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

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.

A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor owensensis with potential applications in production of biofuels and biochemicals

“…These results indicate that CoGH1A has resistance to cations and additional cation is not necessary for activating the enzyme. The effects of cation on CoGH1A were similar to those on some other glycoside hydrolases from Caldicellulosiruptor species, such as the xylanase from C. kronotskyensis [ 22 ] and the xylanase and xylosidase from C. owensensis [ 12 ]. They were very different from the β-galactosidase produced by Lactobacillus delbrueckii [ 23 ].…”

“…Research efforts have recently focused on extremely thermophilic microorganisms for exploring novel cellulases and other GHs to improve the current situation [ 5 – 7 ], due to the advantages of these thermophilic enzymes, such as higher reaction velocity, excellent thermostability, and decreased risk of contamination [ 8 ]. Many thermophilic GHs, such as β- d -glucosidase [ 9 , 10 ], bifunctional cellulolytic enzyme (endo- and exoglucanases) [ 11 ], β- d -xylosidase [ 12 ], and β- d -galactosidase [ 3 , 13 ], have been cloned, heterologously expressed, and biochemically characterized for the purpose of uncovering the catalytic mechanism and evaluating the possibility of industrial applications. Among them, the genus Caldicellulosiruptor has recently attracted high interest for it can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass [ 7 , 14 , 15 ].…”

Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

“…strain 41M-1 and Xyn10B (Protein Data Bank entry 2W5F; sequence identity, 29%) from Clostridium thermocellum, respectively, as the template. Quaternary structures of XynA and XynB were investigated using size exclusion chromatography with a Superdex 200 column (36,37). Blue dextran 2000 (2,000 kDa), ferritin (400 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (all obtained from Sigma-Aldrich) were used as reference proteins.…”

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries

“…It is worth noting that the hydrolysis of GH10-XA alone accumulated xylobiose, confirming that it was a bad substrate, and a third peak released later than 4-O-methyl-glucuronic acids and possibly corresponding to aldouronic acids. It is worth mentioning that GH10-XA and GH67-GC acted in synergy extremely well: if compared to the GH10 and GH67 enzymes from C. lactoaceticus [59], they produced the same amounts of reducing ends from beechwood xylan in 2 h vs 12 h and at much less enzyme concentrations (7-and 9-fold for GH10 and GH67 enzymes, respectively). On the basis of these encouraging results, and to improve the biotransformation and the synergy of GH10-XA and GH67-GC on these substrates, the high-xylose tolerant ␤-xylosidase from Thermotoga thermarum (GH3-XT) [42] was added to the enzymatic mixture.…”

“…This is, at the best of our knowledge, the only ␣-glucuronidase studied so far that is able to recognize as substrate both glucuronoxylan oligomers and 4Np-GlcUA. In fact, most of the GH67 ␣-glucuronidases are only active on 4-O-MeGlcA linked to the non-reducing end of xylo-oligomers [29,30,59] and only few studies demonstrated their high activity against longer polymeric substrates [32,33]. By contrast, the Agu4A ␣-glucuronidase from Thermotoga maritima from family GH4, which efficiently hydrolysed 4Np-GlcUA, was unable to recognize gluronoxylans as substrates [28,75].…”