Discrimination and classification of tobacco wastes by identification and quantification of polyphenols with LC–MS/MS

The chemical composition of polyphenols in tobacco waste was identified by HPLC-PDA–ESI/MS/MS and the contents of chlorogenic acids and rutin in 10 varieties of tobacco wastes were determined by HPLC–UV. The relationships between the contents of active polyphenols and the varieties of tobacco wastes were interpreted by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The results showed that 15 polyphenols were identified in a methanolic extract of dried tobacco waste. The tobacco wastes were characterized by high levels of chlorogenic acids (3-CQA, 5-CQA, and 4-CQA) and rutin; their ranges in the 10 tobacco varieties were 0.116–0.196, 0.686–1.781, 0.094–0.192, and 0.413–0.998 %, respectively. According to multivariate statistics models, two active compound variables can be considered important for the discrimination of the varieties of tobacco wastes: chlorogenic acids and rutin. Consequently, samples of 10 tobacco varieties were characterized into three groups by HCA based on the PCA pattern. In conclusion, tobacco waste could be used as a new pharmaceutical material for the production of natural chlorogenic acids and rutin in the ethnopharmacological industry

Investigation of the kinetics and mechanism of the glycerol chlorination reaction using gas chromatography–mass spectrometry

As a primary by-product in biodiesel production, glycerol can be used to prepare an important fine chemical, epichlorohydrin, by the glycerol chlorination reaction. Although this process has been applied in industrial production, unfortunately, less attention has been paid to the analysis and separation of the compounds in the glycerol chlorination products. In this study, a convenient and accurate method to determine the products in glycerol chlorination reaction was established and based on the results the kinetic mechanism of the reaction was investigated. The structure of main products, including 1,3--dichloropropan-2-ol, 2,3-dichloropropan-1-ol, 3-chloro-1,2-propanediol, 2-chloro-1,3-propanediol and glycerol was ascertained by gas chromatography–mass spectrometry and the isomers of the products were distinguished. Apidic acid was considered as the best catalyst because of its excellent catalytic effect and high boiling point. The mechanism of the glycerol chlorination reaction was proposed and a new kinetic model was developed. Kinetic equations of the process in the experimental range were obtained by data fitting and the activation energies of each tandem reaction were 30.7, 41.8, 29.4 and 49.5 kJ mol-1, respectively. This study revealed the process and mechanism of the kinetics and provides the theoretical basis for engineering problems

Ultrasonic Effects on Protein Salting-out

The addition of inorganic salts to a bovine serum albumin (BSA) colloid to separate protein is a commonly performed method. In this work, we investigate an enhanced ultrasound process for protein separation. The standing time is 4.5 h shorter than that without ultrasound irradiation. The protein can be acquired at a yield of 90.5 % under the conditions of a 20 kHz ultrasound frequency, 60 min of centrifugal separation, and 2 minutes of ultrasound irradiation with a 0.64 W cm–2 sound intensity. Influences of sound field factors such as frequency, sound intensity and irradiation time on this process are discussed. The mechanism of ultrasonic salting-out of protein is construed. Results show that the settling velocity of protein can be accelerated by ultrasound irradiation after the salting-out process

Engineering a Microbial Consortium Based Whole-Cell System for Efficient Production of Glutarate From L-Lysine

Glutarate is an important C5 platform chemical produced during the catabolism of L-lysine through 5-aminovalerate (5-AMV) pathway. Here, we first established a whole-cell biocatalysis system for the glutarate production from L-lysine with the engineered Escherichia coli (E. coli) that co-expressed DavAB and GabDT. However, the accumulation of intermediate 5-AMV was identified as one important factor limiting glutarate production. Meanwhile, the negative interaction of co-expressing DavAB and GabDT in a single cell was also confirmed. Here, we solved these problems through engineering a microbial consortium composed of two engineered E. coli strains, BL21-22AB and BL21-YDT, as the whole-cell biocatalysts, each of which contains a part of the glutarate pathway. After the optimization of bioconversion conditions, including temperature, metal ion additives, pH, and cell ratio, 17.2 g/L glutarate was obtained from 20 g/L L-lysine with a yield of 95.1%, which was improved by 19.2% compared with that in a single cell. Little accumulation of 5-AMV was detected. Even at the high substrate concentration, the reduced 5-AMV accumulation and increased glutarate production were achieved. This synthetic consortium produced 43.8 g/L glutarate via a fed-batch strategy, the highest titer reported to date

One-pot biosynthesis of N-acetylneuraminic acid from chitin via combination of chitin-degrading enzymes, N-acetylglucosamine-2-epimerase, and N-neuraminic acid aldolase

N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources

A Novel Process for Cadaverine Bio-Production Using a Consortium of Two Engineered Escherichia coli

Bio-production of cadaverine from cheap carbon sources for synthesizing bio-based polyamides is becoming more common. Here, a novel fermentation process for cadaverine bio-production from glucose was implemented by using a microbial consortium of two engineered Escherichia coli strains to relieve the toxic effect of cadaverine on fermentation efficiency. To achieve controllable growth of strains in the microbial consortium, two engineered E. coli strains grown separately on different carbon sources were first constructed. The strains were, an L-lysine-producing E. coli NT1004 with glucose as carbon source, and a cadaverine-producing E. coli CAD03 with glucose metabolism deficiency generated by modifying the PTSGlc system with CRISPR-Cas9 technology and inactivating cadaverine degradation pathways. Co-culturing these two engineered E. coli strains with a mixture of glucose and glycerol led to successful production of cadaverine. After optimizing cultivation conditions, a cadaverine titer of 28.5 g/L was achieved with a multi-stage constant-speed feeding strategy

Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis process

N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the equilibrium favoring Neu5Ac cleavage, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a “GRAS” (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor Neu5Ac synthesis the most. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition

Polymer/boron nitride nanosheet composite with high thermal conductivity and sufficient dielectric strength

An efficient method was reported to fabricate boron nitride (BN) nanosheets using a sonication-centrifugation technique in DMF solvent. Then non-covalent functionalization and covalent functionalization of BN nanosheets were performed by octadecylamine (ODA) and hyperbranched aromatic polyamide (HBP), respectively. Then, three different types of epoxy composites were fabricated by incorporation of BN nanosheets, BN-ODA, and BN-HBP. Among all three epoxy composites, the thermal conductivity and dielectric strength of epoxy composites using BN-HBP nanosheets display the highest value, efficiently enhancing to 9.81W/mK at 50vol% and 34.8kV/mm at 2.7vol% (increase by 4057% and 9.4% compared with the neat epoxy), respectively. The significantly improved thermal conductivity and dielectric strength are attributed to the large surface area, which increases the contact area between nanosheets and nanosheets, as well as enhancement of the interfacial interaction between nanosheets and epoxy matrix. Copyright (c) 2015 John Wiley & Sons, Ltd

Enhancing discharged energy density and suppressing dielectric loss of poly(vinylidene fluoride-ter-trifluoroethylene-ter-chlorofluoroethylene) by a sandwiched structure

Polymer dielectrics with high energy density and low dielectric loss are highly desired due to the rapid development of electric devices. Among known polymers, poly(vinylidene fluoride-ter-trifluoroethylene-ter-chlorofluoroethylene) P(VDF-TrFE-CFE) is one of the promising materials for energy storage capacitor applications because of its high dielectric constant. Nevertheless, it suffers from high dielectric loss especially at the high electric field, which suppresses its breakdown strength and energy storage density. Herein, sandwiched structure dielectric films were fabricated by employing polymethyl methacrylate (PMMA) as the outer layer and P(VDF-TrFE-CFE) as the central layer. By modulating the thickness of the central layer, an enhanced discharged energy density of 7.03 J/cm^3 is achieved at a high electric field of 480 MV/m, which is 132% more than that of P(VDF-TrFE-CFE) at its maximum electric field 300 MV/m. Meanwhile, this sandwiched structure film also retains a high discharge efficiency of 78% at 480 MV/m, which is never been seen in polyvinylidene fluoride-based polymers. Results show that PMMA acts as charge barrier and simultaneously enhance the breakdown strength and suppress the dielectric loss of P(VDF-TrFE-CFE)