World Journal
of Microbio/ogy
A 6iotechnofogy
Purification mannanase
11, 310-314
and some properties from f3acih~ sp.
of j?-
T. Ooi* and D. Kikuchi p-Mannanase produced by Bucillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NHJZ SO4 precipitation, adsorption to konjak gel, and calm chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 7O’C and was stable for at least 1 h between pH 5 and 10 and below 6O’C. Its activity was completely inhibited by Hg”. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose. Key
words:
Bacillus sp., mannan, p-mannanase.
p-Mannanase @-1,4-D-mannan endo-I,@-mannosidase; EC 3.2.1.78) catalyses the random hydrolysis of the &l+rnannosidic linkages in p-mannans (i.e. mannan, glucomannan, galactomannan and galactoglucomannan) yielding oligosaccharides (McCleary 1988). p-Mannans have been found in several plants, including the endosperms of copra and ivory palm nuts, guar beans, locust beans, coffee beans and the roots of konjak. Most of these saccharides are only used in the food and feed-processing industries. Recently, however, manno-oligosaccharides produced from konjak glucomannan were found to be one of the best growth factors for lactic bacteria (Kobayashi et aI. 1983). Endo-p-I,4mannanases are produced by various microorganisms, including bacteria and fungi, and also occur in animals and higher plants (Dekker & Rechards 1976). However, there are no commercial p-mannanases available. Various bacteria have been isolated from decayed konjak c&e and one strain, W-2, a Bacih sp., readily degraded fimannan. The present study is on the purification and properties of a p-mannanase from the culture supematant of this bacterium.
of Sugiyama ef al. (1972). Locust bean gum (Sigma) was used as galactomannan. Konjak gel was prepared from commercially available konjak cake by grinding the washed cake in 50 rnM phosphate buffer (pH 6.5). The gel was washed three times in the same buffer before use. Isolation
Enzyme
and Methods
Muferiu1s
Glucomannan was extracted from konjak powder by the procedure The authors ara with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321, Japan: fax: 0266 49 5401. ’ Corresponding author. @ 1995 Rapid Communications
of Oxford
Ltd
Microorganisms
Production
A seed culture (1 ml) of strain W-2 was added to 100 ml of liquid medium, containing I% konjak glucomannan, 0.5% polypeptone, 0.05% KHZP04, and 0.05% MgS04.7HZ0, at pH 6.5, and incubated for 16 h at 37’C in a SO&ml conical flask. The culture broth was then centrifuged for IO min at 13,000 x g and the supematant used as crude enzyme source. Enzyme
Materials
of j$Mamanase-producing
A sample of decayed konjak cake was suspended in sterilized water, spread on agar plates (containing 0.5% konjak powder, 0.5% polypeptone, 0.2% yeast extract, 0.1% KHZP04, 0.05% MgS04.7HZ0, and 1.5% agar, pH 7.0) and incubated for 48 to 72 h at 37°C. The microorganisms from these plates that could decrease the viscosity of the liquid medium without agar were isolated and further screened for mannanase activity. The highest mannanase activity was produced by one strain, W-2, after aerobic growth for 24 h at 37’C in the liquid medium.
Assay
A reaction mixture containing 5 mg locust bean galactomannan in 0.9 ml 0.1 r.4phosphate buffer (pH 7.0) and 0.1 ml enzyme solution was incubated for 10 min at 4O'C. The amount of reducing sugars produced was measured by the Somogyi-Nelson method. One unit was defined as the amount of enzyme which releases I prnol D-mannose equivalents of the reducing sugar per min under the reaction conditions. Protein was determined by the Lowry method, with BSA as standard.
~-Mannanase from Bacillus sp.
-~
0.15
50 40
O.lO 3o
~
20
~
lO
o
,£
0.05
160
180
200
220
240
0 260
Elution volume (ml) Figure 1. Chromatography of the ,8-mannanase on Sephacryl S-200 column. O--fl-Mannanase activity; O--Azso.
Homogeneity of Enzyme Homogeneity of the purified enzyme was investigated by PAGE on 7.5% gel according to standard methods. The gel was stained with 0.25% Coomassie Brilliant Blue G-250 to locate the protein bands.
Measurement of Mr The Mr of the enzyme was estimated by SDS-PAGE and gel filtration. SDS-PAGE was carried out in 12.5% gel using the method of Laemmli. Gel filtration was performed on a column of Sephadex G-IO0 (2.5 x 90 cm) equilibrated with 50 mM phosphate buffer (pH 6.5) containing 0.1% Triton X-IO0.
Figure 2. Estimation of molecular weight of ,8-mannanase by SDS-PAGE. M--Standard marker proteins [1--Phosphorylase b
Purification of ~-Mannanase
equilibrated with 10 mM phosphate buffer (pH 6.0), and eluted with the same buffer. Adsorbed enzyme was eluted in a linear gradient of NaCI (0 to 0.4 M) in the buffer. The active fractions (250 ml), eluted at about 0.1 M NaC1, were collected and concentrated by ultrafiltration. Sephacryl S-200 Column Chromatography.The concentrated enzyme was put on a column (3 x 145 cm) of Sephacryl S-200 (Pharmacia) equilibrated with 50 mM phosphate buffer (pH 6.5) containing 0.1 M NaC1 and eluted with the same buffer (Figure 1). The purified enzyme was confirmed to be homogeneous by PAGE (data not shown).
The enzyme was purified by a six-step process. (NH4)zSO4 Fractionation. Solid (NH4)2SO4 sulphate was added to 2l culture supematant to 80% saturation. After standing overnight, the resulting precipitate was collected by filtration with the aid of Celite and dissolved in 70 ml 50 mM phosphate buffer (pH 6.5) and dialysed against the same buffer for 24 h. Konjak-gel Adsorption. The dialysed solution (100 ml) was adsorbed by mixing with konjak gel (150g) equilibrated with 50 mM phosphate buffer (pH 6.5) at 2:3 (w/v) at O°C. The mixture was stirred slowly on ice. After 2 h, the supematant was removed by centrifugation and the konjak gel washed twice with buffer, resuspended in a small volume of the buffer and gently mixed at 37°C for 12 h to release the enzyme. Insoluble materials were then removed by centrifugation and the supematant (200 ml) dialysed against the buffer. First DEAE-cellulose Column Chromatography. The dialysed enzyme solution (250 ml) was applied to a column (2.2 x 15 cm) of DEAE-cellulose equilibrated with the phosphate buffer and eluted with the buffer. The activity was not absorbed on this column and the unabsorbed fractions (330 m[) were collected and concentrated to 25 ml by ultrafiltration (UK-IO membrane filter; Advanteck, Tokyo). Sephadex G-I00 Column Chromatography. The concentrated enzyme solution was put on a Sephadex G-100 column (3 x 55 cm; Pharmacia) equilibrated with I0 mM phosphate buffer (pH b.O). The activity was eluted near the void fractions, and the active fractions were pooled (115 ml). Second DEAE-cellulose Column Chromatography. The enzyme solution was run through a DEAE-cellulose column (2.2 x 15 cm)
(97.4 kDa); 2--BSA (66.3 kDa); 3--aldolase (42.4 kDa); 4~carbonic anhydrase (30.0 kDa); 5~trypsin inhibitor (20.1 kDa); 6--lysozyme (14.4 kDa)]; C--,8-mannanase.
Results
Identification of Mannolytic Bacterium The isolated bacterial strain, W-2, was classified, according to Claus & Berkeley (1986), as a bacterium belonging to the genus Bacillus.
Purification of ~-Mannanase /~-Mannanase was purified 57-fold from the culture supernatant of Bacillus W-2. A typical purification is shown in Table 1.
Properties of the Enzyme The Mr of the purified/l-mannanase was estimated to be
World Journal of Microbiology & Biotechnology, Vol I I, I995
,~ 1 1
T. Ooi und D. Kikuchi
Table
1. Purification
of /I-mannanase.
Step Culture supernatant (NH&SO, (0 to 80% saturation Konjak-gel adsorption DEAE-cellulose Sephadex G-100 DEAE-cellulose Sephacryl S-200
Total
activity
(U)
Total
9130 8830 4130 3580 2700 1750 994
fraction)
protein
(mg)
Speclflc (U/mg
728 513 188 100 18 6.7 1.4
#
1
m
1
6
6
10
12
Figure 3. Effect of pH (A) and temperature (B) on b-mannanase activity (0) and stability were 0.2 M Mcllvaine buffer (pH 3.5 to 8.5) and 0.2 M glycine /NaOH. (pH 8.5 to 11).
Recovery
12.5 16.8 21.8 35.6 149 261 710
(a).
(%)
100 95 45 39 30 19 10
Temperature
PH
40 kDa by SDS-PAGE (Figure 2) whereas gel filtration on Sephadex G-100 indicated it to be 36 kDa. The effects of pH on the enzyme are shown in Figure 3A. The activity of the enzyme was assayed between pH 3.5 and 12 at ~o’C. Maximal activity was found at pH 7 although the enzyme appeared stable between pH 5 and pH 10 at 40°C for 1 h. The effects of temperature were examined between 30 and t30°C at pH 6.5 (Figure 3B). The highest activity of the enzyme was obtained at 70’C and the enzyme was stable when incubated at pH 7 for 1 h at
of Microbio/ogy
A 6iotechnofogy
Purification mannanase
11, 310-314
and some properties from f3acih~ sp.
of j?-
T. Ooi* and D. Kikuchi p-Mannanase produced by Bucillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NHJZ SO4 precipitation, adsorption to konjak gel, and calm chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 7O’C and was stable for at least 1 h between pH 5 and 10 and below 6O’C. Its activity was completely inhibited by Hg”. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose. Key
words:
Bacillus sp., mannan, p-mannanase.
p-Mannanase @-1,4-D-mannan endo-I,@-mannosidase; EC 3.2.1.78) catalyses the random hydrolysis of the &l+rnannosidic linkages in p-mannans (i.e. mannan, glucomannan, galactomannan and galactoglucomannan) yielding oligosaccharides (McCleary 1988). p-Mannans have been found in several plants, including the endosperms of copra and ivory palm nuts, guar beans, locust beans, coffee beans and the roots of konjak. Most of these saccharides are only used in the food and feed-processing industries. Recently, however, manno-oligosaccharides produced from konjak glucomannan were found to be one of the best growth factors for lactic bacteria (Kobayashi et aI. 1983). Endo-p-I,4mannanases are produced by various microorganisms, including bacteria and fungi, and also occur in animals and higher plants (Dekker & Rechards 1976). However, there are no commercial p-mannanases available. Various bacteria have been isolated from decayed konjak c&e and one strain, W-2, a Bacih sp., readily degraded fimannan. The present study is on the purification and properties of a p-mannanase from the culture supematant of this bacterium.
of Sugiyama ef al. (1972). Locust bean gum (Sigma) was used as galactomannan. Konjak gel was prepared from commercially available konjak cake by grinding the washed cake in 50 rnM phosphate buffer (pH 6.5). The gel was washed three times in the same buffer before use. Isolation
Enzyme
and Methods
Muferiu1s
Glucomannan was extracted from konjak powder by the procedure The authors ara with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321, Japan: fax: 0266 49 5401. ’ Corresponding author. @ 1995 Rapid Communications
of Oxford
Ltd
Microorganisms
Production
A seed culture (1 ml) of strain W-2 was added to 100 ml of liquid medium, containing I% konjak glucomannan, 0.5% polypeptone, 0.05% KHZP04, and 0.05% MgS04.7HZ0, at pH 6.5, and incubated for 16 h at 37’C in a SO&ml conical flask. The culture broth was then centrifuged for IO min at 13,000 x g and the supematant used as crude enzyme source. Enzyme
Materials
of j$Mamanase-producing
A sample of decayed konjak cake was suspended in sterilized water, spread on agar plates (containing 0.5% konjak powder, 0.5% polypeptone, 0.2% yeast extract, 0.1% KHZP04, 0.05% MgS04.7HZ0, and 1.5% agar, pH 7.0) and incubated for 48 to 72 h at 37°C. The microorganisms from these plates that could decrease the viscosity of the liquid medium without agar were isolated and further screened for mannanase activity. The highest mannanase activity was produced by one strain, W-2, after aerobic growth for 24 h at 37’C in the liquid medium.
Assay
A reaction mixture containing 5 mg locust bean galactomannan in 0.9 ml 0.1 r.4phosphate buffer (pH 7.0) and 0.1 ml enzyme solution was incubated for 10 min at 4O'C. The amount of reducing sugars produced was measured by the Somogyi-Nelson method. One unit was defined as the amount of enzyme which releases I prnol D-mannose equivalents of the reducing sugar per min under the reaction conditions. Protein was determined by the Lowry method, with BSA as standard.
~-Mannanase from Bacillus sp.
-~
0.15
50 40
O.lO 3o
~
20
~
lO
o
,£
0.05
160
180
200
220
240
0 260
Elution volume (ml) Figure 1. Chromatography of the ,8-mannanase on Sephacryl S-200 column. O--fl-Mannanase activity; O--Azso.
Homogeneity of Enzyme Homogeneity of the purified enzyme was investigated by PAGE on 7.5% gel according to standard methods. The gel was stained with 0.25% Coomassie Brilliant Blue G-250 to locate the protein bands.
Measurement of Mr The Mr of the enzyme was estimated by SDS-PAGE and gel filtration. SDS-PAGE was carried out in 12.5% gel using the method of Laemmli. Gel filtration was performed on a column of Sephadex G-IO0 (2.5 x 90 cm) equilibrated with 50 mM phosphate buffer (pH 6.5) containing 0.1% Triton X-IO0.
Figure 2. Estimation of molecular weight of ,8-mannanase by SDS-PAGE. M--Standard marker proteins [1--Phosphorylase b
Purification of ~-Mannanase
equilibrated with 10 mM phosphate buffer (pH 6.0), and eluted with the same buffer. Adsorbed enzyme was eluted in a linear gradient of NaCI (0 to 0.4 M) in the buffer. The active fractions (250 ml), eluted at about 0.1 M NaC1, were collected and concentrated by ultrafiltration. Sephacryl S-200 Column Chromatography.The concentrated enzyme was put on a column (3 x 145 cm) of Sephacryl S-200 (Pharmacia) equilibrated with 50 mM phosphate buffer (pH 6.5) containing 0.1 M NaC1 and eluted with the same buffer (Figure 1). The purified enzyme was confirmed to be homogeneous by PAGE (data not shown).
The enzyme was purified by a six-step process. (NH4)zSO4 Fractionation. Solid (NH4)2SO4 sulphate was added to 2l culture supematant to 80% saturation. After standing overnight, the resulting precipitate was collected by filtration with the aid of Celite and dissolved in 70 ml 50 mM phosphate buffer (pH 6.5) and dialysed against the same buffer for 24 h. Konjak-gel Adsorption. The dialysed solution (100 ml) was adsorbed by mixing with konjak gel (150g) equilibrated with 50 mM phosphate buffer (pH 6.5) at 2:3 (w/v) at O°C. The mixture was stirred slowly on ice. After 2 h, the supematant was removed by centrifugation and the konjak gel washed twice with buffer, resuspended in a small volume of the buffer and gently mixed at 37°C for 12 h to release the enzyme. Insoluble materials were then removed by centrifugation and the supematant (200 ml) dialysed against the buffer. First DEAE-cellulose Column Chromatography. The dialysed enzyme solution (250 ml) was applied to a column (2.2 x 15 cm) of DEAE-cellulose equilibrated with the phosphate buffer and eluted with the buffer. The activity was not absorbed on this column and the unabsorbed fractions (330 m[) were collected and concentrated to 25 ml by ultrafiltration (UK-IO membrane filter; Advanteck, Tokyo). Sephadex G-I00 Column Chromatography. The concentrated enzyme solution was put on a Sephadex G-100 column (3 x 55 cm; Pharmacia) equilibrated with I0 mM phosphate buffer (pH b.O). The activity was eluted near the void fractions, and the active fractions were pooled (115 ml). Second DEAE-cellulose Column Chromatography. The enzyme solution was run through a DEAE-cellulose column (2.2 x 15 cm)
(97.4 kDa); 2--BSA (66.3 kDa); 3--aldolase (42.4 kDa); 4~carbonic anhydrase (30.0 kDa); 5~trypsin inhibitor (20.1 kDa); 6--lysozyme (14.4 kDa)]; C--,8-mannanase.
Results
Identification of Mannolytic Bacterium The isolated bacterial strain, W-2, was classified, according to Claus & Berkeley (1986), as a bacterium belonging to the genus Bacillus.
Purification of ~-Mannanase /~-Mannanase was purified 57-fold from the culture supernatant of Bacillus W-2. A typical purification is shown in Table 1.
Properties of the Enzyme The Mr of the purified/l-mannanase was estimated to be
World Journal of Microbiology & Biotechnology, Vol I I, I995
,~ 1 1
T. Ooi und D. Kikuchi
Table
1. Purification
of /I-mannanase.
Step Culture supernatant (NH&SO, (0 to 80% saturation Konjak-gel adsorption DEAE-cellulose Sephadex G-100 DEAE-cellulose Sephacryl S-200
Total
activity
(U)
Total
9130 8830 4130 3580 2700 1750 994
fraction)
protein
(mg)
Speclflc (U/mg
728 513 188 100 18 6.7 1.4
#
1
m
1
6
6
10
12
Figure 3. Effect of pH (A) and temperature (B) on b-mannanase activity (0) and stability were 0.2 M Mcllvaine buffer (pH 3.5 to 8.5) and 0.2 M glycine /NaOH. (pH 8.5 to 11).
Recovery
12.5 16.8 21.8 35.6 149 261 710
(a).
(%)
100 95 45 39 30 19 10
Temperature
PH
40 kDa by SDS-PAGE (Figure 2) whereas gel filtration on Sephadex G-100 indicated it to be 36 kDa. The effects of pH on the enzyme are shown in Figure 3A. The activity of the enzyme was assayed between pH 3.5 and 12 at ~o’C. Maximal activity was found at pH 7 although the enzyme appeared stable between pH 5 and pH 10 at 40°C for 1 h. The effects of temperature were examined between 30 and t30°C at pH 6.5 (Figure 3B). The highest activity of the enzyme was obtained at 70’C and the enzyme was stable when incubated at pH 7 for 1 h at
