Abstract
BackgroundXylan is a major component of plant cells and the most abundant hemicellulose. Xylanases degrade xylan into monomers by randomly cleaving β-1,4-glycosidic bonds in the xylan backbone, and have widespread potential applications in various industries. The purpose of our study was to clone and express the endoxylanase gene xynA of Thermobifida fusca YX in its native form and with a C-terminal histidine (His) tag in Pichia pastoris X-33. We analyzed and compared these two forms of the protein and examined their potential applications in various industries.ResultsThe xynA gene from T. fusca YX was successfully cloned and expressed using P. pastoris X-33. We produced a recombinant native form of the protein (rXyn11A) and a C-terminal His-tagged form of the desired protein (rXyn11A-(His)6). The specific activities of rXyn11A and rXyn11A-(His)6 in culture supernatants approached 149.4 and 133.4 U/mg, respectively. These activities were approximately 4- and 3.5-fold higher than those for the non-recombinant wild-type Xyn11A (29.3 U/mg). Following purification, the specific activities of rXyn11A and rXyn11A-(His)6 were 557.35 and 515.84 U/mg, respectively. The specific activity of rXyn11A was 8% higher than that of rXyn11A-(His)6. Both recombinant xylanases were optimally active at 80°C and pH 8.0, and exhibited greater than 60% activity between pH 6–9 and 60–80°C. They exhibited similar pH stability, while rXyn11A exhibited better thermostability; N-glycosylation enhanced the thermostability of both recombinant xylanases. The products of beechwood xylan hydrolyzed by both xylanases included xylobiose, xylotriose, xylotetraose and xylopentaose.ConclusionsThe C-terminal His tag had adverse effects when added to the Xyn11A protein. The thermostability of both recombinant xylanases was enhanced by N-glycosylation. Their stabilities at a high pH and temperature indicate their potential for application in various industries.
Highlights
Xylan is a major component of plant cells and the most abundant hemicellulose
There were seven possible N-glycosylation sites (Asn-X-Ser/Thr) [33], with very little O-linked glycosylation observed in P. pastoris [34]
Following treatment with endo-H to remove carbohydrate moieties, a protein band was observed at about 32 kDa by SDS-PAGE (Figure 4). These findings suggested that N-glycosylation of recombinant Xyn11A (rXyn11A) and rXyn11A-(His)6 accounted for about 13 kDa
Summary
Xylan is a major component of plant cells and the most abundant hemicellulose. Xylanases degrade xylan into monomers by randomly cleaving β-1,4-glycosidic bonds in the xylan backbone, and have widespread potential applications in various industries. The major component in plant cells and the most abundant hemicellulose, is composed of β-1,4-linked xylopyranosyl residues [1,2]. Degrading all components of lignocellulose requires the synergistic activity of a large variety of enzymes with different specificities, such as exo-1,4-β-glucanases, endo-1,4-β-glucanases, β-glucosidases, endo-xylanases, pectin methyl esterases, and laccase [4]. Thermobifida fusca is a thermophilic actinomycete and a major degrader of plant cell walls in heated organic materials such as compost piles and rotting hay [16]. Xyn11A has thermostable properties, unique among family G xylanases, that are especially useful for industrial purposes [17]
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