Cloning, expression, purification and enzymatic characterization of AfCDA gene from Anoxybacillus flavitheermus WK1

Family 4 carbohydrate esterases deacetylate polymeric carbohydrate substrates such as chitin, acetyl xylan and peptidoglycan. Although some of these enzymes have recently been enzymologically characterized, they have been limited number of study thermophilic bacteria. All members of this enzyme family have a characteristic homolog domain into NodB.The genome project of Anoxybacillus flavithermus WK1 was determined in 2008. When the all genome was screened for carbohydrate deacetylase gene, two genes were determined as candidate. One of these genes is 762 bp long and encodes a protein of 252 aminoacids in length. This gene was amplified by standard PCR to clone into NdeI site of pET-15b expression vector to express with six histidine residues. Recombinant plasmid was transformed into E. coli (Escherichia coli) BL21 (DE3)pLysS cell and expressed. The recombinant protein was purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. Activity of recombinant enzyme was shown on SDS-PAGE by using ?-naphtyl acetate as a substrate and Fast Red. The enzyme was characterized by spectrophotometrically using chromogenic substrate-p-nitrophenyl acetate. Optimum temperature and pH were determined 50 °C and 7.5 respectively. Km and Vmax were determined 4,33 µM and 3333,33 U/mg respectively.Phylogenetic similarity of Anoxybacillus flavithermus WK1 to other species was investigated by using nucleotide and amino acids sequences. Phylogenetic dendogram clearly demonstrated that Anoxybacillus flavithermus WK1 is different from the other species. afcda gene aminoacid sequences were used to find conserved motifs with comparison of other known members of CE-4 family . Five conserved motifs were investigated and all motifs were found as conserved except motif 2.It was shown that the AfCDA enzyme from Anoxybacillus flavithermus WK1 does not need any metal ions to show activity.