Abstract
Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 β-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.
Highlights
Hemicellulose is the second-most abundant structural carbohydrate and accounts for approximately 20% to 30% of the dry weight of the plant cell wall
Sequence analysis revealed that the selected colony contains an ORF of 978 bp (Figure 1B), a 152 bp 5′ untranslated region (UTR), and a 365 bp 3′ UTR (Figure 1B), accounting for 1,494 bp of Acetyl xylan esterase (AXE) sequence and thereby predicting as a nearly full-length cDNA of N. frontalis
The absence of an intron in the DNA sequence of this NfAXE1 suggests the potential for horizontal gene transfer from prokaryotes to anaerobic fungi in the rumen
Summary
Hemicellulose is the second-most abundant structural carbohydrate and accounts for approximately 20% to 30% of the dry weight of the plant cell wall. The main component of its structural backbone is xylan which consists of β-1,4linked xylopyranosyl residues with various substituted side groups, such as arabinose and O-acetyl, and ferulic (4hydroxy-3-methoxycinnamic), p-coumaric, L-arbinofuranosyl, and 4-O-methylglucuronyl residues (Thomson, 1993). Coutinho and Henrissat (1999) classified carbohydrate esterases (CE) into 14 groups, with AXEs classified into CE families 1 to 7 based on significant sequence diversity. This sequence diversity implies functional diversity, including diverse substrate
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