need for the development of novel anticonvulsant drugs. In this study, the anticonvulsant activities and neurotoxicity of 12 cinnamic acid derivatives substituted by fluorine, chlorine, bromine, and trifluoromethyl groups were screened by the maximal electroshock seizure (MES) and rotarod tests (Tox). Three of the tested compounds (compounds 3, 6 and 12) showed better anticonvulsant effects and lower neurotoxicity. They showed respective median effective dose (ED50) of 47.36, 75.72 and 70.65 mg/kg, and median toxic dose (TD50) of them was greater than 500 mg/kg, providing better protective indices. Meanwhile, they showed a pentylenetetrazol (PTZ) ED50 value of 245.2, >300 and 285.2 mg/kg in mice, respectively. Especially, the most active compound 3 displayed a prominent anticonvulsant profile and had lower toxicity. Therefore, the antiepileptic mechanism of 3 on glycosylation changes in chronic epilepsy in mice was further investigated by using glycomics techniques. Lectin microarrays results showed that epilepsy was closely related to abnormal glycosylation, and 3 could reverse the abnormal glycosylation in scPTZ-induced epilepsy in mice. This work can provide new ideas for future discovery of potential biomarkers for evaluation of antiepileptic drugs based on the precise alterations of glycopatterns in epilepsy.

To characterize a novel membrane-bound d -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD). The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with d -alanine the most preferred substrate. The K m and V max values for d -alanine were 9 mM and 20 μmol min−1 mg−1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs. d-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. Km and kcat values for phenyl pyruvate were 0.62mM and 77.38s-1, respectively, and the kcat/Km value was 124.81mM-1s-1. The enzyme properties suggest it may act effectively under cheese ripening conditions.

Rosmarinic acid (RA), as a hydroxycinnamic acid ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL), is a phenylpropanoid-derived plant natural product and has diverse biological activities. This work acts as a modular platform for microbial production using a two-cofactor (ATP and CoA) regeneration system to product RA based on a cell-free biosynthetic approach. Optimal activity of the reaction system was pH 8 and 30 °C. Total turnover number for ATP and CoA was 820.60 ± 28.60 and 444.50 ± 9.65, respectively. Based on the first hour data, the RA productivity reached 320.04 mg L−1 h−1 (0.889 mM L−1 h−1).

Traditional Chinese medicine (TCM) represents a valuable resource for lead compounds discovery. Given the complexity of TCM components, analytical methods play a key role in novel drug development. In our study, we established a high specific and reliable bio-active components screen system, where β2 adrenergic receptor (β2-AR) was immobilized on silica by non-covalent bonds and packed into a stainless steel column (4.6 × 50 mm, 7 μm) to form β2-AR chromatography column. The column was further coupled with high performance liquid chromatography-time of flight tandem mass spectrometry (TOF-MS/MS). By utilizing this strategy, we successfully identified four β2-AR-targeting compounds: tetrahydroberberine, tetrahydrocolumbamine, fumarine and corydaline from Corydalis Rhizome. The association constants between β2-AR and tetrahydroberberine (9.04 × 104/M) as well as fumarine (4.30 × 104/M) were determined by frontal chromatography. We also found that these two compounds shared the identical binding site on immobilized β2-AR with corresponding concentrations of 6.67 × 10−4 M and 5.88 × 10−4 M, respectively. The newly established method represents an efficient tool to identify the target specific natural compounds.

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3. The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K m values of 0.48, 6.3, and 1.9 mM, respectively, and k cat /K m values of 9.3, 3.8, and 3.8 mM−1 s−1, respectively. Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.