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New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate

Paper ID Volume ID Publish Year Pages File Format Full-Text
23480 43441 2013 8 PDF Available
Title
New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate
Abstract

•Four novel N-acyl-d-glucosamine 2-epimerases from cyanobacteria were investigated.•The epimerase from Anabaena variabilis ATCC 29413 was the best enzyme.•It displayed the highest catalytic efficiency ever reported with N-acetyl-d-glucosamine.•It showed a much weaker inhibition by pyruvate than comparable enzymes.•It exhibited exceptionally high activity without allosteric activation by ATP.

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg−1 at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the Km for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.

Keywords
AGE, N-acyl-d-glucosamine 2-epimerase; GlcNAc, N-acetyl-d-glucosamine; ManNAc, N-acetyl-d-mannosamine; NAL, N-acetylneuraminate lyase; Neu5Ac, N-acetylneuraminic acid; TCA, trichloroacetic acidN-Acetylneuraminic acid; N-Acetyl-d-mannosamine; N-Acyl-d-gluc
First Page Preview
New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 168, Issue 3, November 2013, Pages 256–263
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering