Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus

Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h

Effective Enzyme Immobilization onto a Magnetic Chitin Nanofiber Composite

In this study, a novel biocompatible magnetic chitin nanofiber composite (MCNC) was developed as a support for enzyme immobilization, and the enzyme-immobilizing ability was elucidated using chymotrypsin (CT) as a model enzyme. Chitin nanofibers (CNFs) were prepared via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation of chitin and then further modified with magnetic nanoparticles. Glutaraldehyde was used to cross-link the additional CT molecules and aggregate them onto the MCNCs. The CNFs were characterized by transmission electron microscopy, and Fourier transform infrared spectroscopy. The results showed that the CNFs were properly formed and that the CT molecules immobilized on the MCNCs presented excellent properties. After heating the composites at 60 °C for 3 h, the non-cross-linked and cross-linked immobilized CTs retained 51.6% and 70.7% of the initial activity, respectively, whereas the free CTs retained only 29.6% of the initial activity. In addition, non-cross-linked and cross-linked immobilized CTs retained 85.7% and 84.9% of the initial activity, respectively, after 20 days, whereas the free CTs retained only 18.8% of the initial activity. When the MCNCs were used to immobilize the CT molecules, the enzyme loading capacity was enhanced up to 6.3-fold upon cross-linking. Moreover, the immobilized CTs could be easily separated and recycled from the reaction system by a magnetic force

Gastric Protective Activities of Sea Cucumber Fucoidans with Different Molecular Weight and Chain Conformations: A Structure–Activity Relationship Investigation

A variety of bioactivities has been established for fucoidan extracted from sea cucumber, whereas its structure–activity relationships have been seldom investigated. In this study, sea cucumber (<i>Thelenota ananas</i>) fucoidans with different molecular weights were prepared with enzymatic degradation. The chain stiffness and molecular size decreased with the decreasing of molecular weight. Fucoidans with molecular weight of 1380.0, 828.7, and 483.0 kDa exhibited random coil conformations, while fucoidan molecular weight of 215.0 kDa existed as sphere in solution. All examined fucoidans could effectively prevent the ethanol-induced gastric ulcer, of which mechanism involved antioxidation and anti-inflammation. Within the range of the study, the performance of fucoidans adopted random coil conformations declined with the decreasing of molecular weight, and the performance recovered when the chain conformation transited from coil to sphere, indicating the subtle influences of molecular weight and chain conformation on the gastric protective activity of sea cucumber fucoidan

Effective Astaxanthin Extraction from Wet <i>Haematococcus pluvialis</i> Using Switchable Hydrophilicity Solvents

A novel approach based on switchable hydrophilicity solvents (SHS) was developed for extracting astaxanthin from wet microalgae. Dimethylaminocyclohexane (DMCHA) was used to extract astaxanthin from wet Haematococcus pluvialis, and the extraction efficiency reached 87.2%. Astaxanthin was recovered from the DMCHA without distillation by simply adding H₂O and CO₂

Coimmobilization of β‑Agarase and α‑Neoagarobiose Hydrolase for Enhancing the Production of 3,6-Anhydro‑l‑galactose

Here we report a simple and efficient method to produce 3,6-anhydro-l-galactose (l-AHG) and agarotriose (AO3) in one step by a multienzyme system with the coimmobilized β-agarase AgWH50B and α-neoagarobiose hydrolase K134D. K134D was obtained by AgaWH117 mutagenesis and showed improved thermal stability when immobilized via covalent bonds on functionalized magnetic nanoparticles. The obtained multienzyme biocatalyst was characterized by Fourier transform infrared spectroscopy (FTIR). Compared with free agarases, the coimmobilized agarases exhibited a relatively higher agarose-to-l-AHG conversion efficiency. The yield of l-AHG obtained with the coimmobilized agarases was 40.6%, which was 6.5% higher than that obtained with free agarases. After eight cycles, the multienzyme biocatalyst still preserved 46.4% of the initial activity. To the best of our knowledge, this is the first report where two different agarases were coimmobilized. These results demonstrated the feasibility of the new method to fabricate a new multienzyme system onto magnetic nanoparticles via covalent bonds to produce l-AHG

Glycosaminoglycanomics of Cultured Cells Using a Rapid and Sensitive LC-MS/MS Approach

Glycosaminoglycans (GAGs), a family of polysaccharides widely distributed in eukaryotic cells, are responsible for a wide array of biological functions. Quantitative disaccharide compositional analysis is one of the primary ways to characterize the GAG structure. This structural analysis is typically time-consuming (1–2 weeks) and labor intensive, requiring GAG recovery and multistep purification, prior to the enzymatic/chemical digestion of GAGs, and finally their analysis. Moreover, 10⁵–10⁷ cells are usually required for compositional analysis. We report a sensitive, rapid, and quantitative analysis of GAGs present in a small number of cells. Commonly studied cell lines were selected based on phenotypic properties related to the biological functions of GAGs. These cells were lysed using a commercial surfactant reagent, sonicated, and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-aminoacridone, and analyzed by liquid chromatography (LC)-mass spectrometry (MS). Using a highly sensitive MS method, multiple reaction monitoring (MRM), the limit of detection for each disaccharide was reduced to 0.5–1.0 pg, as compared with 1.0–5.0 ng obtained using standard LC-MS analysis. Sample preparation time was reduced to 1–2 days, and the cell number required was reduced to 5000 cells for complete GAG characterization to as few as 500 cells for the characterization of the major GAG disaccharide components. Our survey of the glycosaminoglycanomes of the 20 selected cell lines reveals major differences in their GAG amounts and compositions. Structure–function relationships are explored using these data, suggesting the utility of this method in cellular glycobiology

Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats

<p>We investigated whether fatty liver preceded insulin resistance or vice versa using a long-term orotic acid (OA)-induced nonalcoholic fatty liver disease (NAFLD) model without the confounding effects of obesity and hyperlipidemia and explored the role of the liver in insulin resistance. Male Wistar rats were fed with or without OA supplementation for 30, 60, and 90 days. The NAFLD group showed increased liver lipid at 30, 60, and 90 days; glucose intolerance was noted at 60 and 90 days. Furthermore, partial liver proteins and gene expressions related to upstream signaling of insulin were decreased. However, the liver glycogen content was elevated, and gluconeogenesis genes expressions were obviously decreased at 90 days. The occurrence of fatty liver preceded insulin resistance in OA-induced NAFLD without the interference of obesity and hyperlipidemia, and hepatic insulin resistance may not play a conclusive role in insulin resistance in this model.</p> <p>Whether fatty liver preceded insulin resistance (IR) or vice versa in a long-term orotic acid-induced NAFLD model without the confounding effects of obesity and hyperlipidemia.</p

Sialoglycoprotein Isolated from Eggs of Carassius auratus Ameliorates Osteoporosis: An Effect Associated with Regulation of the Wnt/β-Catenin Pathway in Rodents

In the current study, ovariectomized (OVX) rats and the senescence-accelerated mouse strain P6 (SAMP6) were employed to establish models of postmenopausal osteoporosis and senile osteoporosis, respectively. The effects of treatment with sialoglycoprotein isolated from the eggs of Carassius auratus (<i>Ca</i>-SGP) on these two types of osteoporosis were investigated in vivo. Results showed that <i>Ca</i>-SGP significantly increased bone mineral density, ameliorated trabecular bone microstructure, and improved bone biomechanical properties in both OVX rats and SAMP6. The osteogenesis related Wnt/β-catenin pathway was targeted to study the underlying mechanism of <i>Ca</i>-SGP activity. In postmenopausal osteoporosis, <i>Ca</i>-SGP suppressed the activation of Wnt/β-catenin signal via down-regulating the expression of key genes including LRP5, β-catenin, and Runx2, suggesting that overactive osteogenesis was controlled by <i>Ca</i>-SGP. The bone formation was sharply weakened in senile osteoporosis, whereas <i>Ca</i>-SGP treatment promoted osteoblast activity by stimulating the Wnt/β-catenin signal. In conclusion, <i>Ca</i>-SGP ameliorated these two types of osteoporosis by normalizing bone anabolism

Identification of a Novel Esterase from Marine Environmental Genomic DNA Libraries and Its Application in Production of Free All-<i>trans</i>-Astaxanthin

Astaxanthin is a pigment with various functions. Free astaxanthin is obtained mainly through saponification methods, which could result in many byproducts. Enzymatic methods using lipases have been used in a few cases, while there are no reports on the use of esterases for the production of free astaxanthin. Herein we present the screening and identification of a novel esterase (Est3-14) from a marine mud metagenomic library. Est3-14 is pH-sensitive and keeps good stability in alkaline buffers (residual activity 94%, pH 8.0, 4 °C, and 36 h). Meanwhile, Est3-14 keeps a good stability in the medium temperature condition (residual activity 56.7%, pH 8.0, 40 °C, and 84 h). Est3-14 displayed high hydrolysis activity to prepare free all-<i>trans</i>-astaxanthin in biphasic systems. Furthermore, under optimal conditions (0.5 mL ethanol, 6 mL 0.1 M Tris-HCl buffer, pH 8.0, 0.5% (w/v) <i>H. pluvialis</i> oil, 40 °C), the hydrolytic conversion ratio was 99.3% after 36 h

Bottom-Up Low Molecular Weight Heparin Analysis Using Liquid Chromatography-Fourier Transform Mass Spectrometry for Extensive Characterization

Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-<i>O</i>-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production