Abstract
Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80°C and pH 6.5, as 75°C and pH 6.5 for Agu67A. Xyn10A had good stability at 75°C, 80°C, and pH 4.5–8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.
Highlights
Xylan, the main hemicellulose component of plant cell wall, is a heteropolymeric polysaccharide consisted mostly of linear backbone of b-1,4-D-xylopyranoside units which are commonly decorated with 4-O-methyl-glucuronyl, acetyl, and arabinofuranosyl substituents [1,2]
Xyn10A had the closest relationship with T. saccharolyticum xylanase, and clustered with Alicyclobacillus sp. xylanase as a single evolutionary clade which was distinct from other thermophilic bacteria and fungi
In the reaction of Xyn10A&Agu67A applied together, the produced xylose and xylobiose was increased compared with that of enzyme added separately, indicating the synergism of the two enzymes. All these results clearly showed that Xyn10A acted on both xylan polymer and XOs, and liberate a large number of xylose and xylobiose, indicating Xyn10A was active on XOs with DP $3
Summary
The main hemicellulose component of plant cell wall, is a heteropolymeric polysaccharide consisted mostly of linear backbone of b-1,4-D-xylopyranoside units which are commonly decorated with 4-O-methyl-glucuronyl, acetyl, and arabinofuranosyl substituents [1,2]. The efficient depolymerization of xylan to monosaccharides requires the synergistic function of enzyme system, including endo-b-1,4xylanase (EC 3.2.1.8), b-xylosidase (EC 3.2.1.37), a-L-arabinofuranosidase (EC 3.2.1.55), a-glucuronidase (EC 3.2.1.139), and acetyl xylan esterase (EC 3.2.1.72) [1,3]. Endo-b-1,4-xylanases catalyze the random cleavage of the internal b-1,4-glycosidic linkage between xylose residues in xylan polymer, and have been classified into glycoside hydrolase (GH) families 5, 7, 8, 10, 11 and 43 [4]. The a-glucuronidases (EC 3.2.1.139) cleave the a-1,2linkage between 4-O-methylglucuronic acid (4-O- MeGlcA) and XOs [5]. A large number of xylanolytic enzymes have been identified from a variety of microbial sources
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