Article pubs.acs.org/JAFC
Improved Expression and Characterization of a Multidomain Xylanase from Thermoanaerobacterium aotearoense SCUT27 in Bacillus subtilis Xiongliang Huang,† Zhe Li,† Chenyu Du,§ Jufang Wang,† and Shuang Li*,† †
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China § School of Applied Sciences, The University of Huddersfield, Queensgate, Huddersfield, United Kingdom ABSTRACT: A xylanase gene was cloned and characterized from Thermoanerobacterium aotearoense SCUT27, which was attested to consist of a signal peptide, one glycoside hydrolase family 10 domain, four carbohydrate binding modules, and three surface layer homology domains. The change of expression host from Escherichia coli to Bacillus subtilis resulted in a 4.1-fold increase of specific activity for the truncated XynAΔSLH. Five different versions of secretion signals in B. subtilis indicated that it was preferably routed via a Sec-dependent pathway. Purified XynAΔSLH showed a high activity of 379.8 U/mg on beechwood xylan. XynAΔSLH was optimally active at 80 °C, pH 6.5. Thin layer chromatography results showed that xylobiose and the presumed methylglucuronoxylotriose (MeGlcAXyl3) were the main products liberated from beechwood xylan catalyzed by the recombinant xylanase. All of the results suggest that XynAΔSLH is a suitable candidate for generating xylooligosaccharides from cellulosic materials for industrial uses. KEYWORDS: multidomain xylanase, Thermoanaerobacterium aotearoense SCUT27, high-level expression, thermostability, beechwood xylan
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and basic pI with a β-jelly roll structure. According to their molecular architectures, xylanases can be classified into two types: single-domain and multidomain enzymes (molecular weight >100 kDa). The multidomain xylanases have a modular structure that comprises a catalytic domain (CD), one or more carbohydrate-binding modules (CBMs), and domains with some other functions connected by relatively unstructured linker sequences.2 The CBMs, the most abundant noncatalytic domains in xylanases, may fold and function independently and can increase the rate of catalysis by bringing the biocatalyst into intimate association with the recalcitrant substrate.8 Actually, the enzyme was found to function more efficiently in carbohydrate degradation, and removing the CBM from the enzyme could dramatically decrease the enzyme activity.6 Previously, many single-domain xylanases from various microorganisms, including fungi,9 yeasts,10 and thermophilic bacteria11−13 have been successfully overexpressed in heterologous cells, whereas for the multidomain xylanase, studies were mainly focused on the sequence analysis and enzyme characterization.14−17 Little attention was paid to the efficient multidomain xylanase expression. However, it is desirable to exploit the industrial applications of the enzyme. Thermophilic microorganisms are a potential source of enzymes with novel properties as the enzymes originated from these organisms would function at higher temperature. Thermostable enzymes have obvious advantages as catalysts
INTRODUCTION Xylan is a major structure component of plant cell walls. It is the second most abundant polysaccharide in plants, accounting for approximately one-third of the renewable organic carbon on Earth.1−3 Xylan biodegradation involves the attacks of several hydrolytic enzymes, including two major enzymes, xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and β-xylosidase (exo-1,4-βxylosidase, EC 3.2.1.37).2,4 Xylanase randomly cleaves the internal linkages in xylan, yielding a mixture of xylooligosaccharides.5,6 The potential applications of xylanase cover a wide range of industrial sectors. Currently the most important biotechnological application of xylanase is in prebleaching of pulps to minimize the use of harsh chemicals in the subsequent treatment stages of the paper and pulp industry.6 Another important application of xylanase is its use in the bioconversion of lignocellulosic biomass into fermentative products for the generation of biofuels.2 Thermo- and acid-tolerant xylanase could be used as feed additive to increase the digestibility of animal feeds,1 whereas cold-active xylanase is applied in baking for improving bread quality by increasing specific bread volume.7 Due to the heterogeneity and complexity of xylan, a vast number of diverse xylanases exist in nature. On the basis of amino acid sequence similarity and hydrophobic cluster analysis of the catalytic domains, characterized xylanases (endo-1,4-βxylanase, EC 3.2.1.8) can be classified into glycoside hydrolase (GH) families 10, 11, and 30, especially GH10 and GH11 (http://www.cazy.org/Glycoside-Hydrolases.html). GH10 xylanases generally have a higher molecular weight (>30 kDa) and acidic pI with a (β/α)8 barrel structure, whereas members of GH11 typically have a lower molecular weight (400% when the Ca2+ existed in the reaction mixture.16 Substrate Specificity and Products of Xylan Degradation. Experiments were carried out to study the specificity of XynAΔSLH to various substrates. The XynAΔSLH had the highest specific activity of 379.8 ± 3.2 U/mg toward beechwood xylan (defined as 100%), followed by oat spelt xylan (213.8 ± 1.6 U/mg, 56.3%). No obvious catalytic activity was detected when CMC-Na was used as substrate by XynAΔSLH (16.4 ± 0.4 U/mg). These results are contrary to other previously reported results; for example, the endoxylanases isolated from T. thermarum16 and alkaline wastewater sludge17 showed maximum activity on birchwood xylan, and xylanase from G. thermoleovoranse had maximum activity on oat spelt xylan.13 Using xylooligosaccharides or beechwood xylan as substrate, the purified XynAΔSLH showed relatively uniform hydrolysis capability (Figure 6). Xylotetraose and xylopentaose could be easily hydrolyzed to xylobiose with a trace of xylose by XynAΔSLH in 30 min of catalysis. However, much of xylotriose was not thoroughly degraded to xylobiose after being incubated at 55 °C for 30 min. Moreover, hydrolysis of xylobiose was not observed during the experiment. To ensure the maximum extent of degradation, the beechwood xylan was
T. neapolitana
Journal of Agricultural and Food Chemistry
DOI: 10.1021/acs.jafc.5b01259 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 4. Influence of Metals and EDTA on Enzyme Activity of XynAΔSLHa relative activity (%) compound none KCl NaCl MgCl2 MnCl2 CaCl2 CuCl2 EDTA
1 mM 100.00 101.32 97.75 98.65 92.72 75.25 11.59 37.00
± ± ± ± ± ± ± ±
0.43 4.16 5.87 0.53 2.66 1.31 0.21 1.46 (0.5 mM)
5 mM 98.69 93.84 75.86 70.45 32.47 0.00 22.29
± ± ± ± ±
1.20 0.77 1.94 1.56 0.45
Figure 7. Hydrolysis of sugar cane bagasse by XynAΔSLH and Cellic Ctec2: ●, reaction with XynAΔSLH only; ■, reaction with Cellic Ctec2 only; ▲, reaction with XynAΔSLH and Cellic Ctec2. 0.2 g of sugar cane bagasse was hydrolyzed in 20 mL of 0.1 M Bis-Tris-HCl buffer (pH 6.5) containing 10 U of XynAΔSLH with or without Cellic Ctec2 (0.2 FPU). Experiments were carried out in triplicate, and all standard deviations were
Improved Expression and Characterization of a Multidomain Xylanase from Thermoanaerobacterium aotearoense SCUT27 in Bacillus subtilis Xiongliang Huang,† Zhe Li,† Chenyu Du,§ Jufang Wang,† and Shuang Li*,† †
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China § School of Applied Sciences, The University of Huddersfield, Queensgate, Huddersfield, United Kingdom ABSTRACT: A xylanase gene was cloned and characterized from Thermoanerobacterium aotearoense SCUT27, which was attested to consist of a signal peptide, one glycoside hydrolase family 10 domain, four carbohydrate binding modules, and three surface layer homology domains. The change of expression host from Escherichia coli to Bacillus subtilis resulted in a 4.1-fold increase of specific activity for the truncated XynAΔSLH. Five different versions of secretion signals in B. subtilis indicated that it was preferably routed via a Sec-dependent pathway. Purified XynAΔSLH showed a high activity of 379.8 U/mg on beechwood xylan. XynAΔSLH was optimally active at 80 °C, pH 6.5. Thin layer chromatography results showed that xylobiose and the presumed methylglucuronoxylotriose (MeGlcAXyl3) were the main products liberated from beechwood xylan catalyzed by the recombinant xylanase. All of the results suggest that XynAΔSLH is a suitable candidate for generating xylooligosaccharides from cellulosic materials for industrial uses. KEYWORDS: multidomain xylanase, Thermoanaerobacterium aotearoense SCUT27, high-level expression, thermostability, beechwood xylan
■
and basic pI with a β-jelly roll structure. According to their molecular architectures, xylanases can be classified into two types: single-domain and multidomain enzymes (molecular weight >100 kDa). The multidomain xylanases have a modular structure that comprises a catalytic domain (CD), one or more carbohydrate-binding modules (CBMs), and domains with some other functions connected by relatively unstructured linker sequences.2 The CBMs, the most abundant noncatalytic domains in xylanases, may fold and function independently and can increase the rate of catalysis by bringing the biocatalyst into intimate association with the recalcitrant substrate.8 Actually, the enzyme was found to function more efficiently in carbohydrate degradation, and removing the CBM from the enzyme could dramatically decrease the enzyme activity.6 Previously, many single-domain xylanases from various microorganisms, including fungi,9 yeasts,10 and thermophilic bacteria11−13 have been successfully overexpressed in heterologous cells, whereas for the multidomain xylanase, studies were mainly focused on the sequence analysis and enzyme characterization.14−17 Little attention was paid to the efficient multidomain xylanase expression. However, it is desirable to exploit the industrial applications of the enzyme. Thermophilic microorganisms are a potential source of enzymes with novel properties as the enzymes originated from these organisms would function at higher temperature. Thermostable enzymes have obvious advantages as catalysts
INTRODUCTION Xylan is a major structure component of plant cell walls. It is the second most abundant polysaccharide in plants, accounting for approximately one-third of the renewable organic carbon on Earth.1−3 Xylan biodegradation involves the attacks of several hydrolytic enzymes, including two major enzymes, xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and β-xylosidase (exo-1,4-βxylosidase, EC 3.2.1.37).2,4 Xylanase randomly cleaves the internal linkages in xylan, yielding a mixture of xylooligosaccharides.5,6 The potential applications of xylanase cover a wide range of industrial sectors. Currently the most important biotechnological application of xylanase is in prebleaching of pulps to minimize the use of harsh chemicals in the subsequent treatment stages of the paper and pulp industry.6 Another important application of xylanase is its use in the bioconversion of lignocellulosic biomass into fermentative products for the generation of biofuels.2 Thermo- and acid-tolerant xylanase could be used as feed additive to increase the digestibility of animal feeds,1 whereas cold-active xylanase is applied in baking for improving bread quality by increasing specific bread volume.7 Due to the heterogeneity and complexity of xylan, a vast number of diverse xylanases exist in nature. On the basis of amino acid sequence similarity and hydrophobic cluster analysis of the catalytic domains, characterized xylanases (endo-1,4-βxylanase, EC 3.2.1.8) can be classified into glycoside hydrolase (GH) families 10, 11, and 30, especially GH10 and GH11 (http://www.cazy.org/Glycoside-Hydrolases.html). GH10 xylanases generally have a higher molecular weight (>30 kDa) and acidic pI with a (β/α)8 barrel structure, whereas members of GH11 typically have a lower molecular weight (400% when the Ca2+ existed in the reaction mixture.16 Substrate Specificity and Products of Xylan Degradation. Experiments were carried out to study the specificity of XynAΔSLH to various substrates. The XynAΔSLH had the highest specific activity of 379.8 ± 3.2 U/mg toward beechwood xylan (defined as 100%), followed by oat spelt xylan (213.8 ± 1.6 U/mg, 56.3%). No obvious catalytic activity was detected when CMC-Na was used as substrate by XynAΔSLH (16.4 ± 0.4 U/mg). These results are contrary to other previously reported results; for example, the endoxylanases isolated from T. thermarum16 and alkaline wastewater sludge17 showed maximum activity on birchwood xylan, and xylanase from G. thermoleovoranse had maximum activity on oat spelt xylan.13 Using xylooligosaccharides or beechwood xylan as substrate, the purified XynAΔSLH showed relatively uniform hydrolysis capability (Figure 6). Xylotetraose and xylopentaose could be easily hydrolyzed to xylobiose with a trace of xylose by XynAΔSLH in 30 min of catalysis. However, much of xylotriose was not thoroughly degraded to xylobiose after being incubated at 55 °C for 30 min. Moreover, hydrolysis of xylobiose was not observed during the experiment. To ensure the maximum extent of degradation, the beechwood xylan was
T. neapolitana
Journal of Agricultural and Food Chemistry
DOI: 10.1021/acs.jafc.5b01259 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 4. Influence of Metals and EDTA on Enzyme Activity of XynAΔSLHa relative activity (%) compound none KCl NaCl MgCl2 MnCl2 CaCl2 CuCl2 EDTA
1 mM 100.00 101.32 97.75 98.65 92.72 75.25 11.59 37.00
± ± ± ± ± ± ± ±
0.43 4.16 5.87 0.53 2.66 1.31 0.21 1.46 (0.5 mM)
5 mM 98.69 93.84 75.86 70.45 32.47 0.00 22.29
± ± ± ± ±
1.20 0.77 1.94 1.56 0.45
Figure 7. Hydrolysis of sugar cane bagasse by XynAΔSLH and Cellic Ctec2: ●, reaction with XynAΔSLH only; ■, reaction with Cellic Ctec2 only; ▲, reaction with XynAΔSLH and Cellic Ctec2. 0.2 g of sugar cane bagasse was hydrolyzed in 20 mL of 0.1 M Bis-Tris-HCl buffer (pH 6.5) containing 10 U of XynAΔSLH with or without Cellic Ctec2 (0.2 FPU). Experiments were carried out in triplicate, and all standard deviations were
