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Cloning, Purification and Characterization of Acetyl Xylane Esterase from Anoxybacillus flavithermus DSM 2641T with Activity on Low Molecular-Weight Acetates

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Abstract

Family 4 carbohydrate esterases (CE-4) have deacetylate different forms of acetylated poly/oligosaccharides in nature. This family is recognized with a specific polysaccharide deacetylase domain assigned as NodB homology domain in their secondary structure. Most family 4 carbohydrate esterases have been structurally and biochemically characterized. However, this is the first study about the enzymological function of pdaB-like CE4s from thermophilic bacterium Anoxybacillus flavithermus DSM 2641T. A. flavithermus WK1 genome harbors five putative CE4 family genes. One of them is 762 bp long and encodes a protein of 253 amino acids in length and it was used as reference sequence in this study. It was described as acetyl xylane esterase (AXE) in genome project and this AfAXE gene was amplified without signal sequence and cloned. The recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. The activity of the recombinant enzyme was shown by zymogram analysis with α-naphtyl acetate as a substrate. The enzyme was characterized spectrophotometrically using chromogenic p-nitrophenyl acetate. Optimum temperature and pH were determined as 50 °C and 7.5, respectively. Km and Vmax were determined as 0.43 mM and 3333.33 U/mg, respectively under optimum conditions. To our knowledge this is the first enzymological characterization of a pdaB-like family 4 carbohydrate esterase from the members of Anoxybacillus genus.

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Abbreviations

AXE:

Acetyl xylan esterase

p-NPAc:

para-Nitrophenyl acetate

p-NP:

para-Nitrophenol

α-NAc:

α-Naphtyl acetate

pdaA :

peptidoglycan N-acetylmuramic acid deacetylase A

pdaB :

A polysaccharide deacetylase Gene B

Af :

Anoxybacillus flavithermus

CE4:

Carbohydrate esterases 4

NodB :

Nodulation protein B

MurNAc:

N-acetylmuramic acid

GlcNAc:

N-acetylglucosamine

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Acknowledgments

This work was supported by Recep Tayyip Erdogan University Research Fund Grants BAP- 2011.102.03.3 and BAP- 2012.102.03.4.

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  1. Department of Biology, Molecular Biology Research Laboratory, Recep Tayyip Erdogan University, 53100, Fener, Rize, Turkey

    Ayşenur Eminoğlu, Serdar Ülker & Cemal Sandallı

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  1. Ayşenur Eminoğlu
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  2. Serdar Ülker
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  3. Cemal Sandallı
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Correspondence to Cemal Sandallı.

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Eminoğlu, A., Ülker, S. & Sandallı, C. Cloning, Purification and Characterization of Acetyl Xylane Esterase from Anoxybacillus flavithermus DSM 2641T with Activity on Low Molecular-Weight Acetates. Protein J 34, 237–242 (2015). https://doi.org/10.1007/s10930-015-9618-x

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  • DOI: https://doi.org/10.1007/s10930-015-9618-x

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