Insight into Glycoside Hydrolases for Debranched Xylan Degradation from Extremely Thermophilic Bacterium Caldicellulosiruptor lactoaceticus

et al. (2014) Insight into Glycoside Hydrolases for Debranched Xylan Degradation from Extremely Thermophilic Bacterium Caldicellulosiruptor lactoaceticus. PLoS ONE 9(9): e106482. doi:10.1371/journal.pone.0106482 Insight into Glycoside Hydrolases for Debranched Xylan Degradation from Extremely Thermophilic Bacterium Caldicellulosiruptor lactoaceticus Xiaojing Jia 0 Shuofu Mi 0 Jinzhi Wang 0 Weibo Qiao 0 Xiaowei Peng 0 Yejun Han 0 Jose M. Sanchez-Ruiz, Universidad de Granada, Spain 0 1 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , Beijing , China , 2 Institute of Agro-food Science and Technology, Chinese Academy of Agricultural Sciences , Beijing , China , 3 College of Biosciences and Biotechnology, Shenyang Agricultural University , Shenyang , China 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 a-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 80uC and pH 6.5, as 75uC and pH 6.5 for Agu67A. Xyn10A had good stability at 75uC, 80uC, 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. - Funding: This work was funded by the National High Technology Research and Development Program of China (863 Project, No. 2014AA021905), and 100 Talents Program of Institute of Process Engineering, Chinese Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. In a general sense, 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]. Unlike xylanases, a-glucuronidases cluster into either GH family 67 or family 115 based on amino acid sequences [1,6]. To date, a large number of xylanolytic enzymes have been identified from a variety of microbial sources (CAZy; http://www. cazy.org/). Thermostable hemicelluloytic enzymes, with a number of advantages over mesophilic enzymes, have thus gained worldwide industrial and biotechnological interest. Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic, cellulose and hemicelluloses degrading bacterium, was isolated from an alkaline hot spring in Iceland [7]. It grows efficiently at temperature between 50 and 78uC and pH 5.88.2 within optimum near 68uC and 7.0, respectively. Besides xylan, it utilizes cellulose, starch, pectin, cellobiose, xylose, maltose and lactose as carbon sources. Thus its ability to express highly thermostable carbohydrate-active enzymes makes it an ideal candidate for studying extreme temperature biomass conversion. Currently, the complete genome sequence was available for this species [8], providing new approach to investigate the mechanisms of polysaccharides degradation. The genes encoding thermophilic GHs of C. lactoaceticus 6A provide a platform for degrading natural polysaccharides at higher temperature. In present study, thermophilic degradation of MeGlcA decorated xylan using C. lactoaceticus 6A GHs was studied. The genes of endo-b-1,4-xylanase Xyn10A and a-glucuronidase Agu67A were identified and cloned in the genome of C. lactoaceticus 6A, and heterologously expressed, purified and biochemically characterized. The synergistically hydrolytic properties of the two enzymes on MeGlcA decorated xylan and XOs were also investigated. Results and Discussion Gene cloning and sequence analysis of Xyn10A and Agu67A Through C. lactoaceticus genome sequence analysis, Calla_1331 and Calla_1259 were annotated as putative GH10 endo-b-1,4xylanase (Xyn10A) and a-glucuronidase (Agu67A), respectively. Both Xyn10A and Agu67A had no signal peptide, indicating they are intracellular enzymes. Xyn10A only contained a GH10 motif (Figure 1A), the calculated molecular weight (Mw) and deduced pI of Xyn10A were 46,965 Da and 5.65, respectively. The encoding gene xyn10A was amplified using C. lactoaceticus DNA as template. In genes screening analysis in genome DNA of C. lactoaceticus, no other xylan degradation genes except a putative polysaccharide deacetylase upstream of xyn10A was found (Figure 1B). In amino acids sequence blast analysis, Xyn10A showed high identity with other predicted xylanases from Caldicellulosiruptor sp. In addition to Caldicellulosiruptor, Xyn10A exhibited the highest similarity (79.7%) and identity (65.1%) with Thermoanaerobacterium saccharolyticum GH10 xylanase [GenBank: ADQ57411.2], and similarity (71.5%) and identity (57.8%) with GH10 xylanase from Alicyclobacillus sp. A4 [GenBank: ADK91076.1]. However, Xyn10A showed much lower similarity (27.041.8%) and identity (14.423.5%) with other characterized thermophilic GH10 xy (...truncated)