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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

Characterization of the purified recombinant Ac-Abf51A with 4-nitrophenyl-α-L-arabinofuranoside as the substrate. a The effect of pH on enzyme activity. The activity assays were performed at 60 °C in buffers of pH 3.0–10.0 for 10 min. b pH stability of Ac-Abf51A. After pre-incubating the enzyme at 37 °C for 1 h at pH 3.0–12.0, the residual activities were measured in 100 mM McIlvaine buffer (pH 6.0, 60 °C and 10 min). c The effect of temperature on enzyme activity measured in 100 mM McIlvaine buffer (pH 6.0) for 10 min. d Thermostability of purified Ac-Abf51A. The enzyme was pre-incubated at 70 °C or 80 °C in 100 mM McIlvaine buffer (pH 6.0) for various periods, followed by activity assay at pH 6.0 and 60 °C for 10 min
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Fig3: Characterization of the purified recombinant Ac-Abf51A with 4-nitrophenyl-α-L-arabinofuranoside as the substrate. a The effect of pH on enzyme activity. The activity assays were performed at 60 °C in buffers of pH 3.0–10.0 for 10 min. b pH stability of Ac-Abf51A. After pre-incubating the enzyme at 37 °C for 1 h at pH 3.0–12.0, the residual activities were measured in 100 mM McIlvaine buffer (pH 6.0, 60 °C and 10 min). c The effect of temperature on enzyme activity measured in 100 mM McIlvaine buffer (pH 6.0) for 10 min. d Thermostability of purified Ac-Abf51A. The enzyme was pre-incubated at 70 °C or 80 °C in 100 mM McIlvaine buffer (pH 6.0) for various periods, followed by activity assay at pH 6.0 and 60 °C for 10 min

Mentions: With 4-nitrophenyl-α-l-arabinofuranoside as the substrate, Ac-Abf51A exhibited the highest Abf activity at pH 6.0 (Fig. 3a). The enzyme retained more than 80 % of the initial activity after incubation at pH 4.0–11.0, 37 °C for 1 h (Fig. 3b). When assayed at pH 6.0, Ac-Abf51A was most active at 60 °C (Fig. 3c). The enzyme retained full activity after 12-h incubation at 60 °C and 65 °C, more than 80 % of the initial activity at 70 °C for 1 h, and more than 30 % of the activity at 80 °C for 30 min (Fig. 3d).Fig. 3


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)

Characterization of the purified recombinant Ac-Abf51A with 4-nitrophenyl-α-L-arabinofuranoside as the substrate. a The effect of pH on enzyme activity. The activity assays were performed at 60 °C in buffers of pH 3.0–10.0 for 10 min. b pH stability of Ac-Abf51A. After pre-incubating the enzyme at 37 °C for 1 h at pH 3.0–12.0, the residual activities were measured in 100 mM McIlvaine buffer (pH 6.0, 60 °C and 10 min). c The effect of temperature on enzyme activity measured in 100 mM McIlvaine buffer (pH 6.0) for 10 min. d Thermostability of purified Ac-Abf51A. The enzyme was pre-incubated at 70 °C or 80 °C in 100 mM McIlvaine buffer (pH 6.0) for various periods, followed by activity assay at pH 6.0 and 60 °C for 10 min
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Characterization of the purified recombinant Ac-Abf51A with 4-nitrophenyl-α-L-arabinofuranoside as the substrate. a The effect of pH on enzyme activity. The activity assays were performed at 60 °C in buffers of pH 3.0–10.0 for 10 min. b pH stability of Ac-Abf51A. After pre-incubating the enzyme at 37 °C for 1 h at pH 3.0–12.0, the residual activities were measured in 100 mM McIlvaine buffer (pH 6.0, 60 °C and 10 min). c The effect of temperature on enzyme activity measured in 100 mM McIlvaine buffer (pH 6.0) for 10 min. d Thermostability of purified Ac-Abf51A. The enzyme was pre-incubated at 70 °C or 80 °C in 100 mM McIlvaine buffer (pH 6.0) for various periods, followed by activity assay at pH 6.0 and 60 °C for 10 min
Mentions: With 4-nitrophenyl-α-l-arabinofuranoside as the substrate, Ac-Abf51A exhibited the highest Abf activity at pH 6.0 (Fig. 3a). The enzyme retained more than 80 % of the initial activity after incubation at pH 4.0–11.0, 37 °C for 1 h (Fig. 3b). When assayed at pH 6.0, Ac-Abf51A was most active at 60 °C (Fig. 3c). The enzyme retained full activity after 12-h incubation at 60 °C and 65 °C, more than 80 % of the initial activity at 70 °C for 1 h, and more than 30 % of the activity at 80 °C for 30 min (Fig. 3d).Fig. 3

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