(R)-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester ((R)-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester ((R)-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the (R,S)-DMPM to obtain (R)-DMPM. (2) Up to 20sg/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1smM and 2.8smM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 degC and 8.0, and substrate concentration was up to 0.35sM. After 15sh reaction, the conversion rate from (R,S)-DMPM to (R)-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200sg/L substrate and 12.5sg immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12sh, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of (R,S)-DMPM to prepare (R)-Metalaxyl.
BACKGROUND: It has been established that the dipeptidyl peptidase-4 (DPP-4) inhibitor Diprotin A TFA can reduce vascular endothelial (VE)-cadherin disruption by inhibiting the increase in cleaved beta-catenin in response to hypoxia, thereby protecting the vascular barrier of human umbilical vein endothelial cells. In this study, we sought to investigate the possible effect of Diprotin A TFA on the VE barrier after cerebral ischemic stroke in mice. METHODS: C57BL/6J mice were divided into five groups, namely, (1) sham, (2) stroke, (3) stroke + dimethyl sulfoxide (DMSO), (4) stroke + Diprotin A TFA, and (5) stroke + Diprotin A TFA + XAV-939. First, the cerebral ischemia model was established by photothrombotic ischemia, followed by intraperitoneal injection with Diprotin A TFA and XAV-939 at doses of 70 microg/kg and 40 mg/kg 30 min once in the morning and once in the evening for 3 days. Immunofluorescence staining and Western blot methods were used to analyze the expression of vascular and blood-brain barrier (BBB)-associated molecular markers in the peri-infarct area. RESULTS: Compared with the vehicle control group, we found that mice injected with Diprotin A TFA exhibited reduced cerebral infarction volume, increased vascular area and length around the brain injury, increased pericyte and basement membrane coverage, upregulated expression of BBB tight junction proteins, and improved their BBB permeability, whereas the group injected with both drug and inhibitor exhibited significantly aggravated vascular injury and BBB permeability. CONCLUSION: Diprotin A TFA can reduce VE-cadherin disruption by inhibiting ischemia-hypoxia-induced beta-catenin cleavage to protect blood vessels.
Plastic waste poses an ecological challenge1. While current plastic waste management largely relies on unsustainable, energy-intensive, or even hazardous physicochemical and mechanical processes, enzymatic degradation offers a green and sustainable route for plastic waste recycling2. Poly(ethylene terephthalate) (PET) has been extensively used in packaging and for the manufacture of fabrics and single-used containers, accounting for 12% of global solid waste3. The practical application of PET hydrolases has been hampered by their lack of robustness and the requirement for high processing temperatures. Here, we use a structure-based, deep learning algorithm to engineer an extremely robust and highly active PET hydrolase. Our best resulting mutant (FAST-PETase: Functional, Active, Stable, and Tolerant PETase) exhibits superior PET-hydrolytic activity relative to both wild-type and engineered alternatives, (including a leaf-branch compost cutinase and its mutant4) and possesses enhanced thermostability and pH tolerance. We demonstrate that whole, untreated, post-consumer PET from 51 different plastic products can all be completely degraded by FAST-PETase within one week, and in as little as 24 hours at 50 C. Finally, we demonstrate two paths for closed-loop PET recycling and valorization. First, we re-synthesize virgin PET from the monomers recovered after enzymatic depolymerization. Second, we enable in situ microbially-enabled valorization using a Pseudomonas strain together with FAST-PETase to degrade PET and utilize the evolved monomers as a carbon source for growth and polyhydroxyalkanoate production. Collectively, our results demonstrate the substantial improvements enabled by deep learning and a viable route for enzymatic plastic recycling at the industrial scale.
Title: In Situ Generation of Prussian Blue by MIL-53 (Fe) for Point-of-Care Testing of Butyrylcholinesterase Activity Using a Portable High-Throughput Photothermal Device Guo L, Zhang YJ, Yu YL, Wang JH Ref: Analytical Chemistry, 92:14806, 2020 : PubMed
Butyrylcholinesterase (BuChE), the primary source of serum cholinesterase activity, is an indispensable biochemical marker for clinical diagnosis of liver function and organophosphorus poisoning. The requirement for bulky and expensive instruments represents a huge hindrance for point-of-care testing (POCT) of BuChE, especially in resource-limited settings. Herein, an easy-operated, economic, and portable photothermal (PT) biosensing platform for high-throughput BuChE detection was rationally designed. BuChE could "light up" the PT signal through in situ generation of Prussian blue (PB) by MIL-53 (Fe), which allowed us to translate biological signals into temperature signals. Such temperature change signals could be monitored at high throughput (six samples for a single measurement) by a miniature self-made integrated PT device via combining separable 96-well plates, a three-dimensional (3D) printed sample bracket, 808 nm lasers, and thermometers, satisfying the requirement for rapid on-site detection in a large batch with low cost. In addition, the large specific surface area, 3D network structure, and high porosity of MIL-53 (Fe) offered a beneficial platform for its reaction with enzymatic hydrolysate, resulting in high sensing sensitivity and low detection limit (0.3 U L(-1)), which was at least 20 000 times lower than the normal human serum BuChE activity. This facile, affordable, and broad applicability PT sensing platform provides a beneficial reference for the rational design of other disease diagnostic approaches suitable for POCT.
Title: New Flavoalkaloids with Potent alpha-Glucosidase and Acetylcholinesterase Inhibitory Activities from Yunnan Black Tea 'Jin-Ya' Li N, Zhu HT, Wang D, Zhang M, Yang CR, Zhang YJ Ref: Journal of Agricultural and Food Chemistry, 68:7955, 2020 : PubMed
As the subgroup of flavoalkaloids, N-ethyl-2-pyrrolidinone substituted flavan-3-ols are reported to possess various biological activities that may play important roles in the beneficial healthcare functions of tea. The HPLC and LC-MS analyses showed the existence of N-ethyl-2-pyrrolidinone substituted flavan-3-ols in 'Jin-Ya', which is a Yunnan black tea produced only from buds of tea plant, Camellia sinensis var. assamica. Further phytochemical study on this precious black tea led to the identification of eight flavoalkaloids, 1-8, along with 11 known flavan-3-ols (9-14) and flavonol glycosides (15-19). The new compounds, (-)-6-(5''S)-N-ethyl-2-pyrrolidinone-epiafzelechin (1), (-)-8-(5''R)-N-ethyl-2-pyrrolidinone-epiafzelechin-3-O-gallate (2a) and (-)-8-(5''S)-N-ethyl-2-pyrrolidinone-epiafzelechin-3-O-gallate (2b), were identified based on extensive spectroscopic analysis. Flavoalkaloids 2-6 showed inhibitory activity on alpha-glucosidase with IC50 values ranging from 2.09 to 8.47 muM, comparing to those of quercetin and acarbose (IC50 = 6.87 and 228.9 muM, resp.). Moreover, compounds 2, 3 and 6 displayed inhibitory effect on acetyl-cholinesterase with IC50 values of 14.23, 33.79 and 34.82 muM, respectively, comparing to tacrine (IC50 = 0.223 muM).
A series of genistein derivatives were synthesized and evaluated as multifunctional anti-Alzheimer agents. The results showed that these derivatives had significant acetylcholinesterase (AChE) inhibitory activity; compound 5a exhibited the strongest inhibition to AChE with an IC50 value (0.034 muM) much lower than that of rivastigmine (6.53 muM). A Lineweaver-Burk plot and molecular modeling study showed that compound 5a targeted both the catalytic active site and the peripheral anionic site of AChE. These compounds also showed potent peroxy scavenging activity and metal-chelating ability. The compounds did not show obvious effect on HepG2 and PC12 cell viability at the concentration of 100 muM. Therefore, these genistein derivatives can be utilized as multifunctional agents for the treatment of AD.
Title: Purification, identification and characterization of an esterase with high enantioselectivity to (S)-ethyl indoline-2-carboxylate Zhang YJ, Chen CS, Liu HT, Chen JL, Xia Y, Wu SJ Ref: Biotechnol Lett, 41:1223, 2019 : PubMed
OBJECTIVE: To purify an esterase which can selectively hydrolyze (R,S)-ethyl indoline-2-carboxylate to produce (S)-indoline-2-carboxylic acid and characterize its enzymatic properties. RESULTS: An intracellular esterase from Bacillus aryabhattai B8W22 was isolated and the purified protein was identified as a carboxylesterase by MALDI-TOF mass spectrometry. The enzyme (named BaCE) was 59.03-fold purification determined to be of approximately 35 kDa. Its specific activity was 0.574 U/mL with 20% yield. The enzyme showed maximum activity at pH 8.5 and 30 degrees C and was stable at 20-30 degrees C using pNPB as the substrate. The Km, Vmax, kcat and kcat/Km of the esterase were 0.52 mM, 6.39 muM/min, 26.87 min(-1) and 51.67 mM/min, respectively. The esterase demonstrated high enantioselectivity toward (S)-ethyl indoline-2-carboxylate with 96.55% e.e.p at 44.39% conversion, corresponding to an E value of 133.45. CONCLUSIONS: In this study, a new esterase BaCE with an apparent molecular mass of 35 kDa was purified to homogeneity for the first time. The esterase from Bacillus aryabhattai B8W22 was isolated with a purification more than 59-fold and a yield of 20% by anion exchange chromatography and hydrophobic interaction chromatography. And its biochemical characterization were described in detail with pNPB as substrate. It displayed high enantioselectivity toward (S)-ethyl indoline-2-carboxylate. We next plan to highly express esterase BaCE in Escherichia coli, and apply it to industrial production of (S)-indoline-2-carboxylic acid.
Title: High-level expression and characterization of a stereoselective lipase from Aspergillus oryzae in Pichia pastoris Zheng JY, Lan X, Li XJ, Huang LJ, Zhang YJ, Wang Z Ref: Protein Expr Purif, 155:1, 2019 : PubMed
Pichia pastoris expression is a mature and efficient eukaryotic expression system. In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)-methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P. pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose FF ion-exchange chromatography and phenyl Seflnose 6 FF hydrophobic interaction chromatography. The purified lipase activity reached 5509 U/mg. AOL showed high activity toward short-chain triacylglyceride (C(4)), and the optimum temperature and pH were 40 degreesC and 8.0, respectively. The purified enzyme activity was inhibited by Zn(2+) and Cu(2+). Moreover, the K(m) and V(max) values were 1 mM and 32.89 mmol/min, respectively.
Title: Intracerebroventricular injection of Abeta1-42 combined with two-vessel occlusion accelerate Alzheimer's disease development in rats Dai SJ, Zhang JY, Bao YT, Zhou XJ, Lin LN, Fu YB, Zhang YJ, Li CY, Yang YX Ref: Pathol Res Pract, 214:1583, 2018 : PubMed
Numerous experimental studies and clinical observations suggest that cerebral ischemia may contribute to the pathogenesis of Alzheimer's disease (AD). Two-vessel occlusion caused cerebral ischemia model is often used in the study of vascular dementia (VaD). But how cerebral ischemia works on AD rat model which induced by intracerebroventricular injection of Abeta1-42 remains unclear. In the following study, we investigated the characteristics of rat model caused by intracerebroventricular injection of Abeta1-42 or two-vessel occlusion (2-VO) only and by both of the two operations. The animal cognitive functions were accessed by the Morris water maze. Regional cerebral blood flow was detected by Laser Doppler Blood Flowmeter. HE&Nissl staining, Congo red staining and immunohistochemistry were used to observe the status of neuronal loss, Abeta deposition and the phosphorylated tau expression in hippocampus, respectively. We also measured the contents of AchE and ChAT in serum and hippocampus by Enzyme Linked Immunosorbent Assay. The MWM results showed that rats of Abeta1-42+2-VO group had a disorder in cognitive functions, at an early stage of one week after modeling, comparing with rats of sham group. The regional cerebral blood flow (rCBF) was significantly reduced in Abeta1-42+2-VO and 2-VO group one week after modeling, and still maintained low perfusion levels four weeks after modeling. HE and Nissl staining showed that Abeta1-42+2-VO rats' hippocampal CA1 neurons were in disorder, degeneration and necrosis, severe neuronal loss from the first week to the fourth week, while this phenomenon only appeared in the fourth week after modeling in rats of Abeta1-42 group and 2-VO group. Congo red staining showed that Abeta1-42 + 2-VO group rats' hippocampus CA1 had amyloid deposits from the first week to the fourth week, Abeta1-42 group were not find amyloid deposition significantly until four weeks after modeling, however, 2-VO group had no significant amyloid deposition all the time. Notably, IHC showed that, two weeks after modeling, the p-tau positive total area and integrated optical density of hippocampal CA1 region were significantly increased in Abeta1-42 + 2-VO group rats, while 2-VO group and Abeta1-42 group rats had no significantly changes all the time. We also found that the content of AchE was increased both in serum and hippocampus of Abeta1-42 + 2-VO group rats, and ChAT was decreased. However, there was no significantly change in cortex of content of AchE: acetylcholinesterase (AchE) and choline acetylase (ChAT) all three groups. Together, our study suggest that intracerebroventricular injection of Abeta1-42 combined with two-vessel occlusion may accelerate Alzheimer's disease development in rats. Also, this may serve as a less-time consuming new model to study the Alzheimer's disease and especially AD accompanied by cerebral ischemia.
Title: C-8 N-Ethyl-2-pyrrolidinone-Substituted Flavan-3-ols from the Leaves of Camellia sinensis var. pubilimba Meng XH, Zhu HT, Yan H, Wang D, Yang CR, Zhang YJ Ref: Journal of Agricultural and Food Chemistry, 66:7150, 2018 : PubMed
Camellia sinensis var. pubilimba, one variety of the genus Camellia sect. Thea (Theaceae), has been used for producing green tea mainly by the local people of its growing areas of Guangxi province, China. Forty compounds, including eight C-8 N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (1-8) and their substituted unit N-ethyl-5-hydroxy-2-pyrrolidinone (9), four flavan-3-ol monomers (10-13) and one dimer (14), nine flavonoids (15-23), three hydrolyzable tannins (24-26), two lignans (27-28), 11 simple phenolics (29-39), and caffeine (40), were first isolated and identified from the leaves. Their structures were determined by detailed spectroscopic analysis and comparison with the literature data and authentic samples. Both 1 and 4 were obtained as a mixture of the N-ethyl-2-pyrrolidinone C-5 enantiomers (1a and 1b and 4a and 4b), respectively, while the resolution of another three pairs of enantiomers (2 and 3, 5 and 6, and 7 and 8) was achieved. Among them, 1b is a new compound whose NMR data together with its enantiomer (1a) were reported for the first time, while 2 and 3 are two new natural products. Most of the isolates exhibited significant antioxidant activities, stronger than ascorbic acid and trolox, while parts of the isolates, particularly C-8 N-ethyl-2-pyrrolidinone-substituted flavan-3-ols, showed obvious inhibitory effects on acetylcholinesterase (AChE). The results indicated that C. sinensis var. pubilimba is a valuable plant resource for tea production.
To what extent the genomes of different species belonging to one genus can be diverse and the relationship between genomic differentiation and environmental factor remain unclear for oceanic bacteria. With many new bacterial genera and species being isolated from marine environments, this question warrants attention. In this study, we sequenced all the type strains of the published species of Glaciecola, a recently defined cold-adapted genus with species from diverse marine locations, to study the genomic diversity and cold-adaptation strategy in this genus.The genome size diverged widely from 3.08 to 5.96 Mb, which can be explained by massive gene gain and loss events. Horizontal gene transfer and new gene emergence contributed substantially to the genome size expansion. The genus Glaciecola had an open pan-genome. Comparative genomic research indicated that species of the genus Glaciecola had high diversity in genome size, gene content and genetic relatedness. This may be prevalent in marine bacterial genera considering the dynamic and complex environments of the ocean. Species of Glaciecola had some common genomic features related to cold adaptation, which enable them to thrive and play a role in biogeochemical cycle in the cold marine environments.
Title: Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport Gu SH, Wu KM, Guo YY, Pickett JA, Field LM, Zhou JJ, Zhang YJ Ref: BMC Genomics, 14:636, 2013 : PubMed
BACKGROUND: One of the challenges in insect chemical ecology is to understand how insect pheromones are synthesised, detected and degraded. Genome wide survey by comparative sequencing and gene specific expression profiling provide rich resources for this challenge. A. ipsilon is a destructive pest of many crops and further characterization of the genes involved in pheromone biosynthesis and transport could offer potential targets for disruption of their chemical communication and for crop protection. RESULTS: Here we report 454 next-generation sequencing of the A. ipsilon pheromone gland transcriptome, identification and expression profiling of genes putatively involved in pheromone production, transport and degradation. A total of 23473 unigenes were obtained from the transcriptome analysis, 86% of which were A. ipsilon specific. 42 transcripts encoded enzymes putatively involved in pheromone biosynthesis, of which 15 were specifically, or mainly, expressed in the pheromone glands at 5 to 120-fold higher levels than in the body. Two transcripts encoding for a fatty acid synthase and a desaturase were highly abundant in the transcriptome and expressed more than 40-fold higher in the glands than in the body. The transcripts encoding for 2 acetyl-CoA carboxylases, 1 fatty acid synthase, 2 desaturases, 3 acyl-CoA reductases, 2 alcohol oxidases, 2 aldehyde reductases and 3 acetyltransferases were expressed at a significantly higher level in the pheromone glands than in the body. 17 esterase transcripts were not gland-specific and 7 of these were expressed highly in the antennae. Seven transcripts encoding odorant binding proteins (OBPs) and 8 encoding chemosensory proteins (CSPs) were identified. Two CSP transcripts (AipsCSP2, AipsCSP8) were highly abundant in the pheromone gland transcriptome and this was confirmed by qRT-PCR. One OBP (AipsOBP6) were pheromone gland-enriched and three OBPs (AipsOBP1, AipsOBP2 and AipsOBP4) were antennal-enriched. Based on these studies we proposed possible A. ipsilon biosynthesis pathways for major and minor sex pheromone components. CONCLUSIONS: Our study identified genes potentially involved in sex pheromone biosynthesis and transport in A. ipsilon. The identified genes are likely to play essential roles in sex pheromone production, transport and degradation and could serve as targets to interfere with pheromone release. The identification of highly expressed CSPs and OBPs in the pheromone gland suggests that they may play a role in the binding, transport and release of sex pheromones during sex pheromone production in A. ipsilon and other Lepidoptera insects.
Title: Genome sequence of the protease-producing bacterium Rheinheimera nanhaiensis E407-8T, isolated from deep-sea sediment of the South China Sea Zhang XY, Zhang YJ, Qin QL, Xie BB, Chen XL, Zhou BC, Zhang YZ Ref: Journal of Bacteriology, 194:7001, 2012 : PubMed
The protease-producing bacterium E407-8(T) was isolated from deep-sea sediment of the South China Sea and has been identified recently as representing a new species, Rheinheimera nanhaiensis. The draft genome of R. nanhaiensis E407-8(T) consists of 3,987,205 bp and contains 3,730 predicated protein-coding genes, including 82 extracellular peptidase genes.
Deep-sea sediment is one of the most important microbial-driven ecosystems, yet it is not well characterized. Genome sequence analyses of deep-sea sedimentary bacteria would shed light on the understanding of this ecosystem. In this study, the complete genome of deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913 (SM9913) is described and compared with that of the closely related Antarctic surface sea-water ecotype Pseudoalteromonas haloplanktis TAC125 (TAC125). SM9913 has fewer dioxygenase genes than TAC125, indicating a possible sensitivity to reactive oxygen species. Accordingly, experimental results showed that SM9913 was less tolerant of H(2)O(2) than TAC125. SM9913 has gene clusters related to both polar and lateral flagella biosynthesis. Lateral flagella, which are usually present in deep-sea bacteria and absent in the related surface bacteria, are important for the survival of SM9913 in deep-sea environments. With these two flagellar systems, SM9913 can swim in sea water and swarm on the sediment particle surface, favoring the acquisition of nutrients from particulate organic matter and reflecting the particle-associated alternative lifestyle of SM9913 in the deep sea. A total of 12 genomic islands were identified in the genome of SM9913 that may confer specific features unique to SM9913 and absent from TAC125, such as drug and heavy metal resistance. Many signal transduction genes and a glycogen production operon were also present in the SM9913 genome, which may help SM9913 respond to food pulses and store carbon and energy in a deep-sea environment.
Title: Phenolic compounds from the whole plants of Gentiana rhodantha (Gentianaceae) Xu M, Zhang M, Wang D, Yang CR, Zhang YJ Ref: Chem Biodivers, 8:1891, 2011 : PubMed
Gentiana rhodantha Franch. ex Hemsl. (Gentianaceae), an annual herb widely distributed in the southwest of China, has been medicinally used for the treatment of inflammation, cholecystitis, and tuberculosis by the local people of its growing areas. Chemical investigation on the whole plants led to the identification of eight new phenolic compounds, rhodanthenones A-D (1-4, resp.), apigenin 7-O-glucopyranosyl-(1-->3)-glucopyranosyl-(1-->3)-glucopyranoside (5), 1,2-dihydroxy-4-methoxybenzene 1-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (6), 1,2-dihydroxy-4,6-dimethoxybenzene 1-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (7), and methyl 2-O-beta-D-glucopyranosyl-2,4,6-trihydroxybenzoate (8), together with eleven known compounds, 9-19. Their structures were determined on the basis of detailed spectroscopic analyses and chemical methods. Acetylcholinesterase (AChE) inhibition and cytotoxicity tests against five human cancer cell lines showed that only rhodanthenone D (4) and mangiferin (12) exhibited 18.4 and 13.4% of AChE inhibitory effects at a concentration of 10(-4) M, respectively, while compounds 1-5 and the known xanthones lancerin (11), mangiferin (12), and neomangiferin (13) displayed no cytotoxicity at a concentration of 40 muM.
