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A novel bifunctional GH51 exo-α-l-arabinofuranosidase/endo-xylanase from Alicyclobacillus sp. A4 with significant biomass-degrading capacity.

Yang W, Bai Y, Yang P, Luo H, Huang H, Meng K, Shi P, Wang Y, Yao B - Biotechnol Biofuels (2015)

Bottom Line: Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually.It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts.The distinguished synergy might be ascribed to its bifunctional α-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.

ABSTRACT

Background: Improving the hydrolytic performance of xylanolytic enzymes on arabinoxylan is of importance in the ethanol fermentation industry. Supplementation of debranching (arabinofuranosidase) and depolymerizing (xylanase) enzymes is a way to address the problem. In the present study, we identified a bifunctional α-l-arabinofuranosidase/endo-xylanase (Ac-Abf51A) of glycoside hydrolase family 51 in Alicyclobacillus sp. strain A4. Its biochemical stability and great hydrolysis efficiency against complex biomass make it a potential candidate for the production of biofuels.

Results: The gene encoding Ac-Abf51A was cloned. The comparison of its sequence with reference proteins having resolved 3D-structures revealed nine key residues involved in catalysis and substrate-binding interaction. Recombinant Ac-Abf51A produced in Escherichia coli showed optimal activity at pH 6.0 and 60 °C with 4-nitrophenyl-α-l-arabinofuranoside as the substrate. The enzyme exhibited an exo-type mode of action on polyarabinosides by catalyzing the cleavage of α-1,2- and α-1,3-linked arabinofuranose side chains in sugar beet arabinan and water-soluble wheat arabinoxylan and α-1,5-linked arabinofuranosidic bonds in debranched sugar beet arabinan. Surprisingly, it had capacity to release xylobiose and xylotriose from wheat arabinoxylan and was active on xylooligosaccharides (xylohexaose 1.2/mM/min, xylopentaose 6.9/mM/min, and xylotetraose 19.7/mM/min), however a lower level of activity. Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually.

Conclusions: This study for the first time reports a GH51 enzyme with both exo-α-l-arabinofuranosidase and endo-xylanase activities. It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts. The distinguished synergy might be ascribed to its bifunctional α-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings.

No MeSH data available.


Related in: MedlinePlus

Amino acid sequence alignment of Ac-Abf51A from Alicyclobacillus sp. A4 with CelA4 from Alicyclobacillus sp. A4 (ADI82825.1) and other GH51 Abfs from Bifidobacterium longum B667 (PDB: 2Y2 W), Geobacillus stearothermophilus T6 (PDB: 1PZ3), Clostridium thermocellum ATCC 27405 (PDB: 2C7F), Thermobacillus xylanilyticus D3 (PDB: 2VRK), Thermotoga maritima MSB8 (PDB: 3UG3), and Thermotoga petrophila RKU-1 (PDB: 3S2C), using the ClustalW program. Identical and similar amino acids are indicated by black and gray shades, respectively. The catalytic glutamate residues are indicated by asterisk, and the conserved residues are indicated by hash
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Fig1: Amino acid sequence alignment of Ac-Abf51A from Alicyclobacillus sp. A4 with CelA4 from Alicyclobacillus sp. A4 (ADI82825.1) and other GH51 Abfs from Bifidobacterium longum B667 (PDB: 2Y2 W), Geobacillus stearothermophilus T6 (PDB: 1PZ3), Clostridium thermocellum ATCC 27405 (PDB: 2C7F), Thermobacillus xylanilyticus D3 (PDB: 2VRK), Thermotoga maritima MSB8 (PDB: 3UG3), and Thermotoga petrophila RKU-1 (PDB: 3S2C), using the ClustalW program. Identical and similar amino acids are indicated by black and gray shades, respectively. The catalytic glutamate residues are indicated by asterisk, and the conserved residues are indicated by hash

Mentions: The full-length Abf gene, Ac-abf51A (GenBank accession no.KT781102), contains 1509 bp and encodes a 502-residue polypeptide with a calculated molecular mass of 56.7 kDa. The deduced amino acid sequence of Ac-Abf51A is most similar to a putative Abf of Alicyclobacillus hesperidum (99 % identity; WP_006446014.1), and 68 % identical with the crystal structure-resolved Abf from G. stearothermophilus T6 (1PZ3). Using the Accelrys Discovery Studio software with 1PZ3 as the template, modeled Ac-Abf51A folds into two modules: the N-terminal catalytic module of the frequently encountered (α/β)8 barrel (TIM barrel) of GH51 and the C-terminal module of 12-stranded β-sandwich with a jelly-roll topology (Additional file 1). The structure of the (α/β)8 barrel domain places Ac-Abf51A into the superfamily of clan GH-A, in which two conserved glutamates (Glu175 and Glu294) were found on strands β-4 (acid/base) and β-7 (nucleophile), respectively (Fig. 1). Based on homology analysis of amino acid sequences with the characterized GH51 Abf with crystal structure [17], nine key residues including Glu29, Arg69, Asn74, Asn174, Glu175, His244, Tyr246, Glu294, and Gln351 are conserved and probably involved in catalysis and substrate-binding interaction (Fig. 1).Fig. 1


A novel bifunctional GH51 exo-α-l-arabinofuranosidase/endo-xylanase from Alicyclobacillus sp. A4 with significant biomass-degrading capacity.

Yang W, Bai Y, Yang P, Luo H, Huang H, Meng K, Shi P, Wang Y, Yao B - Biotechnol Biofuels (2015)

Amino acid sequence alignment of Ac-Abf51A from Alicyclobacillus sp. A4 with CelA4 from Alicyclobacillus sp. A4 (ADI82825.1) and other GH51 Abfs from Bifidobacterium longum B667 (PDB: 2Y2 W), Geobacillus stearothermophilus T6 (PDB: 1PZ3), Clostridium thermocellum ATCC 27405 (PDB: 2C7F), Thermobacillus xylanilyticus D3 (PDB: 2VRK), Thermotoga maritima MSB8 (PDB: 3UG3), and Thermotoga petrophila RKU-1 (PDB: 3S2C), using the ClustalW program. Identical and similar amino acids are indicated by black and gray shades, respectively. The catalytic glutamate residues are indicated by asterisk, and the conserved residues are indicated by hash
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666033&req=5

Fig1: Amino acid sequence alignment of Ac-Abf51A from Alicyclobacillus sp. A4 with CelA4 from Alicyclobacillus sp. A4 (ADI82825.1) and other GH51 Abfs from Bifidobacterium longum B667 (PDB: 2Y2 W), Geobacillus stearothermophilus T6 (PDB: 1PZ3), Clostridium thermocellum ATCC 27405 (PDB: 2C7F), Thermobacillus xylanilyticus D3 (PDB: 2VRK), Thermotoga maritima MSB8 (PDB: 3UG3), and Thermotoga petrophila RKU-1 (PDB: 3S2C), using the ClustalW program. Identical and similar amino acids are indicated by black and gray shades, respectively. The catalytic glutamate residues are indicated by asterisk, and the conserved residues are indicated by hash
Mentions: The full-length Abf gene, Ac-abf51A (GenBank accession no.KT781102), contains 1509 bp and encodes a 502-residue polypeptide with a calculated molecular mass of 56.7 kDa. The deduced amino acid sequence of Ac-Abf51A is most similar to a putative Abf of Alicyclobacillus hesperidum (99 % identity; WP_006446014.1), and 68 % identical with the crystal structure-resolved Abf from G. stearothermophilus T6 (1PZ3). Using the Accelrys Discovery Studio software with 1PZ3 as the template, modeled Ac-Abf51A folds into two modules: the N-terminal catalytic module of the frequently encountered (α/β)8 barrel (TIM barrel) of GH51 and the C-terminal module of 12-stranded β-sandwich with a jelly-roll topology (Additional file 1). The structure of the (α/β)8 barrel domain places Ac-Abf51A into the superfamily of clan GH-A, in which two conserved glutamates (Glu175 and Glu294) were found on strands β-4 (acid/base) and β-7 (nucleophile), respectively (Fig. 1). Based on homology analysis of amino acid sequences with the characterized GH51 Abf with crystal structure [17], nine key residues including Glu29, Arg69, Asn74, Asn174, Glu175, His244, Tyr246, Glu294, and Gln351 are conserved and probably involved in catalysis and substrate-binding interaction (Fig. 1).Fig. 1

Bottom Line: Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually.It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts.The distinguished synergy might be ascribed to its bifunctional α-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.

ABSTRACT

Background: Improving the hydrolytic performance of xylanolytic enzymes on arabinoxylan is of importance in the ethanol fermentation industry. Supplementation of debranching (arabinofuranosidase) and depolymerizing (xylanase) enzymes is a way to address the problem. In the present study, we identified a bifunctional α-l-arabinofuranosidase/endo-xylanase (Ac-Abf51A) of glycoside hydrolase family 51 in Alicyclobacillus sp. strain A4. Its biochemical stability and great hydrolysis efficiency against complex biomass make it a potential candidate for the production of biofuels.

Results: The gene encoding Ac-Abf51A was cloned. The comparison of its sequence with reference proteins having resolved 3D-structures revealed nine key residues involved in catalysis and substrate-binding interaction. Recombinant Ac-Abf51A produced in Escherichia coli showed optimal activity at pH 6.0 and 60 °C with 4-nitrophenyl-α-l-arabinofuranoside as the substrate. The enzyme exhibited an exo-type mode of action on polyarabinosides by catalyzing the cleavage of α-1,2- and α-1,3-linked arabinofuranose side chains in sugar beet arabinan and water-soluble wheat arabinoxylan and α-1,5-linked arabinofuranosidic bonds in debranched sugar beet arabinan. Surprisingly, it had capacity to release xylobiose and xylotriose from wheat arabinoxylan and was active on xylooligosaccharides (xylohexaose 1.2/mM/min, xylopentaose 6.9/mM/min, and xylotetraose 19.7/mM/min), however a lower level of activity. Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually.

Conclusions: This study for the first time reports a GH51 enzyme with both exo-α-l-arabinofuranosidase and endo-xylanase activities. It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts. The distinguished synergy might be ascribed to its bifunctional α-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings.

No MeSH data available.


Related in: MedlinePlus