N-Acetyl-glucosamine fatty acid esters were synthesized by a lipase-catalyzed transesterification with methyl hexanoate and N-acetyl-glucosamine (GlcNAc), which resulted in the formation of 2-(acetylamino)-2-deoxy-6-O-hexanoate-D-glucose, a novel glycolipid. Additionally N-butyryl-glucosamine (GlcNBu) was used for a similar synthesis, leading to the formation of 2-(butyrylamino)-2-deoxy-6-O-hexanoate-D-glucose. The higher hydrophobicity of GlcNBu led to an increase in the overall yield and the initial reaction rate when compared to the reaction with GlcNAc. By pre-dissolving GlcNAc and GlcNBu in dimethyl sulfoxide (DMSO), it was possible to completely dissolve both sugars in the organic solvent, thus further enhancing the initial reaction rate and yield respectively.Practical applications: Glycolipids are used in a wide range of applications, ranging from food, cosmetic, and pharmaceutical formulations, where they can be used as emulsifiers or foaming agents to classic cleaning products, utilizing their good detergent properties. Further applications may include fields like membrane protein extraction, bioremediation, or tertiary oil recovery. Novel glycolipids with tailor-made properties might be useful to improve any of the named applications and widen the diversity of available environmentally friendly surfactants, often termed "green surfactants." Glycolipids are the most prominent example therefrom.
Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.
A novel substrate, 6-(4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (pNO2PheDU), was chemically synthesized and analytically verified for the potential biocatalytic synthesis of enantiopure β-amino acids. The hydantoinase (EC 3.5.2.2) from Arthrobacter crystallopoietes DSM20117 was chosen to prove the enzymatic hydrolysis of this substrate, since previous investigations showed activities of this enzyme toward 6-monosubstituted dihydrouracils. Whole cell biotransformations with recombinant Escherichia coli expressing the hydantoinase showed degradation of pNO2PheDU. Additionally, the corresponding N-carbamoyl-β-amino acid (NCarbpNO2βPhe) was chemically synthesized, an HPLC-method with chiral stationary phases for detection of this product was established and thus (S)-enantioselectivity toward pNO2PheDU has been shown. Consequently this novel substrate is a potential precursor for the enantiopure β-amino acid para-nitro-β-phenylalanine (pNO2βPhe).Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0174-8) contains supplementary material, which is available to authorized users.
The hydantoinase process is applied for the industrial synthesis of optically pure amino acids via whole cell biocatalysis, providing a simple and well-established method to obtain the catalyst. Nevertheless, whole cell approaches also bear disadvantages like intracellular degradation reactions, transport limitations as well as low substrate solubility. In this work the hydantoinase and carbamoylase from Arthrobacter crystallopoietes DSM 20117 were investigated with respect to their applicability in a cell-free hydantoinase process. Both enzymes were heterologously expressed in Escherichia coli BL21DE3. Cultivation and induction of the hydantoinase under oxygen deficiency resulted in markedly higher specific activities and a further increase in expression was achieved by codon-optimization. Further expression conditions of the hydantoinase were tested using the microbioreactor system BioLector®, which showed a positive effect upon the addition of 3% ethanol to the cultivation medium. Additionally, the hydantoinase and carbamoylase were successfully purified by immobilized metal ion affinity using Ni Sepharose beads as well as by functionalized magnetic beads, while the latter method was clearly more effective with respect to recovery and purification factor. Immobilization of both enzymes via functionalized magnetic beads directly from the crude cell extract was successful and resulted in specific activities that turned out to be much higher than those of the purified free enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0420-3) contains supplementary material, which is available to authorized users.
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