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

Eminoğlu A., Ulker S., Sandallı C.

PROTEIN JOURNAL, vol.34, no.4, pp.237-242, 2015 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 34 Issue: 4
  • Publication Date: 2015
  • Doi Number: 10.1007/s10930-015-9618-x
  • Journal Name: PROTEIN JOURNAL
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.237-242
  • Keywords: pdaB-like CE4 superfamily, Acetyl xylan esterase, Anoxybacillus flavithermus DSM2641(T), Enzymatic characterization, CHITIN DEACETYLASE, CARBOHYDRATE ESTERASE, ENZYMES, GENE, MECHANISM, REVEALS
  • Recep Tayyip Erdoğan University Affiliated: No


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 2641(T). 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 alpha-naphtyl acetate as a substrate. The enzyme was characterized spectrophotometrically using chromogenic p-nitrophenyl acetate. Optimum temperature and pH were determined as 50 A degrees 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.