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The efficiency of whole-cell biotransformation is often affected by genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for synthesis of phenylpyruvic acid (PPA) from L-phenylalanine. Firstly, L-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into E. coli BL21 (DE3) chromosome and the copy-numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). 59 copies of pmLAAD was obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of Isopropyl-β-D-thiogalactopyranoside (IPTG), the resulting strain, E. coli BL8△recA△lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37 % increase in PPA production compared to E. coli BL8△recA. Finally, the engineered E. coli BL8△recA△lacI strain achieved 19.14 g/L PPA at 24 h in 5 L bioreactor. The culture of the strain does not require the addition of antibiotics and inducers. The production level of CIChE strains can catch up with plasmid expression strains. This work extends production methods for whole-cell biotransformation. This article is protected by copyright. All rights reserved.

l-DOPA is a major drug for the treatment of Parkinson’s disease, and microbial enzymatic synthesis is an essential way for its industrial production. The expression of tyrosine phenol-lyase (TPL) derived from Erwinia herbicola under six different constitutive promoters in Escherichia coli was studied. The recombinant strain 5 can express TPL well with growth. The optimal medium composition of this strain was as follows: tryptone 18 g/L, yeast extract 28 g/L, glycerol 10 g/L. After the cell culture is completed, the collected cells were used to catalyze the production of l-DOPA, and the concentration can reach 67.9 g/L.

A novel feruloyl esterase (BpFae12) with rosmarinic acid (RA) hydrolysis activity was isolated from Bacillus pumilus W3 and expressed in Escherichia coli BL21 (DE3). With RA as a substrate, the optimal pH and temperature of BpFae12 were pH 8.0 and 50 °C, respectively. The specific enzyme activity was 12.8 U·mg−1. BpFae12 showed the highest activity and substrate affinity toward RA (Vmax of 13.13 U·mg−1, Km of 0.41 mM). Moreover, it also presented strong hydrolysis performance against chlorogenic acid (190.17 U·mg−1). RA was effectively Hydrolyzed into more bioactive caffeic acid and 3,4-dihydroxyphenyllactic acid by BpFae12, which have potential applications in the food industry.

Proteus mirabilis could convert l-phenylalanine into 2-phenylethanol (2-PE) via the Ehrlich pathway, the amino acid deaminase pathway, and the aromatic amino acid decarboxylase pathway. The aromatic amino acid decarboxylase pathway was proved for the first time in P. mirabilis. In this pathway, l-aromatic amino acid transferase demonstrated a unique catalytic property, transforming 2-penylethylamine into phenylacetaldehyde. Eleven enzymes were supposed to involve in 2-phenylethanol synthesis. The mRNA expression levels of 11 genes were assessed over time by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in vivo. As a result, the expression of 11 genes was significantly increased, suggesting that P. mirabilis could transform l-phenylalanine into 2-phenylethanol via three pathways under aerobic conditions; nine genes were significantly overexpressed, suggesting that P. mirabilis could synthesize 2-phenylethanol via the Ehrlich pathway under anaerobic conditions. This study reveals the multipath synthetic metabolism for 2-phenylethanol in P. mirabilis and will enrich the new ideas for natural (2-PE) synthesis.

Alcohol dehydrogenases convert aldehydes causing volatile aromas in cheese into corresponding alcohols. In this study, we cloned and recombinantly produced five novel ADHs from Lactobacillus reuteri DSM20016 in Escherichia coli BL21 (DE3). The resulting proteins have molecular weights between 35 and 39 kDa. The optimum pH for enzyme activity of ADH-1, ADH-2 and ADH-3 was weakly acidic (pH 5, 6 and 5, respectively), compared with pH 7 for ADH-4 and ADH-5. The optimum temperature range for ADH-1, ADH-3, ADH-4 and ADH-5 was 40 °C ―55 °C, compared with 35 °C for ADH-2. These enzymes reduced a broad range of substrates; the highest catalytic efficiency and substrate affinity for ADH-1 was observed with heptaldehyde (Kcat/Km = 39.4 s−1·mM−1, Km = 0.459 mM), while 3-methylbutanal was the best substrate for ADH-5 (Kcat/Km = 24.0 s−1·mM−1, Km = 1.35 × 10−2 mM). ADH-2, ADH-3 and ADH-4 exhibited the highest substrate affinity for valeraldehyde, propionaldehyde, and hexanal, respectively (Km = 1.19 × 10−2 mM, 2.81 × 10−2 mM, 1.35 × 10−2 mM). The enzymatic properties indicated that these enzymes effectively converted fragrant compounds during cheese ripening.

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.

A new method for imatinib synthesis is described by using the C–N coupling reaction of 4-(4-methylpiperazine-1-methyl)benzamide with N-(5-bromo-2-tolyl)-4-(3-pyridyl)pyrimidin-2-amine to form imati...

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.

Herein, novel luminol functional gold nanoparticles (lum-AuNPs) were quickly prepared in an alkaline luminol solution with HAuCl4, which had the unique characteristics of uniform size and excellent luminescence properties. A self-made flow injection-chemiluminescence (FI-CL) system was established to study the interaction between pepsin (Pep) and five alkaloids (anisodamine, berberine, reserpine, jatrorrhizine and matrine) using lum-AuNPs as the CL probe. Based on the abovementioned home-made CL system, the possible interaction mechanisms of Pep with five alkaloids have been comprehensively discussed by molecular docking simulation, chemical thermodynamics and kinetic studies. The results indicated that there were obvious CL enhancement and inhibition effects on the lum-AuNPs CL system for the Pep and the complex of Pep/alkaloids, respectively. The possible mechanism for the interaction of Pep–five alkaloids was mainly mediated by the hydrophobic force. The binding constant K and binding site n for the Pep–alkaloid interaction are consistent with the list of Ber > Res > Ani, Jat > Mat, which is relative to the potential of groups of alkaloids interacting with the active site of Pep.

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.

In this article, a novel series of (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (TMCA) amide derivatives 1-18 were designed and synthesized by a facile and one-pot step, which were achieved with good yields using 1-hydroxybenzotriazole (HOBT) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as activation system. All the synthesized derivatives were biologically evaluated for their anticonvulsant, sedative activity and neurotoxicity using the maximal electroshock (MES) model, sc-pentylenetetrazol (PTZ) model, pentobarbital sodium-induced sleeping model, and locomotor activity tests, respectively. Among them, compounds 4, 9 and 16 exhibited good anticonvulsant activity in primary evaluation. Furthermore, compound 4 is the most effective anticonvulsant and sedative agent in subsequent tests, while the low threshold of toxicity of compound 4 is vigilant. Compounds 9 and 16 also performed significantly anticonvulsant activity in subsequent tests with weak toxicity. The molecular modeling experiments also predicted good binding interactions of the obtained active molecules with the GABA transferas. Therefore, it could be concluded that the synthesized derivatives 4, 9 and 16 would represent useful lead compounds for further investigation in the development of anticonvulsant and sedative agents.

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.

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.

In this study, based on homology modeling and molecular docking, the key residues involved in substrate recognition in the active center of NADH-dependent d -lactate dehydrogenase (LF-d-LDH0653) from Lactobacillus fermentum were analyzed. The site-directed saturation mutagenesis of the key Tyr53 site was carried out to study the substrate specificity of mutants. 19 variants were obtained, of which the most significant change in substrate specificity was observed for variant Y53A. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of Y53A to α-ketoisocaproate were 0.13 mM, 112.60 s−1, and 856.67 mM−1 s−1, respectively. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of Y53A to phenylpyruvate were 0.16 mM, 78.17 s−1, and 481.62 mM−1 s−1, respectively. The results indicated that Tyr53 was the key site that determines the substrate specificity of the enzyme, and single-point mutagenesis can dramatically affect the catalytic efficiency for various other substrates.

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.

Hydroxytyrosol is a high-value-added compound with a variety of biological and pharmacological activities. In this study, a whole-cell catalytic method for the synthesis of hydroxytyrosol was developed: aromatic amino acid aminotransferase (TyrB), l-glutamate dehydrogenase (GDH), α-keto acid decarboxylase (PmKDC), and aldehyde reductase (YahK) were co-expressed in Escherichia coli to catalyze the synthesis of hydroxytyrosol from l-3,4-dihydroxyphenylalanine (l-DOPA). The plasmids with different copy numbers were used to balance the expression of the four enzymes, and the most appropriate strain (pRSF- yahK- tyrB and pCDF- gdh- Pmkdc) was identified. After determination of the optimum temperature (35 °C) and pH (7.5) for whole-cell catalysis, the yield of hydroxytyrosol reached 36.33 mM (5.59 g/L) and the space-time yield reached 0.70 g L-1 h-1.

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.

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.

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.

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.

α-Asaronol is one of trace metabolites of α-asarone formed in vivo and in vitro and exhibits good anticonvulsant activities with low neurotoxicity. The present study was mainly to describe the pharmacokinetics and tissue distribution of α-asaronol and its metabolite E-2,4,5-trimethoxy cinnamic acid (E-2,4,5-TMCA), in rat after oral and intravenous administration of α-asaronol. The results indicate that α-asaronol can be absorbed (tmax = 5–10 min) and transformed to E-2,4,5-TMCA (tmax = 10–15 min) rapidly after oral administration. Presumably due to hepatic first-pass effect, α-asaronol shows a low bioavailability (about 25.9%). Furthermore, α-asaronol is distributed rapidly and widely in various tissues with the order of brain > heart > kidney > spleen > liver > lung, and eliminated quickly following the intravenous administration. The maximal concentration of α-asaronol in the brain is about 1.603 ± 0.221 μg/g at 5 min. In comparison, the concentrations of E-2,4,5-TMCA, except brain, are all higher than that of α-asaronol in the tested tissues with the order of kidney > liver > lung > heart ≈ spleen > brain. Current study results will contribute to interpretation and understanding preclinical PK properties of α-asaronol and its antiepileptic effects in animals.

BACKGROUND: Cheese ripening involves a complex series of metabolic reactions and numerous concomitant secondary transformations. Alcohol dehydrogenase (ADH) converts aldehydes into their corresponding alcohols, which enrich cheese aroma. RESULTS: In this study, we identified five ADH genes in Proteus mirabilis JN458, and these genes were overexpressed and characterized in Escherichia coli BL21 (DE3). The optimum pH was 7.0 for the purified recombinant ADH‐1, ADH‐2, and ADH‐3 and 8.0 for ADH‐4 and ADH‐5. The optimum temperature was 40 °C for ADH‐1, ADH‐3, and ADH‐5 and 45 °C for ADH‐2 and ADH‐4. The Kₘ value of ADH‐1, ADH‐2, and ADH‐3 was 34.45, 16.90, and 10.01 µmol L⁻¹ for phenylacetaldehyde, respectively. The Kₘ value of ADH‐4 and ADH‐5 was 14.81 and 24.62 µmol L⁻¹ for 2‐methylbutanal, respectively. CONCLUSION: Proteus species play important roles during cheese ripening. The results of our study are important for further research on cheese flavor and for quality control during cheese production. © 2019 Society of Chemical Industry

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q-TOF-MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid-methanol solution (v:v, 5:95), chromatographic separation by an Agilent Eclipse XDB-C18 column (150 × 4.6 mm i.d., 5 µm) and online identification of the metabolites by Q-TOF-MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including 4 phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O-methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. This article is protected by copyright. All rights reserved.

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.

Phenylpropanoids comprise a broad spectrum of biologically active natural products. As part of our ongoing research on antiepileptic active compounds from traditional Chinese herb, Acorus calamus var. angustatus Besser, three undescribed phenylpropanoids and twenty-two known ones were isolated. All the undescribed structures were determined by a combination of 1D and 2D NMR, HRMS. In addition, γ-asaronol was identified as racemates and its absolute configuration were determined by the modified Mosher's method and ECD spectral data. Furthermore, some selected isolated compounds were evaluated for their cell viability and neuroprotective activities in H2O2-induced SH-SY5Y cells. α-Asaronol, β-asaronol, 3-(2,4,5-trimethoxyphenyl)propan-1-ol and 1,2,4-trimethoxy-5-(3-methoxypropyl)benzene exerted potential protective activity from neuronal oxidative stress in all test concentrations ranging from 0.01 to 100 μM, in which the neuroprotective activity of β-asaronol was the best.

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.

A pilot-scale process has been developed for green and scalable synthesis of (±)-β-(3,4-dihydroxyphenyl) lactic acid ((±)-DSS) and their two important derivatives, namely, (±)-IDHP [(±)-isopropyl 2-hydroxy-3-(3,4-dihydroxyphenyl)propanoate] and (±)-DBZ [(±)-bornyl 2-hydroxy-3-(3,4-dihydroxyphenyl)propanoate]. Subsequent hydrogenation has been carried out by employing Raney Ni as catalyst. The improved process results in higher yields of 47.5% for (±)-DBZ and 49.2% for (±)-IDHP compared to the initial process with a yield of 12% for (±)-DBZ and 18% for (±)-IDHP in our original medicinal chemistry route. Furthermore, kilograms of optical DBZ [(−)-S-DBZ and (+)-R-DBZ, >99% ee] and IDHP [(−)-S-IDHP and (+)-R-IDHP, >99% ee] have been produced by chiral high-performance liquid chromatography in good yield (>84%).

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.

A sensitive and specific gas chromatographic-mass spectrometry with selected ion monitoring (GC-MS/SIM) method has been developed for simultaneous identification and quantification ofα-asarone,β-asarone, and methyl eugenol ofAcorus tatarinowiiSchott in rat plasma. Chromatographic separation was performed on a Restek Rxi-5MS capillary column (30 m × 0.32 mm × 0.25 μm), using 1-naphthol as internal standard (IS). MS detection of these compounds and IS was performed atm/z178, 208, 208, and 144. Intra- and interday precisions of all compounds of interest were less than 10%. The recoveries are situated in the range of 92.4–105.2%. Pharmacokinetics of methyl eugenol confirmed to be one-compartment open model,α-asarone andβ-asarone was two-compartment open model, respectively. The method will probably be an alternative to simultaneous determination and pharmacokinetic study of volatile ingredients inAcorus tatarinowiiSchott.

Imatinib and its analogues were successfully synthesized by an improved method in 19.5– 46.2% total yield of six main steps. Pyrimidinyl amine was prepared by the reaction of enaminone and guanidine nitrate without the use of a toxic cyanamide. N-(2-Methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine as a key intermediate for the synthesis of imatinib was prepared by copper-catalyzed N-arylation of heteroarylamine in 82% yield. The copper salts were used instead of the expensive palladium compounds in this C−N bond-forming reaction. The intermediate nitro compound was reduced by a N2H4·H2O/FeCl3/C system using water as a solvent in good yield.

Simple and inexpensive copper-mediated N-arylation ofheteroarylamines was achieved by using N,N′-dimethylethylenediamine as a ligand and K2CO3 as a base in dioxane heated at 100 °C. In this coupling reaction, the influence of the copper species, ligand, base and solvent was investigated in detail. N-Arylated derivatives of several heteroarylamines were synthesized under optimized reaction conditions, and all the products were isolated in good yields. By controlling the amount of CuI/DMEDA added, heteroarylamines with weak nucleophilic activity were coupled with aryl iodides or aryl bromides. The activity of the copper catalyst for this C–N bond-forming reaction follows the order CuI > Cu0 > CuII. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)