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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.

Chromatography is an important branch of analytical chemistry that focuses on the separation and analysis of complex structures. Following more than 100 years of development and improvement, chromatography theory and technology have gradually become sophisticated. It has become a coalition of science, technology, and art. Recently, chromatography has been successfully used in combination with mass spectrometry, nuclear magnetic resonance spectroscopy, and atomic emission spectroscopy. Chromatography and the combination with other techniques has significantly improved the analysis of complex systems, such as the environment, food, petrochemicals, biological specimens, and medicine. As one of the oldest healing systems, Traditional Chinese Medicine (TCM) has served to maintain the health of people in China and worldwide for thousands of years. Therefore, it has become a core representative of traditional Chinese culture. In the past two years, TCM has been widely used to treat COVID-19, especially in patients with mild symptoms. Recently, Chinese government emphasized the inheritance and innovation of TCM and stepped up efforts to promote its modernization. TCM includes herbal medicine, acupuncture, moxibustion, massage, food therapy, and physical exercise, such as Tai Chi. In most cases, the patients are administered a mixture of TCM formulas containing more than two herbal medicines, resulting in a highly complicated compound mixture. There is no doubt that long-term clinical practices have demonstrated the safety and therapeutic effect of TCM. However, the compound mixture must be simplified to identify the active compounds. This is mainly because of the existence of carcinogenic compounds, pesticides, and heavy metal residues introduced through plantation and production processes. Moreover, enzymes within the human system generate further new compounds in response to the entry of the TCM containing thousands of components. Consequently, the complex TCM and organism systems interact with each other, constituting a giant complex drug-organism system. The analysis of this giant complex system is acknowledged as a key aspect in the modernization process of TCM. In the last 20 years, many studies have been conducted to screen and identify effective compounds in TCM. These effective compounds can be either the original compounds or new metabolic components generated in vivo. All these efforts are aimed at simplifying the components of TCM and elucidating the therapeutic mechanism. It is well known that chromatography can provide technical support for complex systems owing to its unique advantage of outstanding separation and analysis capabilities. Therefore, chromatography and its combination with other technologies have become mainstream technologies for promoting the compilation of molecular structure, information, digitalization, and modernization of TCM. This paper reviews the research and application of chromatography and combination technologies in a giant complex TCM formula-organism system. Furthermore, the authors briefly introduce and summarize the understanding, research ideas, and activities of the authors' team on the modernization of TCM. "Liang Guanxi" and "He strategy" are proposed as novel strategies for studying the giant complex drug-organism system. A distinguished technology integrated with mathematical model of causal relation, combined receptor chromatography, identification of chemical molecular structure and evaluating of pharmacological activities was established. It was successfully employed to determine the core effector-response substances of "Liang Guanxi" herb pairs in a giant complex drug-organism system. Subsequently, utilizing the proposed technology of Combination of Traditional Chinese Medicine Molecular Chemistry, the author's team designed and developed four series of innovative drugs. Inspired by the hundred years of chromatography history and thousands of years of TCM culture, the future development of chromatographic technology is expected. Furthermore, the mechanisms of TCM in medical healthcare, prevention, and treatment of diseases are likely be explained through chromatography, leading to a new strategy to realize the molecularization and digitalization of TCM, which is beneficial to the development of original new drugs.

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.

Spermidine is an important polyamine that can be used for the synthesis of various bioactive compounds in the food and pharmaceutical fields. In this study, a novel efficient whole-cell biocatalytic method with an NADPH self-sufficient cycle for spermidine biosynthesis was designed and constructed by co-expressing homoserine dehydrogenase (HSD), carboxyspermidine dehydrogenase (CASDH), and carboxyspermidine decarboxylase (CASDC). First, the enzyme-substrate coupled cofactor regeneration system from co-expression of NADP

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.

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...

Preterm white matter injury (PWMI) is the most common form of brain damage in premature infants caused by hypoxia-ischemia (HI), inflammation, or excitotoxicity. It is characterized by oligodendrocyte precursor cell (OPC) differentiation disorder and dysmyelination. Our previous study confirmed that alpha-asarone (α-asaronol), a major compound isolated from the Chinese medicinal herb Acorus gramineus by our lab, could alleviate neuronal overexcitation and improve the cognitive function of aged rats. In the present study, we investigated the effect and mechanism of α-asaronol on myelination in a rat model of PWMI induced by HI. Notably, α-asaronol promoted OPC differentiation and myelination in the corpus callosum of PWMI rats. Meanwhile, the concentration of glutamate was significantly decreased, and the levels of PPARγ and glutamate transporter 1 (GLT-1) were increased by α-asaronol treatment. In vitro, it was also confirmed that α-asaronol increased GLT-1 expression and recruitment of the PPARγ coactivator PCG-1a in astrocytes under oxygen and glucose deprivation (OGD) conditions. The PPARγ inhibitor GW9662 significantly reversed the effect of α-asaronol on GLT-1 expression and PCG-1a recruitment. Interestingly, the conditioned medium from α-asaronol-treated astrocytes decreased the number of OPCs and increased the number of mature oligodendrocytes. These results suggest that α-asaronol can promote OPC differentiation and relieve dysmyelination by regulating glutamate levels via astrocyte PPARγ-GLT-1 signaling. Although whether α-asaronol binds to PPARγ directly or indirectly is not investigated here, this study still indicates that α-asaronol may be a promising small molecular drug for the treatment of myelin-related diseases.

Tanshinol borneol ester (DBZ) exerts anti-atherosclerotic and anti-inflammatory effects. However, its effects on cardiac hypertrophy are not well understood. In this work, we investigated the treatment effects and potential mechanisms of DBZ on the hypertrophic heart under oxidative stress and endoplasmic reticulum (ER) stress. A hypertrophic model was established in rats using transverse-aortic constriction (TAC) surgery and in neonatal rat cardiomyocytes (NRCMs) using angiotensin II (Ang II). Our results revealed that DBZ remarkably inhibited oxidative stress and ER stress, blocked autophagy flow, and decreased apoptosis in vivo and in vitro through nuclear NRF2 accumulation, and enhanced NRF2 stability via regulating the mTOR/β-TrcP/NRF2 signal pathway. Thus, DBZ may serve as a promising therapeutic for stress-induced cardiac hypertrophy.

α-Asaronol from

Carboxyspermidine (C-Spd) is a potentially valuable polyamine carboxylate compound and an excellent building block for spermidine synthesis, which is a critical polyamine with significant implications for human health and longevity. C-Spd can also be used to prepare multivalent cationic lipids and modify nucleoside probes. Because of these positive effects on human health, C-Spd is of considerable interest as a food additive and pharmaceutical target.A putative gene afcasdh from Agrobacterium fabrum str. C58, encoding carboxyspermidine dehydrogenase with C-Spd biosynthesis activity, was synthesized and transformed into Escherichia coli BL21 (DE3) for overexpression. The recombinant AfCASDH was purified and fully characterized. The optimum temperature and pH for the recombinant enzyme were 30 °C and 7.5, respectively. The coupled catalytic strategy of AfCASDH and various NADPH regeneration systems were developed to enhance the efficient production of C-Spd compound. Finally, the maximum titer of C-Spd production successfully achieved 1.82 mmol LA novel AfCASDH from A. fabrum str. C58 was characterized that could catalyze the formation of C-Spd from putrescine and l-aspartate-β-semialdehyde (L-Asa). A whole-cell catalytic strategy coupled with NADPH regeneration was established successfully for C-Spd biosynthesis for the first time. The coupled system indicated that AfCASDH might provide a feasible method for the industrial production of C-Spd. © 2021 Society of Chemical Industry.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×10

α-Asaronol [or (E)-3′-hydroxyasarone; systematic name: (E)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-ol; C12H16O4] was synthesized towards the development of a potential antiepileptic drug. Following purification by recrystallization, single crystals of α-asaronol were obtained by a liquid interface diffusion method at room temperature. The product was characterized by 1H and 13C NMR, and FT–IR spectroscopic analysis. X-ray crystallography revealed the title crystal to belong to the orthorhombic space group P212121. Preliminary bioassays with mouse neuroblastoma N2a cells demonstrated the neuroprotective activities of the synthesized α-asaronol.

L-lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum. L-lactate dehydrogenase gene (LF-L-LDH0845) with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248. To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates, site-directed mutation of Tyr221 was performed by analyzing the amino acids in active center. Kinetic parameters show that the k cat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate, 4-methyl-2-oxopentanoate, and glyoxylate are 1.21 s -1 , 1.35 s -1 , and 0.72 s -1 , respectively, which are 420%, 150% and 130% of the wild-type LDH0845. This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845, making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.

A novel short-chain alcohol dehydrogenase from Tarenaya hassleriana labeled as putative tropinone reductase was heterologously expressed in Escherichia coli. Purified recombinant protein had molecular weight of approximately 30 kDa on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T. hassleriana tropinone reductase-like enzyme (ThTRL) had not detected oxidative activity. The optimum pH for enzyme activity of ThTRL was weakly acidic (pH 5.0). 50°C was the optimum temperature for ThTRL. The highest catalytic efficiency and substrate affinity for recombinant ThTRL were observed with (+)-camphorquinone (k

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...

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.

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.

The analysis and separation of chiral compounds with wide polar range by supercritical fluid chromatography is of major importance in the process of drug development and quality control. In this work, a fast and reliable enantioselective method for the simultaneous quantitative determination of 8 DBZ-related enantiomers has been successfully developed by supercritical fluid chromatography using an amylose-based reversed-chiral stationary phase. Within less than seven minutes all target compounds could be baseline resolved, using a mobile phase comprising supercritical carbon dioxide and methanol with 0.05 % H

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the 7 variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat /Km ) for COBE was 51.36 s-1 ·mM-1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197. This article is protected by copyright. All rights reserved.

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.

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.

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.

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.

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.

Inspired by the traditional Chinese herbal pair of Polygala tenuifolia-Acori Tatarinowii for treating epilepsy, 33 novel substituted cinnamic α-asaronol esters and analogues were designed by Combination of Traditional Chinese Medicine Molecular Chemistry (CTCMMC) strategy, synthesized and tested systematically not only for anticonvulsant activity in three mouse models but also for LDH inhibitory activity. Thereinto, 68–70 and 75 displayed excellent and broad spectra of anticonvulsant activities with modest ability in preventing neuropathic pain, as well as low neurotoxicity. The protective indices of these four compounds compared favorably with stiripentol, lacosamide, carbamazepine and valproic acid. 68–70 exhibited good LDH1 and LDH5 inhibitory activities with noncompetitive inhibition type, and were more potent than stiripentol. Notably, 70, as a representative agent, was also shown as a moderately positive allosteric modulator at human α1β2γ2 GABAA receptors (EC50 46.3 ± 7.3 μM). Thus, 68–70 were promising candidates for developing into anti-epileptic drugs, especially for treatment of refractory epilepsies such as Dravet syndrome.

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 β

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.

Ethnopharmacological relevance Sophora japonica (Fabaceae), also known as Huai (Chinese: 槐), is a medium-sized deciduous tree commonly found in China, Japan, Korea, Vietnam, and other countries. The use of this plant has been recorded in classical medicinal treatises of ancient China, and it is currently recorded in both the Chinese Pharmacopoeia and European Pharmacopoeia. The flower buds and fruits of S. japonica , also known as Flos Sophorae Immaturus and Fructus Sophorae in China, are most commonly used in Asia (especially in China) to treat hemorrhoids, hematochezia, hematuria, hematemesis, hemorrhinia, uterine or intestinal hemorrhage, arteriosclerosis, headache, hypertension, dysentery, dizziness, and pyoderma. To discuss feasible trends for further research on S. japonica , this review highlights the botany, ethnopharmacology, phytochemistry, biological activities, and toxicology of S. japonica based on studies published in the last six decades. Materials and methods Information on the S. japonica was collected from major scientific databases (SciFinder, PubMed, Elsevier, SpringerLink, Web of Science, Google Scholar, Medline Plus, China Knowledge Resource Integrated (CNKI), and “ Da Yi Yi Xue Sou Suo ( http://www.dayi100.com/login.jsp )” for publications between 1957 and 2015 on S. japonica . Information was also obtained from local classic herbal literature, government reports, conference papers, as well as PhD and MSc dissertations. Results Approximately 153 chemical compounds, including flavonoids, isoflavonoids, triterpenes, alkaloids, polysaccharides, amino acids, and other compounds, have been isolated from the leaves, branches, flowers, buds, pericarps, and/or fruits of S. japonica . Among these compounds, several flavonoids and isoflavonoids comprise the active constituents of S. japonica , which exhibit a wide range of biological activities in vitro and in vivo such as anti-inflammatory, antibacterial, antiviral, anti-osteoporotic, antioxidant, radical scavenging, antihyperglycemic, antiobesity, antitumor, and hemostatic effects. Furthermore, flavonoids and isoflavonoids can be used as quality control markers for quality identification and evaluation of medicinal materials and their preparations. Information on evaluating the safety of S. japonica is very limited, so further study is required. To enable safer, more effective, and controllable therapeutic preparations, more in-depth information is urgently needed on the quality control, toxicology data, and clinical value of crude extract and active compounds of S. japonica . Conclusions S. japonica has long been used in traditional Chinese medicine (TCM) due to its wide range of biological activities, and is administered orally. Phytochemical and pharmacological studies of S. japonica have increased in the past few years, and the extract and active components of this plant can be used to develop new drugs based on their traditional application as well as their biological activities. Therefore, this review on the ethnopharmacology, phytochemistry, biological activities, and toxicity of S. japonica offers promising data for further studies as well as the commercial exploitation of this traditional medicine.