scholarly journals Optimization of enzyme cascades for the in vitro synthesis of hyaluronic acid

2018 ◽  
Vol 90 (9) ◽  
pp. 1291-1291
Author(s):  
J. Gottschalk ◽  
A. Eisele ◽  
L. Elling
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Synthesis ◽  
Enzyme Cascades

In vitro synthesis of glycosaminoglycans in endocrine ophthalmopathy

1992 ◽  
Vol 127 (5) ◽  
pp. 397-402 ◽  
Author(s):  
G Kahaly ◽  
C Stover ◽  
J Beyer ◽  
E Otto
Keyword(s):  
Hyaluronic Acid ◽  
Acid Concentration ◽  
Cetylpyridinium Chloride ◽  
Acid Synthesis ◽  
Cell Mediated Immunity ◽  
Glycosaminoglycan Synthesis ◽  
In Vitro Synthesis ◽  
Significant Difference ◽  
Endocrine Ophthalmopathy

The effects of humoral and cell-mediated immunity on the glycosaminoglycan synthesis of retrobulbar fibroblasts was evaluated in patients with endocrine ophthalmopathy. After incubation with IgG and sera, secreted glycosaminoglycans, radiolabeled with D-6-3H-glucosamine and 35sulfate, were precipitated with cetylpyridinium chloride and ethanol. Hyaluronic acid synthesis of human retrobulbar fibroblasts after incubation with sera and IgG and after co-culture with lymphocytes was assessed by means of a radiometric test. Patients' IgG, compared to controls', accounted for a higher secretory stimulation of porcine retrobulbar fibroblasts (as measured by cetylpyridinium chloride precipitation) after 24 and 48 h. Contrasting with 24 h incubation time, glycosaminoglycan values after 48 h were increased two to threefold. Patients' and controls' sera caused earlier and stronger, yet indistinguishable glycosaminoglycan production. Non-sulfated hyaluronic acid was the preponderant glycosaminoglycan secreted into the media by retrobulbar fibroblasts. As assessed with the radiometric test, incubation with patients' and controls' sera and IgG did not reveal a significant difference in stimulating the hyaluronic synthesis of patients' and controls' retrobulbar fibroblasts. When measuring the hyaluronic acid synthesis of controls' and patients' retrobulbar fibroblasts after co-cultivation of lymphocytes, however, patients' lymphocytes had a marked ability to increase the hyaluronic acid concentration compared to controls' lymphocytes. The hyaluronic acid concentration after incubation of a patient's retrobulbar fibroblasts with autologous lymphocytes was markedly more elevated than the intrinsic hyaluronic acid production of retrobulbar fibroblasts. In conclusion, though a significant in vitro influence of patients' IgG and sera on the glycosaminoglycan release of both porcine and human (patients' as well as controls') retrobulbar fibroblasts could not be observed in this study, the indications of a marked stimulatory influence of lymphocytes on the hyaluronic acid secretion of retrobulbar fibroblasts demand further investigation.

scholarly journals Multi‐enzyme cascades for the in vitro synthesis of guanosine diphosphate L‐fucose

ChemCatChem ◽  
2020 ◽  
Author(s):  
Reza Mahour ◽  
Pavel A. Marichal-Gallardo ◽  
Thomas Rexer ◽  
Udo Reichl
Keyword(s):  
In Vitro Synthesis ◽  
Guanosine Diphosphate ◽  
Enzyme Cascades

scholarly journals Key Factors for A One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid

2019 ◽  
Vol 20 (22) ◽  
pp. 5664 ◽  
Author(s):  
Gottschalk ◽  
Zaun ◽  
Eisele ◽  
Kuballa ◽  
Elling
Keyword(s):  
Molecular Weight ◽  
Hyaluronic Acid ◽  
Pasteurella Multocida ◽  
Ph Value ◽  
Key Factors ◽  
One Pot ◽  
Data Set ◽  
In Vitro Synthesis ◽  
Enzyme Cascade

In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to produce defined HA. Here we present a one-pot enzyme cascade with six enzymes for the synthesis of HA from the cheap monosaccharides glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). The combination of two enzyme modules, providing the precursors UDP–GlcA and UDP–GlcNAc, respectively, with hyaluronan synthase from Pasteurella multocida (PmHAS), was optimized to meet the kinetic requirements of PmHAS for high HA productivity and molecular weight. The Mg2+ concentration and the pH value were found as key factors. The HA product can be tailored by different conditions: 25 mM Mg2+ and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-NaOH pH 8 result into an HA product with high Mw HA (1.55 MDa) and low dispersity (1.05). Whereas with 15 mM Mg2+ and HEPES–NaOH pH 8.5, we reached the highest HA concentration (2.7 g/L) with a yield of 86.3%. Our comprehensive data set lays the basis for larger scale enzymatic HA synthesis.

scholarly journals Nucleotide Sugars in Chemistry and Biology

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5755
Author(s):  
Satu Mikkola
Keyword(s):  
Drug Development ◽  
Building Blocks ◽  
Synthesis Methods ◽  
Bacterial Cells ◽  
Nucleotide Sugar ◽  
In Vitro Synthesis ◽  
Nucleotide Sugars ◽  
Potential Inhibitors ◽  
Enzyme Cascades

Nucleotide sugars have essential roles in every living creature. They are the building blocks of the biosynthesis of carbohydrates and their conjugates. They are involved in processes that are targets for drug development, and their analogs are potential inhibitors of these processes. Drug development requires efficient methods for the synthesis of oligosaccharides and nucleotide sugar building blocks as well as of modified structures as potential inhibitors. It requires also understanding the details of biological and chemical processes as well as the reactivity and reactions under different conditions. This article addresses all these issues by giving a broad overview on nucleotide sugars in biological and chemical reactions. As the background for the topic, glycosylation reactions in mammalian and bacterial cells are briefly discussed. In the following sections, structures and biosynthetic routes for nucleotide sugars, as well as the mechanisms of action of nucleotide sugar-utilizing enzymes, are discussed. Chemical topics include the reactivity and chemical synthesis methods. Finally, the enzymatic in vitro synthesis of nucleotide sugars and the utilization of enzyme cascades in the synthesis of nucleotide sugars and oligosaccharides are briefly discussed.

In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling

2005 ◽  
Vol 32 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Dieter Hoffmann ◽  
Bernd Buchberger ◽  
Cordula Nemetz
Keyword(s):  
In Vitro Synthesis ◽  
Phenotypic Resistance ◽  
Hiv 1
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