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

Background: Thermophilic enzymes have attracted much attention for their advantages of high reaction velocity, exceptional thermostability, and decreased risk of contamination. Exploring efficient thermophilic glycoside hydrolases will accelerate the industrialization of biofuels and biochemicals

The synergism of hot water pretreatment and enzymatic hydrolysis in depolymerization of lignocellulosic content of palm kernel cake

Palm kernel cake (PKC), mainly composed of mannan, lignin and protein, is abundant renewable resource with commercial value. To develop clean and efficient way for PKC refinery, the method based on the synergism of hot water pretreatment (HWP), steam pretreatment (SP) and enzymatic hydrolysis were developed. HWP of 180 degrees C, 20 min and SP of 121 degrees C, 20min showed similar performance for sugar release from PKC. The main saccharides produced from PKC by HWP and SP were mannose and manno-oligosaccharides, while no furfural formed. The surface structure analyzed by SEM showed that HWP enhanced the microporosity of PKC, and the accessibility of which was increased thereafter. When HWP pretreated PKC was further hydrolyzed with enzyme cocktail (cellulase, xylanase, endo-mannanase), 45% of PKC was solubilized compared with the control. The manno-oligosaccharides produced by HWP and SP were converted to mannose and mannobiose by endo-mannanase. The results suggested that both HWP and SP promote enzymatic hydrolysis of PKC by releasing oligosaccharides and enhancing microporosity, and the synergism of which was effective for PKC decomposition. (C) 2016 Elsevier B.V. All rights reserved.</p

Thermostable pectate lyase from Caldicellulosiruptor kronotskyensis provides an efficient addition for plant biomass deconstruction

To understand the enzymological basis for extremely thermophilic, biomass-degrading genus Caldicellulosiruptor metabolize pectin, a thermostable pectate lyase Pel-863 encoded by a gene cluster for hexose-containing polysaccharide metabolism in genome of C. kronotskyensis was studied. The pectate lyase of Caldicellulosiruptor was highly conserved and the representative Pel-863 was biochemically characterized, and the application for pectin containing biomass degradation was also studied. Pel-863 exhibited an optimal activity at 70 degrees C and pH 9.0 with Ca2+ as cofactor. It degraded polygalacturonic acid (PGA), methylated pectin and pectic biomass through endo-cleaving action. The respective V-max and K-m for Pel-863 were 172.8 U/mg and 0.60 g/L on PGA. FTIR and SEM analysis indicated that Pel-863 could remove most of pectin in hemp fiber with less damage compared to alkaline degumming. In addition, pre-digestion with Pel-863 improved glucose and xylose yield by 14.2% and 311.6% respectively for corn stalk, 6.5% and 55% for rice stalk compared with sole action of Novozymes Cellic CTec2. (C) 2015 Elsevier B.V. All rights reserved

J. Mol. Catal. B-Enzym.

The extreme thermophilic bacterium Caldicellulosiruptor kronotskyensis can use hemicelluloses and cellulose as carbohydrate source. The gene Calkro_0081 encoded a novel GH11 xylanase (Xyn11A) with a catalytic domain (GH-CD) and a carbohydrate binding module (CBM6). The native Xyn11A and two corresponded truncations Xyn11A-CD (catalytic domain of Xyn11A) and Xyn11A-CBM (carbohydrate binding module of Xyn11A) were respectively cloned, heterologously expressed, and biochemically characterized. The recombinant Xyn11A is active in a wide temperature range from 40 to 95 degrees C with the highest activity at 75 degrees C. Xyn11A was completely stable at 70 degrees C for 6 h and more than 50% activity was retained after incubation for 6 h at 75 degrees C. The optimum pH of Xyn11A was 6.0, and it retained 100% activity after 15 h incubation in pH 5.5-7.5 at 70 degrees C. As for Xyn11A-CD, the optimal pH value and temperature were 6.0 and 65 degrees C, the residual activity retained 30% after 6h at 60 degrees C. The respective specific activities of Xyn11A, Xyn11A-CD, and Xyn11A-CBM were 1752.0, 986.8, and 0 IU/mg on beechwood xylan (BWX) at optimum conditions. The activity of Xyn11A is the highest among the reported thermostable xylanases at 75 C. Using BWX as substrate, the final products were xylose and xylobiose after hydrolysis with Xyn11A or Xyn11A-CD. No hydrolytic activity of CBM6 was found, while which shows crucial functions on thermostability and activity for Xyn11A. The characteristics of thermostability and high activity make Xyn11A a potential enzyme for industry application. (C) 2014 Elsevier B.V. All rights reserved.The extreme thermophilic bacterium Caldicellulosiruptor kronotskyensis can use hemicelluloses and cellulose as carbohydrate source. The gene Calkro_0081 encoded a novel GH11 xylanase (Xyn11A) with a catalytic domain (GH-CD) and a carbohydrate binding module (CBM6). The native Xyn11A and two corresponded truncations Xyn11A-CD (catalytic domain of Xyn11A) and Xyn11A-CBM (carbohydrate binding module of Xyn11A) were respectively cloned, heterologously expressed, and biochemically characterized. The recombinant Xyn11A is active in a wide temperature range from 40 to 95 degrees C with the highest activity at 75 degrees C. Xyn11A was completely stable at 70 degrees C for 6 h and more than 50% activity was retained after incubation for 6 h at 75 degrees C. The optimum pH of Xyn11A was 6.0, and it retained 100% activity after 15 h incubation in pH 5.5-7.5 at 70 degrees C. As for Xyn11A-CD, the optimal pH value and temperature were 6.0 and 65 degrees C, the residual activity retained 30% after 6h at 60 degrees C. The respective specific activities of Xyn11A, Xyn11A-CD, and Xyn11A-CBM were 1752.0, 986.8, and 0 IU/mg on beechwood xylan (BWX) at optimum conditions. The activity of Xyn11A is the highest among the reported thermostable xylanases at 75 C. Using BWX as substrate, the final products were xylose and xylobiose after hydrolysis with Xyn11A or Xyn11A-CD. No hydrolytic activity of CBM6 was found, while which shows crucial functions on thermostability and activity for Xyn11A. The characteristics of thermostability and high activity make Xyn11A a potential enzyme for industry application. (C) 2014 Elsevier B.V. All rights reserved

Biochemical characterization of two thermostable xylanolytic enzymes encoded by a gene cluster of Caldicellulosiruptor owensensis.

The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (kcat/Km) of Coxyn A was 366 mg ml(-1) s(-1) on beechwood xylan, and the catalytic efficiency (kcat/Km) of Coxyl A was 2253 mM(-1) s(-1) on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro

PLoS One

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 alpha-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 degrees C and pH 6.5, as 75 degrees C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 degrees C, 80 degrees 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.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 alpha-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 degrees C and pH 6.5, as 75 degrees C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 degrees C, 80 degrees 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

Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment

Background: Pretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars

Biochemical characterization of extra- and intracellular endoxylanse from thermophilic bacterium Caldicellulosiruptor kronotskyensis

Caldicellulosiruptor kronotskyensis grows on lignocellulosic biomass by the catalysis of intrinsic glycoside hydrolase, and has potential application for consolidated bioprocessing. In current study, two predicted extra-(Xyn10A) and intracellular (Xyn10B) xylanase from C. kronotskyensis were comparatively characterized. Xyn10A and Xyn10B share GH10 catalytic domain with similarity of 41%, while the former contains two tandem N-terminus CBM22s. Xyn10A showed higher hydrolytic capability than Xyn10B on both beechwood xylan (BWX) and oat spelt xylan (OSX). Truncation mutation experiments revealed the importance of CBMs for hydrolytic activity, substrate binding and thermostability of Xyn10A. While the quantity of CBM was not directly related to bind and thermostability. Although CBM was considered to be crucial for substrate binding, Xyn10B and Xyn10A as well as truncations performed similar binding affinity to insoluble substrate OSX. Analysis of point mutation revealed similar key residues, Glu493, Glu601 and Trp658 for Xyn10A and Glu139, Glu247 and Trp305 for Xyn10B. Both Xyn10A and Xyn10B exhibited hydrolytic activity on the mechanical pretreated corncob. After pre-digested by Xyn10A or Xyn10B, the micropores in the the mechanical pretreated corncob were observed, which enhanced the accessibility for cellulase. Compared with corncob hydrolyzed with cellulase alone, enhanced hydrolytic performance of was observed after predigestion by Xyn10A or Xyn10B.</p

Temperature and pH profile of Coxyn A and Coxyl A.

<p><b>A</b> Effect of temperature on Coxyn A activity. <b>B</b> Effect of pH on Coxyn A activity. <b>C</b> Effect of temperature on Coxyl A activity. <b>D</b> Effect of pH on Coxyl A activity. <b>E</b> Effect of temperature on Coxyn A stability. <b>F</b> Effect of temperature on Coxyl A stability.</p