Increased enzymatic hydrolysis of sugarcane bagasse by a novel glucose- and xylose-stimulated β-glucosidase from Anoxybacillus flavithermus subsp. yunnanensis E13T
© The Author(s). 2017
Received: 22 November 2016
Accepted: 13 March 2017
Published: 16 March 2017
β-Glucosidase is claimed as a key enzyme in cellulose hydrolysis. The cellulosic fibers are usually entrapped with hemicelluloses containing xylose. So there is ongoing interest in searching for glucose- and xylose-stimulated β-glucosidases to increase the efficiency of hydrolysis of cellulosic biomass.
A thermostable β-glucosidase gene (Bglp) was cloned from Anoxybacillus flavithermus subsp. yunnanensis E13T and characterized. Optimal enzyme activity was observed at 60 °C and pH 7.0. Bglp was relatively stable at 60 °C with a 10-h half-life. The kinetic parameters V max and K m for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 771 ± 39 μmol/min/mg and 0.29 ± 0.01 mM, respectively. The activity of Bglp is dramatically stimulated by glucose or xylose at concentrations up to 1.4 M. After Bglp was added to Celluclast® 1.5 L, the conversion of sugarcane bagasse was 48.4 ± 0.8%, which was much higher than of Celluclast® 1.5 L alone. Furthermore, Bglp showed obvious advantages in the hydrolysis when initial concentrations of glucose and xylose are high.
The supplementation of BglP significantly enhanced the glucose yield from sugarcane bagasse, especially in the presence of high concentrations of glucose or xylose. Bglp should be a promising candidate for industrial applications.
KeywordsConversion of sugarcane bagasse β-Glucosidase Glucose-stimulated Thermostability Xylose-stimulated
The production of biofuels from renewable cellulosic biomass is important for the development of alternative energy. The efficient hydrolysis of cellulose requires the synergistic action of endoglucanases, cellobiohydrolases and β-glucosidases (EC 126.96.36.199). β-Glucosidase is responsible for the hydrolysis of oligosaccharides and cellobiose into glucose. Because oligosaccharides and cellobiose act as strong inhibitors of both endoglucanases and cellobiohydrolases, β-glucosidase is considered as a key enzyme for efficient cellulose hydrolysis .
However, the efficient hydrolysis of cellulosic biomass is more difficult than that of pure cellulose, because the cellulosic fibers are usually entrapped in other structural biopolymers, mainly hemicelluloses and lignin. Hemicelluloses consist of an available bulk source of xylose. In this context, glucose- and xylose-stimulated β-glucosidases appear to be particularly well suited to maximize the overall efficiency of hydrolysis of cellulosic biomass, acting in association with endo- and exocellulases and xylanases . Recently, many β-glucosidases with different glucose-tolerant or glucose-stimulated have been reported [3–6]. However, only a few glucose- and xylose-stimulated β-glucosidases have been characterized [7–9]. The advantage of the β-glucosidase group in practical cellulosic biomass hydrolysis is not experimentally studied.
Anoxybacillus flavithermus ssp. yunnanensis E13T is a thermophilic bacterium that grows at 60 °C . We determined the whole genome sequence of the strain E13T  and noted that a gene sequence (KF453503, BglP), defined as β-glucosidase in function annotation based on Genbank database, exhibited similarity with some putative β-glucosidases without being biochemically characterized. In this paper, the gene BglP was cloned, expressed and characterized in detail. The novel thermostable BglP is a glucose- and xylose-stimulated β-glucosidase. The supplementation of BglP significantly enhanced the glucose yield from sugarcane bagasse, especially in the presence of high concentrations of glucose or xylose.
Sequence analysis of Bglp
A β-glucosidase gene (Bglp) of 1347 bp with a predicted molecular weight of 52 kDa was revealed by the whole genome sequencing of A. flavithermus subsp. yunnanensis E13T. No signal peptide was found. The amino acid sequence of Bglp showed the highest sequence similarity of 91% with a glucose-tolerant β-glucosidase from Anoxybacillus sp. DT3-1 , 60% with a β-glucosidase from Bacillus bogoriensis (WP_026675503), 56% with a β-glucosidase from Caldicoprobacter oshimai (WP_025747479) and 55–57% with some β-glucosidases from Thermoanaerobacter sp. strains (ADD25173 and CAA91220). The sequence alignment of Bglp with the homologous is shown in Additional file 1: Figure S1. Two conserved catalytic nucleophile regions (Asn163-Glu164 and Glu354-Asn355-Gly356) which are highly conserved throughout the glycoside hydrolase family 1 (GH1) are identified in Bglp.
Enzymatic properties of the purified BglP
The recombinant BglP was expressed in soluble form in E. coli BL21 (DE3) cells and purified with Ni-NTA affinity chromatography. The purified BglP was a single band on 10% SDS-PAGE (Additional file 1: Figure S2). The apparent molecular mass of BglP was approximately 52 kDa, which corresponded to the calculated mass.
Hydrolysis activities of BglP on various substrates
Linkage of glycosyl group
Relative activityb (%)
51 ± 1.6
15.3 ± 0.6
13.2 ± 0.4
8.2 ± 0.2
2.0 ± 0.3
17.5 ± 0.4
7.9 ± 0.1
6.7 ± 0.4
4.9 ± 0.2
11.0 ± 0.6
5.6 ± 0.2
The effects of various metal ions and reagents on the activity of BglP
Relative activity (%)
101 ± 1.3
102 ± 1.2
99 ± 0.7
96 ± 0.5
95 ± 1.6
92 ± 1.3
98 ± 1.0
92 ± 0.9
99 ± 0.5
86 ± 1.6
98 ± 1.0
83 ± 0.4
91 ± 1.4
44 ± 0.5
77 ± 0.8
20 ± 0.6
99 ± 0.5
98 ± 0.9
98 ± 1.5
96 ± 1.3
96 ± 0.9
82 ± 1.4
99 ± 1.2
92 ± 1.6
97 ± 0.7
90 ± 1.6
91 ± 1.0
80 ± 0.5
82 ± 0.9
73 ± 1.4
3.4 ± 0.4
Effect of sugars on the activity and thermostability of BglP
To fully exploit the effect of glucose on BglP, the enzymatic thermostability at 60-70 °C was determined in the presence of a final concentration of 1 M glucose (Fig. 2b). The result suggested that the stability significantly increased in the presence of glucose. The BglP preserved 65% of its original activity after 15-h incubation at 60 °C in the presence of glucose. The half-life of BglP at 65 °C was approximately 2 h, while the half-life prolonged to approximately 5.5 h in the presence of glucose.
Conversion of sugarcane bagasse under high glucose and xylose concentrations
The conversion of sugarcane bagasse decreased to 19.9 ± 0.8% (Celluclast® 1.5 L alone) in the presence of 50 mM of glucose in the initial reaction mixture. The conversions further decreased with increasing initial concentrations of glucose. The lowest 10.5 ± 0.1% of conversion was observed at 500 mM initial glucose. The β-glucosidase in Celluclast® 1.5 L showed a lower hydrolytic efficiency in the presence of high concentrations of glucose. The supplementation of BglP significantly enhanced the conversion under all the concentrations of glucose. The conversion of the synergistic action at 500 mM was 35.6 ± 0.5% (Fig. 3a), which was 3.4-fold higher than that of Celluclast® 1.5 L alone.
Although the addition of 50 mM xylose had no effect on the conversion that performed by Celluclast® 1.5 L alone, a significant inhibition was observed when the xylose concentration was over 100 mM (Fig. 3b), which suggested the β-glucosidase in Celluclast® 1.5 L is only sensitive to high concentrations of xylose. The conversion by Celluclast® 1.5 L alone at 200 mM decreased to 15.2 ± 0.7%. When BglP was added to the reaction mixture, the conversion only weakly decreased from 48.4 ± 0.8 to 45.8 ± 0.9% with increasing initial concentrations of xylose from 0 to 200 mM. The conversion at 200 mM was 3.0-fold higher than that of Celluclast® 1.5 L alone.
β-Glucosidase is an important component of the cellulose enzyme system. A strong inhibition of β-glucosidase by glucose is most commonly observed . Searching for novel β-glucosidases with insensitive to glucose attracts more interest in recent years. Based on the different influences of glucose on the activity, Cao et al.  concluded that β-glucosidases can be divided roughly into three groups: (1) the glucose-sensitive β-glucosidases; (2) the glucose-tolerant β-glucosidases that are not activated by glucose; (3) the glucose-stimulated β-glucosidases. The glucose-stimulated β-glucosidases are less common, as compared with two previous groups. The BglP activity is stimulated 2.6-fold by 0.4 M glucose (Fig. 2a), therefore BglP should belong to the last group.
The stimulatory specific activity of BglP was as high as 2163 ± 11.2 U/mg at 0.4 M glucose, and this level was higher than most of glucose-stimulated β-glucosidases. The β-glucosidases from uncultured bacterium [13, 14], Thermoanaerobacterium aotearoense , Anoxybacillus sp. DT3-1  and Neotermes koshunensis  were stimulated about 4.0-, 2.7-, 1.4-, 1.3- and 1.3-fold by glucose, respectively and the stimulatory specific activity were about 11, 10.8, 145, 1505 and 16 U/mg, respectively. One of the reasons for the stimulation could be transglycosylation [6, 16]. Therefore, we analyzed the reaction products of BglP after incubation with 10 mM pNPG and glucose (100, 250 and 500 mM, respectively). No other product except glucose was determined, suggesting a lack of transglycosylation activity in BglP. Souza et al.  speculated that the stimulatory effect of glucose may be attributed to its binding to modulator binding sites, probably inducing conformational changes that stimulate the hydrolyzing activity. Further work will be necessary to determine whether the same applies to BglP.
Characteristics of BglP from A. flavithermus subsp. yunnanensis E13T and other glucose- and xylose-stimulated β-glucosidases
Relative activity (%) a
Inhibitory concentration (M)
Specific activity (pNPG, U/mg)
Source of strain
A. flavithermus subsp. yunnanensis
258 ± 5.1
182 ± 3.4
2.2 and 1.4 for glucose and xylose, respectively
842 ± 15.5
~10 h at 60 °C;
204 ± 12
191 ± 14
0.7 and 0.5 for glucose and xylose, respectively
20 min at 55 °C
180 ± 9
200 ± 11
44 min at 55 °C
H. grisea var. thermoidea
7 min at 60 °C
135.0 ± 4.3
131.0 ± 0.2
30 min at 50 °C
0.3 for glucose
Marinomonas sp. MWYL1
~11 min at 45 °C
In the case without initial sugar, the conversion of sugarcane bagasse by BglP was improved 1.9-fold than of Celluclast® 1.5 L alone. The supplement of β-glucosidases from a metagenomic library  and Thermoanaerobacterium aotearoense  to the conversion of sugarcane bagasse resulted in about 1.5- and 1.2-fold enhancement, respectively. The enhancement levels were lower than that of BglP, which may be due to the higher specific activity of BglP. In the case with initial sugar, since the β-glucosidases in Celluclast® 1.5 L is sensitive to both glucose and xylose, the advantages of BglP were more obvious. The conversion supplemented with BglP was 3.4- and 3.0-fold higher than that of Celluclast® 1.5 L alone at 500 mM of initial glucose and 200 mM of initial xylose, respectively. The results not only clearly revealed the significant role of the glucose- and xylose-stimulated β-glucosidases in the practical hydrolysis of cellulosic biomass, but also showed that BglP should have important practical implication in cellulose refining.
BglP from A. flavithermus subsp. yunnanensis E13T is a thermostable β-glucosidase, whose hydrolysis rate (V max value) is very fast. The activity of Bglp is dramatically stimulated by glucose and xylose, and has high tolerance to glucose and xylose. The supplement of Bglp to the hydrolysis mixture of sugarcane bagasse containing Celluclast® 1.5 L resulted in great enhancement of the conversion, especially in the presence of high concentrations of glucose and xylose.
Homologues of AmyP were obtained by searching the NCBI protein database using the BLAST tools . Sequence alignments were performed using the ClustalW . BLAST analyses suggested that Bglp belongs to GH1. A GH1 domain (pfam00232) was served as definition criteria for conserved nucleophile regions.
Cloning, expression and purification of β-glucosidase BglP
The strain A. flavithermus ssp. yunnanensis E13T was isolated from a hot spring in our previous study . It is available from the China Center for Type Culture Collection (AB2010187T) and the Korean Collection for Type Cultures (13759T). The β-glucosidase gene BglP was amplified by PCR using the primers (see Additional file 1). The PCR product was digested and ligated into pET22b, and then the plasmid pET22b-Bglp was transformed into E. coli BL21 (DE3) (Novagen). The recombinant BglP was induced by adding a final concentration of 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at OD600 about 0.5–0.6, and incubated further for 5 h. The cells were harvested and lysed by sonication. The lysate was heat treated at 60 °C for 30 min to denature the thermolabile proteins in E. coli, and then centrifuged (20,000 × g, 30 min). The supernatant was loaded on a Ni-NTA agarose gel column for purification. The protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined according to Bradford method.
β-Glucosidase activity was determined with p-nitrophenyl-β-D-glucopyranoside (pNPG, Sigma) as a substrate. The reaction mixture contained 10 μl of enzyme and 290 μl of 50 mM Na2HPO4-citric acid buffer (pH 7.0) with 5 mM pNPG. The reaction was incubated at 60 °C for 10 min and stopped by adding 1 ml of 1 M Na2CO3. The p-nitrophenol (pNP) released was measured at A 410. Other aryl-glycoside substrates were assayed under the same conditions. BglP activity against various saccharides was estimated with 1% substrate concentration, and the glucose released was quantified by a glucose oxidase-peroxidase assay (Sangon, China). The hydrolysis of carboxymethyl cellulose (CMC) and microcrystalline cellulose (Avicel®) was measured after 30 min reaction, employing 20.0 U of pNPG activity. The reducing sugar released was determined according to the classical method . In all analyses, 1 U was defined as the amount of enzyme that releases 1 μmol of product per min under the assay conditions. All experiments were performed at least three times and three independent reproductions were carried out.
Properties of recombinant BglP
The optimal temperature was measured over a temperature range from 0 to 80 °C at pH 7.0 (50 mM, Na2HPO4-citric acid buffer). Thermostability was determined by incubated the protein at 55–70 °C during 0.5 - 15 h. The residual activities were determined in standard reaction condition. The optimum pH of BglP was determined at 60 °C. To measure the pH stability, the purified BglP was incubated at pH 6.0–9.0 at 25 °C. Samples were removed at varying time intervals, and the residual activities were measured.
The influence of various metal ions (5 and 10 mM) on the BglP activity was investigated using MgCl2, CaCl2, FeCl3, CoCl2, ZnCl2, MnCl2, CuCl2 and FeCl2. The effects of dithiothreitol (DTT), urea, ethylenediaminetetraacetic acid (EDTA), β-mercaptoethanol, dimethyl sulfoxide (DMSO), Triton X-100, Tween 80 and sodium dodecyl sulfate (SDS) were determined. All enzyme activity was determined after pre-incubation of the purified BglP with above compounds in at 60 °C for 30 min. The BglP activity without any additive was taken as a percentage of the activity.
Determination of kinetic parameters
The Michaelis-Menten constants (K m) and maximum velocities (V max) were determined from Lineweaver-Burk plots by using pNPG or cellobiose as substrates. The purified BglP was incubated in Na2HPO4-citric acid buffer (pH 7.0) with the substrates in concentrations ranging from 0.05 to1.6 mM of pNPG or 5–70.0 mM of cellobiose at 60 °C. The kinetic parameters of pNPG in the presence of 0.4 M of glucose or 0.6 M xylose were established for the stimulation of activity.
The inhibition constants (K i) for the pNPG hydrolysis inhibition by glucose and xylose were estimated using Dixon plots. The concentration ranges of glucose and xylose were 3.0–3.7 and 1.8–2.3 M, respectively. Two concentrations of pNPG (5 and 8 mM) were used. All experimental kinetic curves were repeated three times, and three independent reproductions were carried out. The kinetic parameters are given as the mean ± SD of the values calculated for six different experiments (n = 6).
Effects of sugars on BglP activity and stability
The effects of various sugars (100 mM each of glucose, xylose, galactose, arabinose, mannose, maltose, fructose, sucrose, cellobiose and ribose) on the BglP activity were studied. The stimulatory effects of glucose and xylose on the activity were further tested using varying concentrations of glucose (0.05–4.0 M) and xylose (0.05–3.0 M). The BglP was pre-incubated with sugars at 14 °C for 24 h. The residual activities were quantified, and the control activity without sugar was taken as a percentage of the activity.
In determination of the effect of glucose on thermostability, glucose (1 M) was added into the reaction solution and incubated at different temperatures (60, 65 and 70 °C) for different time (0.5–15 h), and then the residual activities were determined.
Conversion of sugarcane bagasse by BglP
Sugarcane bagasse was preformed according to the previous study . In brief, the sugarcane bagasse (Jing Hui Sugar Group Co. Ltd, China) pretreated with 1% (v/v) H2SO4 with a solid to liquid ratio of 1:20. The solids were carried out in an autoclave at 120 °C for 70 min by using 4% NaOH. Then the alkaline solids were washed with water using until pH changed to 7.0. The dried solids were used for subsequent experiments.
Enzymatic conversion of sugarcane bagasse was performed with 10% (w/v, dry basis) substrate concentration in 20 ml of Na2HPO4-citric acid buffer (pH 7.0). The commercial Cellulast® 1.5 L (Novozymes, Denmark) load was 40 filter paper unite (FPU) per gram of sugarcane bagasse according to the previous study . The purified BglP load was 50 μg per gram of sugarcane bagasse. The flasks with the reaction mixture were carried out in a rotary shaker at 60 °C and 120 rpm. After a 48-h hydrolysis, the glucose released was quantified by HPLC according to method of Lei et al. . The conversion of sugarcane bagasse was calculated according to Cao et al. . In determination of the effects of glucose and xylose on the hydrolysis of sugarcane bagasse, glucose (the final concentration of 50–500 mM) or xylose (50–200 mM) was added into the reaction mixture. A final concentration of 0.2% (v/v) toluene was used to prevent microbial contamination during the conversion .
Glycoside hydrolase family 1
- K i :
- K m :
Sodium dodecyl sulfate
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- V max :
This work was supported by the Natural Science Foundation of China (31270081) and (31470207) and the Science Fund for Distinguished Young Scholars of Anhui Province, China (170808 J04).
Availability of data and materials
All data generated or analysed during this study are included in this published article and its Additional file 1.
YL, XZ and HP designed experiments. YL, JW, XZ and RL performed experiments. RL, YG and HP analyzed the data. YL, RJ and HP wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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