title: High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli.
abstract: N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained.
authors: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Electronic address: l.klermund@lrz.tum.de.
authors: Ludwig
authors: L
authors: Klermund
authors: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Electronic address: amelie.riederer@tum.de.
authors: Amelie
authors: A
authors: Riederer
authors: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Electronic address: a.groher@lrz.tum.de.
authors: Anna
authors: A
authors: Groher
authors: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Electronic address: k.castiglione@lrz.tum.de.
authors: Kathrin
authors: K
authors: Castiglione
mesh_terms: D046870:Anabaena variabilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase
keywords: Chaperones; Epimerization; N-acetylneuraminic acid; N-acyl-d-glucosamine 2-epimerase; Sialic acid; Soluble expression