# Purification and Characterization of a Thermostableβ-Mannanase fromBacillus subtilis BE-91: Potential Application in Inflammatory Diseases

Authors: Lifeng Cheng; Shengwen Duan; Xiangyuan Feng; Ke Zheng; Qi Yang; Zhengchu Liu
Journal: BioMed Research International
 (2016)

---

## Abstract

β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase fromBacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5–7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. K
m and V
m
a
x values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.


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## Body

## 1. Introduction

Mannan consists of a series of complex polysaccharides, which are found in the cell wall of marine algae [1]. The backbone is comprised of β-1,4-linked mannose residues. Konjac glucomannan is a randomly arranged polymer of β-1,4-linked glucose and mannose residues at ratio of 1.0 : 1.6. Both the backbones of mannan and Konjac are modified by α-1,6-linked galactosyl residues to form galactomannan and galactoglucomannan, respectively [2].β-mannanase (EC 3.2.1.78) is a hemicellulase that attacks the internal glycosidic bonds of mannan backbone to release the condensed β-1,4-manno-oligosaccharides [3]. β-mannanases are widely applied in pulp and paper processing [4], feed [5], food [6], pharmaceutical [7], oil, and textile industries [8] to randomly hydrolyze the β-1,4 mannopyranoside linkage in mannan, galactomannan, glucomannan, and galactoglucomannan.β-mannanase is widely produced by bacteria [9, 10], actinomycetes [11], fungi [12], plants, and animals [13]. Among them, β-mannanase from bacteria is wildely used because of numerous advantages, including extracellular secretion, economic production and purification, and novel characteristics, such as tolerance to heat and alkaline conditions [14].Although multipleβ-mannanase-producing bacteria have been reported [15, 16], they are far from the diverse industry needs. Currently, acidic and alkaline β-mannanase has been proposed to meet the industrial demands [17]. However, the requirements of high energy in production and the environmental impact limit their development. Neutral and weakly acidic β-mannanase with lower energy for production has attracted considerable interest over the past few years; however, it has rarely been characterized. It is clarified that β-mannanase with high activity in short fermentation time confers lower costs during the production procedures. Therefore, the exploitation of strains producing high β-mannanases activity is valuable and profitable. In current study, we isolated and preserved a powerful hemicellulose-degrading bacterium (BE-91). Then we explored the efficient purification process and characterized the enzymatic properties of its β-mannanase.


## 2. Materials and Methods

### 2.1. Microorganism, Media, and Fermentation Conditions

B. subtilis BE-91, a strain used for herbaceous fiber extraction, was identified and preserved by the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science (Changsha, Hunan, China).B. subtilis BE-91 was cultured in Petri dish containing 0.5% yeast extract, 1% NaCl, 0.5% Konjac glucomannan, 1% bacto tryptone, 0.05% trypan blue, and 1.5% agar. The seed medium was mainly composed of 0.1% glucose, 0.4% Konjac glucomannan, 0.3% beef extract, 0.2% yeast extract, 0.5% peptone, and 0.5% NaCl. The fermentation medium primarily consisted of 0.2% yeast extract, 0.7% Konjac glucomannan, 0.5% peptone, 0.3% beef extract, and 0.5% NaCl.B. subtilis BE-91 was first activated in the seed medium at 35
±
1°C for 5.5 h. Subsequently, the suspension was serially diluted, spread onto Petri dishes, and incubated at 35
±
1°C for 18 h. The single colony exhibiting the largest hydrolytic halo was transferred into an Erlenmeyer flask with the seed medium and cultured at 35
±
1°C for 6 h at 180 rpm. Consequently, 2% culture was inoculated in the fermentation medium and cultured for 6 h at 35
±
1°C at 180 rpm [18].


### 2.2. Classification of Strain BE-91

The 16S rDNA of strain BE-91 was PCR amplified from genomic DNA using the Bacterial Identification PCR Kit (TaKaRa, Japan). The obtained 16S rDNA was sequenced by the ABI 3730XL 96-capillary DNA analyzer. The primers were as follows: P1 5′-AGAGTTTGATCMTGGCTCAG-3′ and P2 5′-TACGGYTACCTTGTTACGACTT-3′. The resulting sequence aligned closely with the related standard sequences of other bacteria retrieved from GenBank. Neighbor-joining clusters were constructed by Mega 6.0 [19].


### 2.3. Enzymatic Assays

β-mannanase activity was estimated by initiating the reaction at 65°C for 10 min in 0.05 mol/L citric acid/0.1 mol/L Na2HPO4 buffer (pH 6.0) with 0.2% (w/v) Konjac glucomannan as substrate. The amounts of reducing sugar in the reaction were quantified based on a standard curve generated with mannose using the 3,5-dinitrosalicylic acid (DNS) method. One unit (IU) of β-mannanase activity was defined as the amount of protein producing 1 μmol/L of reducing sugar per minute (e.g., mannose) under standard conditions [20].


### 2.4. Purification ofβ-Mannanase

The bacterialβ-mannanase was purified using a two-step process involving ultrafiltration (Sartorius, Germany) and gel filtration. The fermentation liquid was fractionated orderly by 100 kDa, 50 kDa, and 5 kDa membrane thresholds. The solution filtered with 5 kDa < MW < 50 kDa was further purified on a Sephadex G-100 gel column (Ф1.6 cm × 100 cm, Pharmacia). The eluate was obtained at a rate of 0.5 mL/min and collected in 5 mL fractions. β-mannanase activity was determined by the DNS method, whereas the protein was quantified by the Coomassie brilliant blue staining against bovine serum albumin (BSA) standard [21].


### 2.5. The Determination of Apparent Molecular Weight

The molecular mass of theβ-mannanase was determined by SDS-PAGE (Bio-Rad, USA), with 3% stacking gel and 12% separating gel [22]. The protein bands were stained with 0.01% Coomassie brilliant blue R-250 and destained with a water-methanol-acetic acid (9 : 9 : 2) solvent. Zymogram analysis was performed by the method of Chanhan [17]. The molecular weight of β-mannanase was derived from the relative mobility of molecular weight markers resolved simultaneously.


### 2.6. The Effect of Temperature on the Activity and Stability ofβ-Mannanase

The activity ofβ-mannanase was assayed at a range of temperatures between 50 and 70°C in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at pH 6.0. The thermostability was assessed by preincubating the enzyme, without a substrate, at different temperatures varying over 20–80°C for 30 min. The residual activity was promptly measured by the DNS method. The β-mannanase activity was considered to be 100% when preincubated at 4°C.


### 2.7. The Effect of pH on the Activity and Stability ofβ-Mannanase

β-mannanase activity was evaluated by incubating the purified enzyme at different pH conditions ranging from 4.0 to 8.0 in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at 4°C. The stability at a particular pH was tested by preincubating the purified enzyme, without a substrate, for 30 min in various 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffers at pH 3.0–8.5 at 4°C. The residual β-mannanase activity was immediately measured after treatment by the DNS procedure.


### 2.8. The Effect of Metal Ions on the Activity ofβ-Mannanase

In order to examine the effects of metal ions on the activity ofβ-mannanase, the enzyme was incubated for 30 min at 4°C in the presence of various 1.0 mmol/L metal ions, CaCl2·2H2O, ZnCl2, FeCl3, PbCl2·2H2O, MnCl2·4H2O, MgCl2·6H2O, KCl, CuCl2·2H2O, AlCl3, BaCl2, and NH4Cl. The residual β-mannanase activity was measured at a specific condition and that of the treatment in the absence of additives as a control.


### 2.9. Substrate Specificity and Kinetic Parameters

Various glycans, such as Konjac glucomannan [23], locust bean gum fromCeatonia siliqua seeds (Sigma, G0753), carob galactomannan (Megazyme, P-GALML), guar galactomannan (Megazyme, P-GGMMV), ivory nut mannan (Megazyme, P-MANIV), 1,4-beta-D-mannan (Megazyme, P-MANCB), wheat arabinoxylan (Megazyme, P-120601a), beechwood xylan (Megazyme, P-141101a), and carboxymethyl cellulose (Megazyme, P-CMC4M) were examined. In brief, 0.2% (w/v) glycans were incubated with β-mannanase at 65°C for 10 min in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at pH 6.0, and the reducing sugars were measured by DNS. The Michaelis-Menten kinetic parameters, V
m
a
x and K
m, were calculated for β-mannanase. The assays of the purified enzyme were carried out by the standard DNS procedure, as described above, using 1–5 mg/mL locust bean gum and 0.5–2.5 mg/mL Konjac glucomannan as substrates. The kinetic constants were determined from the Michaelis-Menten equation by directly inputting the initial rates from Lineweaver-Burk plots or the nonlinear regression [24].


### 2.10. Statistical Analysis

Eachβ-mannanase activity experiment was performed in triplicate and expressed as mean ± SD (standard deviation). The statistical analyses were performed with SPSS 15.0 (SPSS Inc., Chicago IL, USA). One-way or two-way analysis of variance (ANOVA) was used to compare various treatment groups.


## 2.1. Microorganism, Media, and Fermentation Conditions

B. subtilis BE-91, a strain used for herbaceous fiber extraction, was identified and preserved by the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science (Changsha, Hunan, China).B. subtilis BE-91 was cultured in Petri dish containing 0.5% yeast extract, 1% NaCl, 0.5% Konjac glucomannan, 1% bacto tryptone, 0.05% trypan blue, and 1.5% agar. The seed medium was mainly composed of 0.1% glucose, 0.4% Konjac glucomannan, 0.3% beef extract, 0.2% yeast extract, 0.5% peptone, and 0.5% NaCl. The fermentation medium primarily consisted of 0.2% yeast extract, 0.7% Konjac glucomannan, 0.5% peptone, 0.3% beef extract, and 0.5% NaCl.B. subtilis BE-91 was first activated in the seed medium at 35
±
1°C for 5.5 h. Subsequently, the suspension was serially diluted, spread onto Petri dishes, and incubated at 35
±
1°C for 18 h. The single colony exhibiting the largest hydrolytic halo was transferred into an Erlenmeyer flask with the seed medium and cultured at 35
±
1°C for 6 h at 180 rpm. Consequently, 2% culture was inoculated in the fermentation medium and cultured for 6 h at 35
±
1°C at 180 rpm [18].


## 2.2. Classification of Strain BE-91

The 16S rDNA of strain BE-91 was PCR amplified from genomic DNA using the Bacterial Identification PCR Kit (TaKaRa, Japan). The obtained 16S rDNA was sequenced by the ABI 3730XL 96-capillary DNA analyzer. The primers were as follows: P1 5′-AGAGTTTGATCMTGGCTCAG-3′ and P2 5′-TACGGYTACCTTGTTACGACTT-3′. The resulting sequence aligned closely with the related standard sequences of other bacteria retrieved from GenBank. Neighbor-joining clusters were constructed by Mega 6.0 [19].


## 2.3. Enzymatic Assays

β-mannanase activity was estimated by initiating the reaction at 65°C for 10 min in 0.05 mol/L citric acid/0.1 mol/L Na2HPO4 buffer (pH 6.0) with 0.2% (w/v) Konjac glucomannan as substrate. The amounts of reducing sugar in the reaction were quantified based on a standard curve generated with mannose using the 3,5-dinitrosalicylic acid (DNS) method. One unit (IU) of β-mannanase activity was defined as the amount of protein producing 1 μmol/L of reducing sugar per minute (e.g., mannose) under standard conditions [20].


## 2.4. Purification ofβ-Mannanase

The bacterialβ-mannanase was purified using a two-step process involving ultrafiltration (Sartorius, Germany) and gel filtration. The fermentation liquid was fractionated orderly by 100 kDa, 50 kDa, and 5 kDa membrane thresholds. The solution filtered with 5 kDa < MW < 50 kDa was further purified on a Sephadex G-100 gel column (Ф1.6 cm × 100 cm, Pharmacia). The eluate was obtained at a rate of 0.5 mL/min and collected in 5 mL fractions. β-mannanase activity was determined by the DNS method, whereas the protein was quantified by the Coomassie brilliant blue staining against bovine serum albumin (BSA) standard [21].


## 2.5. The Determination of Apparent Molecular Weight

The molecular mass of theβ-mannanase was determined by SDS-PAGE (Bio-Rad, USA), with 3% stacking gel and 12% separating gel [22]. The protein bands were stained with 0.01% Coomassie brilliant blue R-250 and destained with a water-methanol-acetic acid (9 : 9 : 2) solvent. Zymogram analysis was performed by the method of Chanhan [17]. The molecular weight of β-mannanase was derived from the relative mobility of molecular weight markers resolved simultaneously.


## 2.6. The Effect of Temperature on the Activity and Stability ofβ-Mannanase

The activity ofβ-mannanase was assayed at a range of temperatures between 50 and 70°C in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at pH 6.0. The thermostability was assessed by preincubating the enzyme, without a substrate, at different temperatures varying over 20–80°C for 30 min. The residual activity was promptly measured by the DNS method. The β-mannanase activity was considered to be 100% when preincubated at 4°C.


## 2.7. The Effect of pH on the Activity and Stability ofβ-Mannanase

β-mannanase activity was evaluated by incubating the purified enzyme at different pH conditions ranging from 4.0 to 8.0 in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at 4°C. The stability at a particular pH was tested by preincubating the purified enzyme, without a substrate, for 30 min in various 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffers at pH 3.0–8.5 at 4°C. The residual β-mannanase activity was immediately measured after treatment by the DNS procedure.


## 2.8. The Effect of Metal Ions on the Activity ofβ-Mannanase

In order to examine the effects of metal ions on the activity ofβ-mannanase, the enzyme was incubated for 30 min at 4°C in the presence of various 1.0 mmol/L metal ions, CaCl2·2H2O, ZnCl2, FeCl3, PbCl2·2H2O, MnCl2·4H2O, MgCl2·6H2O, KCl, CuCl2·2H2O, AlCl3, BaCl2, and NH4Cl. The residual β-mannanase activity was measured at a specific condition and that of the treatment in the absence of additives as a control.


## 2.9. Substrate Specificity and Kinetic Parameters

Various glycans, such as Konjac glucomannan [23], locust bean gum fromCeatonia siliqua seeds (Sigma, G0753), carob galactomannan (Megazyme, P-GALML), guar galactomannan (Megazyme, P-GGMMV), ivory nut mannan (Megazyme, P-MANIV), 1,4-beta-D-mannan (Megazyme, P-MANCB), wheat arabinoxylan (Megazyme, P-120601a), beechwood xylan (Megazyme, P-141101a), and carboxymethyl cellulose (Megazyme, P-CMC4M) were examined. In brief, 0.2% (w/v) glycans were incubated with β-mannanase at 65°C for 10 min in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at pH 6.0, and the reducing sugars were measured by DNS. The Michaelis-Menten kinetic parameters, V
m
a
x and K
m, were calculated for β-mannanase. The assays of the purified enzyme were carried out by the standard DNS procedure, as described above, using 1–5 mg/mL locust bean gum and 0.5–2.5 mg/mL Konjac glucomannan as substrates. The kinetic constants were determined from the Michaelis-Menten equation by directly inputting the initial rates from Lineweaver-Burk plots or the nonlinear regression [24].


## 2.10. Statistical Analysis

Eachβ-mannanase activity experiment was performed in triplicate and expressed as mean ± SD (standard deviation). The statistical analyses were performed with SPSS 15.0 (SPSS Inc., Chicago IL, USA). One-way or two-way analysis of variance (ANOVA) was used to compare various treatment groups.


## 3. Results and Discussion

### 3.1. Screening of the Highβ-Mannanase Activity Producing Strain

Four bacteria were stochastically selected for theβ-mannanase activity assay. Figure 1 exhibited the halos produced on the screening plate. Table 1 summarized the β-mannanase activity of the four bacteria (strain BE-23 without β-mannanase activity was used as a negative control). Strain BE-91 fermented for 9 h exhibited the highest activity, up to 273.7 IU/mL. Wild-typeB. subtilis MA139 yielded a maximum β-mannanase activity of 170 IU/mL after 3 days of fermentation, and the maximum enzyme activity ofB. subtilis TJ-102 was 205.3 IU/mL at 38 h [25, 26]. Notably, BE-91 secreted β-mannanase with higher activity in shorter time.Table 1
β-mannanase activities of five bacteria.
Bacterium number
Activity (IU/mL)a
Specific activity (IU/mg)a
BE-23
0
0
BE-78
191.5 ± 4.5
879.8 ± 13.2
BE-46
83.2 ± 2.1
311.6 ± 9.4
BE-83
70.7 ± 1.6
119.7 ± 25.5
BE-91
273.7 ± 6.5
2,319.2 ± 26.3
aData are mean ± SD, n
=
3.Figure 1
Clear halos produced by control and active colonies withβ-mannanase activity 1, BE-78; 2, BE-46; 3, BE-83; 4, BE-23 (negative control); 5, BE-91.


### 3.2. Classification ofB. subtilis BE-91

The 1,508 bp sequence of 16S rDNA of strain BE-91 was analyzed by a phylogenic tree (Figure2). The homology between BE-91 16S rDNA (gi 260159552) andB. subtilis 16S rDNA (gi 530330588 and gi 341831474) was 99%. It was confirmed that the similarity ofB. subtilis type strains about 16S rRNA gene sequence is higher than 98% [27, 28]. We also obtained ≥98% similarity to 16S rRNA gene sequences ofB. subtilis isolates.Figure 2
Phylogenetic tree based on 16S rDNA sequences of strain BE-91 and other bacteria by Mega 6.0 using neighbor-joining analysis with 1000 bootstrap replicates.


### 3.3. Isolation and Purification ofβ-Mannanase

2,000 mL of fermentation liquor was purified by ultrafiltration and chromatography. Specific activity, recovery, and multiple purifications at each step were summarized in Table2. The recovery of β-mannanase inB. subtilis BE-91 exceeded 66.0%; multiple purifications achieved 32.9-fold pure β-mannanase activity, and the specific activity of the purified enzyme reached 79,859.2 IU/mg. The purified β-mannanase was shown to be homogeneous judged by SDS-PAGE analysis (Figure 3). Compared with the previous separation and purification methods [29, 30], the two-step method has the advantages of high efficiency, high yield, and easy operation.Table 2
Purification ofβ-mannanase by ultrafiltration and gel chromatography.
Purification step
Total activity (IU)
Total protein (mg)
Specific activity (IU/mg)
Recovery (%)
Purification multiple (fold)
Fermentation liquor
429,650.8
176.7
2,431.4
100
1
Ultrafiltration
328,317.4
8.6
38,070.2
76.4
15.6
Gel chromatography
283,500.2
3.6
79,859.2
66.0
32.9Figure 3
SDS-PAGE analysis ofβ-mannanase. Lane M: protein molecular weight standard; Lane 1: culture broth; Lane 2: purified β-mannanase; Lane 3: zymogram of purified β-mannanase.


### 3.4. Apparent Molecular Weight ofβ-Mannanase

The apparent molecular weight ofβ-mannanase was 28.2 kDa (Figure 3), lower than those of the most known β-mannanases fromBacillus spp. (Bacillus licheniformisTHCM 3.1, 40 kDa;B. subtilis WY34, 39.6 kDa;B. subtilis Z-2, 38 kDa;Bacillus circulans CGMCC1554, 32 kDa) [28, 31–34]. Similarly, the molecular weights of β-mannanases fromPenicillium occitanis Po16 andBacillus halodurans PPKS-2 were 22 and 18 kDa, respectively [30, 31]. Due to low molecular weights, these enzymes may rapidly penetrate the lignocellulose systems and depolymerize the mannans more efficiently [35].


### 3.5. Optimal Temperature and Thermostability ofβ-Mannanase

The purifiedβ-mannanase was maximally active at 65°C (Figure 4) and remained more than 80% active at 70°C (Figure 5). Compared with the optimal temperatures obtained for other β-mannanases (40°C forPenicillium occitanis Pol6; 50°C for bothBacillus circulansTN-31 andB. subtilisB36; 60°C forPaenibacillus sp. DZ3) [29, 31, 36], β-mannanase of BE-91 showed a pronounced activity at higher temperatures. As compared to the thermostability of the β-mannanase from wild-typeB. subtilis BCC41051 (60°C for 30 min) [37], this β-mannanase retains 80% residual activity after incubation at 20–70°C for 30 min, indicating enhanced thermostability.Figure 4
Optimum temperature curve ofβ-mannanase.Figure 5
Thermal stability curve ofβ-mannanase.


### 3.6. Optimal pH and Stability ofβ-Mannanase

The optimal pH and the stability of BE-91β-mannanase were measured at various pHs. The optimum enzyme activity was obtained at pH 6.0 (Figure 6), and more than 80% maximal activity was retained at pH 4.5–7.0 (Figure 7). Interestingly, the optimal pH of BE-91 β-mannanase was the same as that ofB. subtilis MA139 (pH 6.0), an enzyme that can potentially be used as a feed additive for monogastric animals [25]. At pH < 4.0, the β-mannanase activity was negligible, retaining <80% of its maximal value obtained after incubation at pH > 7.5, 4°C for 30 min. A relatively broad zone of optimum activity was observed. Therefore, BE-91 β-mannanase can be considered a weakly acidic and neutral enzyme, thereby rendering suitability for animal feed industry [38].Figure 6
Optimum pH curve ofβ-mannanase.Figure 7
pH stability curve ofβ-mannanase.


### 3.7. The Effect of Metal Ions onβ-Mannanase Stability

The effect of a variety of metal ions onβ-mannanase activity was measured (Table 3). The highest induction was achieved with Mn2+, which showed 168% baseline activity, followed by Al3+, Ca2+, Cu2+, Zn2+, Mg2+, and N
H
4
+, respectively. K+ and Fe3+ had no obvious effects on β-mannanase activity in these conditions. Ba2+ and Pb2+ greatly inhibited the enzyme activity to a final rate of 83% and 74%, respectively. This suggests that BE-91 β-mannanase should not be contaminated by Ba2+ and Pb2+.Table 3
Effects of different metal ions (1 mmol/L) onβ-mannanase activity.
Metal ions
Relative activity (%)a
Blank
100
K+
99 ± 3.2
N
H
4
+
103 ± 2.7
Ca2+
117 ± 3.6
Zn2+
115 ± 2.9
Mn2+
168 ± 4.5
Cu2+
116 ± 2.1
Mg2+
107 ± 2.8
Ba2+
83 ± 3.1
Pb2+
74 ± 2.9
Fe3+
99 ± 3.6
Al3+
121 ± 4.3
aData are mean ± SD, n
=
3.


### 3.8. Kinetic Parameters

The purified enzyme hydrolyzed Konjac glucomannan but only slightly hydrolyzed ivory nut mannan, guar galactomannan, and 1,4-beta-D-mannan. Wheat arabinoxylan, beechwood xylan, and CMC were barely hydrolyzed, as shown in Table4. This β-mannanase exhibited the highest activity with Konjac glucomannan, enriched in glucose units. This finding suggests that β-mannanase of BE-91 preferentially hydrolyzes the β-1,4-linkage of the glucosylated mannan backbone.Table 4
Hydrolytic activity of the purified enzyme on different polysaccharides.
Substrate (0.5%, w/v)
Relative activity (%)a
Konjac glucomannan
100
Locust bean gum
88.15 ± 1.8
Carob galactomannan
91.85 ± 1.7
Guar galactomannan
35.70 ± 0.6
Ivory nut mannan
32.74 ± 0.3
1,4-Beta-D-mannan
46.22 ± 0.4
Wheat arabinoxylan
0
Beechwood xylan
0
Carboxymethyl cellulose
0
Assays were carried out at 65°C at pH 6.0 for 10 min in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer.
aData are mean ± SD, n
=
3.K
m and V
m
a
x values of this β-mannanase estimated by the Lineweaver-Burk plot were 7.14 mg/mL and 107.5 μmol/min/mL, respectively, for locust bean gum, versus 1.749 mg/mL and 33.45 μmol/min/mL for Konjac glucomannan, respectively. These results displayed higher affinity of β-mannanase towards natural Konjac glucomannan (V
m
a
x/K
m, 19.1 μmol/min/mg) than the locust bean gum (V
m
a
x/K
m, 15.0 μmol/min/mg), similar to the values obtained forPenicillium pinophilum C1 andPenicillium freii F63, hence constituting it as an adequate candidate in food industry for the production of oligosaccharides [17, 18, 39].


## 3.1. Screening of the Highβ-Mannanase Activity Producing Strain

Four bacteria were stochastically selected for theβ-mannanase activity assay. Figure 1 exhibited the halos produced on the screening plate. Table 1 summarized the β-mannanase activity of the four bacteria (strain BE-23 without β-mannanase activity was used as a negative control). Strain BE-91 fermented for 9 h exhibited the highest activity, up to 273.7 IU/mL. Wild-typeB. subtilis MA139 yielded a maximum β-mannanase activity of 170 IU/mL after 3 days of fermentation, and the maximum enzyme activity ofB. subtilis TJ-102 was 205.3 IU/mL at 38 h [25, 26]. Notably, BE-91 secreted β-mannanase with higher activity in shorter time.Table 1
β-mannanase activities of five bacteria.
Bacterium number
Activity (IU/mL)a
Specific activity (IU/mg)a
BE-23
0
0
BE-78
191.5 ± 4.5
879.8 ± 13.2
BE-46
83.2 ± 2.1
311.6 ± 9.4
BE-83
70.7 ± 1.6
119.7 ± 25.5
BE-91
273.7 ± 6.5
2,319.2 ± 26.3
aData are mean ± SD, n
=
3.Figure 1
Clear halos produced by control and active colonies withβ-mannanase activity 1, BE-78; 2, BE-46; 3, BE-83; 4, BE-23 (negative control); 5, BE-91.


## 3.2. Classification ofB. subtilis BE-91

The 1,508 bp sequence of 16S rDNA of strain BE-91 was analyzed by a phylogenic tree (Figure2). The homology between BE-91 16S rDNA (gi 260159552) andB. subtilis 16S rDNA (gi 530330588 and gi 341831474) was 99%. It was confirmed that the similarity ofB. subtilis type strains about 16S rRNA gene sequence is higher than 98% [27, 28]. We also obtained ≥98% similarity to 16S rRNA gene sequences ofB. subtilis isolates.Figure 2
Phylogenetic tree based on 16S rDNA sequences of strain BE-91 and other bacteria by Mega 6.0 using neighbor-joining analysis with 1000 bootstrap replicates.


## 3.3. Isolation and Purification ofβ-Mannanase

2,000 mL of fermentation liquor was purified by ultrafiltration and chromatography. Specific activity, recovery, and multiple purifications at each step were summarized in Table2. The recovery of β-mannanase inB. subtilis BE-91 exceeded 66.0%; multiple purifications achieved 32.9-fold pure β-mannanase activity, and the specific activity of the purified enzyme reached 79,859.2 IU/mg. The purified β-mannanase was shown to be homogeneous judged by SDS-PAGE analysis (Figure 3). Compared with the previous separation and purification methods [29, 30], the two-step method has the advantages of high efficiency, high yield, and easy operation.Table 2
Purification ofβ-mannanase by ultrafiltration and gel chromatography.
Purification step
Total activity (IU)
Total protein (mg)
Specific activity (IU/mg)
Recovery (%)
Purification multiple (fold)
Fermentation liquor
429,650.8
176.7
2,431.4
100
1
Ultrafiltration
328,317.4
8.6
38,070.2
76.4
15.6
Gel chromatography
283,500.2
3.6
79,859.2
66.0
32.9Figure 3
SDS-PAGE analysis ofβ-mannanase. Lane M: protein molecular weight standard; Lane 1: culture broth; Lane 2: purified β-mannanase; Lane 3: zymogram of purified β-mannanase.


## 3.4. Apparent Molecular Weight ofβ-Mannanase

The apparent molecular weight ofβ-mannanase was 28.2 kDa (Figure 3), lower than those of the most known β-mannanases fromBacillus spp. (Bacillus licheniformisTHCM 3.1, 40 kDa;B. subtilis WY34, 39.6 kDa;B. subtilis Z-2, 38 kDa;Bacillus circulans CGMCC1554, 32 kDa) [28, 31–34]. Similarly, the molecular weights of β-mannanases fromPenicillium occitanis Po16 andBacillus halodurans PPKS-2 were 22 and 18 kDa, respectively [30, 31]. Due to low molecular weights, these enzymes may rapidly penetrate the lignocellulose systems and depolymerize the mannans more efficiently [35].


## 3.5. Optimal Temperature and Thermostability ofβ-Mannanase

The purifiedβ-mannanase was maximally active at 65°C (Figure 4) and remained more than 80% active at 70°C (Figure 5). Compared with the optimal temperatures obtained for other β-mannanases (40°C forPenicillium occitanis Pol6; 50°C for bothBacillus circulansTN-31 andB. subtilisB36; 60°C forPaenibacillus sp. DZ3) [29, 31, 36], β-mannanase of BE-91 showed a pronounced activity at higher temperatures. As compared to the thermostability of the β-mannanase from wild-typeB. subtilis BCC41051 (60°C for 30 min) [37], this β-mannanase retains 80% residual activity after incubation at 20–70°C for 30 min, indicating enhanced thermostability.Figure 4
Optimum temperature curve ofβ-mannanase.Figure 5
Thermal stability curve ofβ-mannanase.


## 3.6. Optimal pH and Stability ofβ-Mannanase

The optimal pH and the stability of BE-91β-mannanase were measured at various pHs. The optimum enzyme activity was obtained at pH 6.0 (Figure 6), and more than 80% maximal activity was retained at pH 4.5–7.0 (Figure 7). Interestingly, the optimal pH of BE-91 β-mannanase was the same as that ofB. subtilis MA139 (pH 6.0), an enzyme that can potentially be used as a feed additive for monogastric animals [25]. At pH < 4.0, the β-mannanase activity was negligible, retaining <80% of its maximal value obtained after incubation at pH > 7.5, 4°C for 30 min. A relatively broad zone of optimum activity was observed. Therefore, BE-91 β-mannanase can be considered a weakly acidic and neutral enzyme, thereby rendering suitability for animal feed industry [38].Figure 6
Optimum pH curve ofβ-mannanase.Figure 7
pH stability curve ofβ-mannanase.


## 3.7. The Effect of Metal Ions onβ-Mannanase Stability

The effect of a variety of metal ions onβ-mannanase activity was measured (Table 3). The highest induction was achieved with Mn2+, which showed 168% baseline activity, followed by Al3+, Ca2+, Cu2+, Zn2+, Mg2+, and N
H
4
+, respectively. K+ and Fe3+ had no obvious effects on β-mannanase activity in these conditions. Ba2+ and Pb2+ greatly inhibited the enzyme activity to a final rate of 83% and 74%, respectively. This suggests that BE-91 β-mannanase should not be contaminated by Ba2+ and Pb2+.Table 3
Effects of different metal ions (1 mmol/L) onβ-mannanase activity.
Metal ions
Relative activity (%)a
Blank
100
K+
99 ± 3.2
N
H
4
+
103 ± 2.7
Ca2+
117 ± 3.6
Zn2+
115 ± 2.9
Mn2+
168 ± 4.5
Cu2+
116 ± 2.1
Mg2+
107 ± 2.8
Ba2+
83 ± 3.1
Pb2+
74 ± 2.9
Fe3+
99 ± 3.6
Al3+
121 ± 4.3
aData are mean ± SD, n
=
3.


## 3.8. Kinetic Parameters

The purified enzyme hydrolyzed Konjac glucomannan but only slightly hydrolyzed ivory nut mannan, guar galactomannan, and 1,4-beta-D-mannan. Wheat arabinoxylan, beechwood xylan, and CMC were barely hydrolyzed, as shown in Table4. This β-mannanase exhibited the highest activity with Konjac glucomannan, enriched in glucose units. This finding suggests that β-mannanase of BE-91 preferentially hydrolyzes the β-1,4-linkage of the glucosylated mannan backbone.Table 4
Hydrolytic activity of the purified enzyme on different polysaccharides.
Substrate (0.5%, w/v)
Relative activity (%)a
Konjac glucomannan
100
Locust bean gum
88.15 ± 1.8
Carob galactomannan
91.85 ± 1.7
Guar galactomannan
35.70 ± 0.6
Ivory nut mannan
32.74 ± 0.3
1,4-Beta-D-mannan
46.22 ± 0.4
Wheat arabinoxylan
0
Beechwood xylan
0
Carboxymethyl cellulose
0
Assays were carried out at 65°C at pH 6.0 for 10 min in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer.
aData are mean ± SD, n
=
3.K
m and V
m
a
x values of this β-mannanase estimated by the Lineweaver-Burk plot were 7.14 mg/mL and 107.5 μmol/min/mL, respectively, for locust bean gum, versus 1.749 mg/mL and 33.45 μmol/min/mL for Konjac glucomannan, respectively. These results displayed higher affinity of β-mannanase towards natural Konjac glucomannan (V
m
a
x/K
m, 19.1 μmol/min/mg) than the locust bean gum (V
m
a
x/K
m, 15.0 μmol/min/mg), similar to the values obtained forPenicillium pinophilum C1 andPenicillium freii F63, hence constituting it as an adequate candidate in food industry for the production of oligosaccharides [17, 18, 39].


## 4. Conclusion

B. subtilis bacteria are abundant, moderately stable, and mostly nonpathogenic microorganisms. Our results indicated thatB. subtilis BE-91 could be considered a prominent candidate for the production of extracellular β-mannanase. In addition, this study developed an advanced purification approach, “two-step method,” with high efficiency, high yield, and easy operation. Furthermore, the β-mannanase purified from BE-91 was extremely stable at relatively high temperatures and various weak acidic or neutral pHs. Finally, the enzyme showed a higher affinity towards natural Konjac glucomannan, a major functional food material. Therefore, this β-mannanase, purified and characterized fromB. subtilis BE-91 for the first time, is suitable for inflammatory diseases.



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*Source: 6380147-2016-10-27.xml*