Abstract
BackgroundXylan, the major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-l-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes.ResultsHere, we cloned an acetyl xylan esterase (PcAxe) from Penicillium chrysogenum P33 and expressed it in Pichia pastoris GS115. The optimal pH and temperature of the recombinant PcAxe (rPcAxe) for 4-nitrophenyl acetate were 7.0 and 40 °C, respectively. rPcAxe is stable across a broad pH range, retaining 100% enzyme activity om pH 6–9 after a 1 h incubation. The enzyme tolerates the presence of a wide range of metal ions. Sequence alignment revealed a GH62 domain exhibiting α-l-arabinofuranosidase activity with pH and temperature optima of pH 7.0 and 50 °C, in addition to the expected esterase domain. rPcAxe displayed significant synergy with a recombinant xylanase, with a degree of synergy of 1.35 for the hydrolysis of delignified corn stover. Release of glucose was increased by 51% from delignified corn stover when 2 mg of a commercial cellulase was replaced by an equivalent amount of rPcAxe, indicating superior hydrolytic efficiency.ConclusionsThe novel bifunctional enzyme PcAxe was identified in P. chrysogenum P33. rPcAxe includes a carbohydrate esterase domain and a glycosyl hydrolase family 62 domain. This is the first detailed report on a novel bifunctional enzyme possessing acetyl xylan esterase and α-l-arabinofuranosidase activities. These findings expand our current knowledge of glycoside hydrolases and pave the way for the discovery of similar novel enzymes.
Highlights
Xylan, the major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups
Compared with enzymes possessing a single function, multifunctional enzymes can hydrolyze a variety of different substrates simultaneously, which reduces the amount of enzyme types needed for the hydrolysis of lignocellulose, decreases their cost in biotechnological processes, and has additional advantages
The relative activity of recombinant PcAxe (rPcAxe) was maintained at 95.8 and 92.7% in the presence of 5 mM Mn2+ and Fe2+, respectively. rPcAxe could maintain a relative activity of more than 80% with the presence of most of the tested metal ions except F e3+ (10 mM). This resistance to metal ions is advantageous in industrial applications, since metal ions are inevitably present in industrial production. rPcAxe displayed optimal α-larabinofuranosidase activity at pH 7, which is typical for bacterial arabinofuranosidases [24, 36], and the same was true for its acetyl xylan esterase activity, which differs from PcAxe2 from P. chrysosporium [12] and most other fungal arabinofuranosidases [37]
Summary
The major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-l-arabinofuranosidases are two important accessory enzymes that remove side chain residues from the hemicellulose backbone and function synergistically with other xylanolytic enzymes [4, 5]. Compared with enzymes possessing a single function, multifunctional enzymes can hydrolyze a variety of different substrates simultaneously, which reduces the amount of enzyme types needed for the hydrolysis of lignocellulose, decreases their cost in biotechnological processes, and has additional advantages. In this sense, multifunctional enzymes are more intriguing than traditional enzymes, especially those involved in heteroxylan hydrolysis [6, 7]. MTCC5214 and Lactobacillus sp. based on the elution profile and zymogram analysis [13], a bifunctional enzyme possessing acetyl xylan esterase and α-l-arabinofuranosidase activities has not been studied in detail, and would be potentially useful for hydrolyzing lignocellulose
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