Characterization of mannanase from Bacillus circulans NT 6.7 and its application in mannooligosaccharides preparation as prebiotic
© Pangsri et al. 2015
Received: 13 December 2014
Accepted: 26 November 2015
Published: 14 December 2015
This study focused on the characterization of mannanase from Bacillus circulans NT 6.7 for mannooligosaccharides (MOS) production. The enzyme from B. circulans NT 6.7 was produced using defatted copra meal as a carbon source. The mannanase was purified by ultrafiltration and column chromatography of Q-Sepharose. The purified protein (M1) was a dimeric protein with a 40 kDa subunit. The purified M1 exhibited optimum pH and temperature at pH 6.0 and 60 °C, respectively. It was activated by Mn2+, Mg2+, and Cu2+, and as inhibited by EDTA (45–65 %). The purified enzyme exhibited high specificity to beta-mannan: konjac (glucomannan), locust bean gum (galactomannan), ivory nut (mannan), guar gum (galactomannan) and defatted copra meal (galactomannan). The defatted copra meal could be hydrolyzed by purified M1 into mannooligosaccharides which promoted beneficial bacteria, especially Lactobacillus group, and inhibited pathogenic bacteria; Shigella dysenteria DMST 1511, Staphylococcus aureus TISTR 029, and Salmonella enterica serovar Enteritidis DMST 17368. Therefore, the mannanase from B. circulans NT 6.7 would be a novel source of enzymes for the mannooligosaccharides production as prebiotics.
In 2004, “prebiotic” was defined as ‘‘selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” (Gibson et al. 2004). Bifidobacteria and Lactobacilli are probiotic microbials that benefit the host by improving intestinal microbial balance (Gibson and Roberfroid 1995). Pathogenic bacteria (Clostridia bacteroides, Escherichia coli and Salmonella) produce toxins which can affect human gastrointestinal tracts, resulting in diarrhea, vomiting and nausea (Manning 2004). Some prebiotics, such as inulin, galactooligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharides (XOS), and mannooligosaccharides (MOS), are widely used in the market.
Mannooligosaccharides are derived from the cell wall of the yeast Saccharomyces cerevisiae is commercially available as a feed supplement (Ferket et al. 2002). However, they can also be produced from plant mannan, such as konjac, ivory nut, locust bean, palm kernel, coffee bean, and copra meal. Copra meal is a by-product of the coconut milk process and contains a large amount of mannose in the form of mannan. Mannan consists of repeating β-1, 4 mannose units and a few α-1, 6-galactose units attached to a β-1, 4 mannose backbone (Mohammad et al. 1996).
Mannanase was classified, based on the site of lysis in hydrolytic process, into two types: endo-β-1, 4-mannanase EC 18.104.22.168) and β-mannosidase (EC 22.214.171.124). Endo-β-1, 4-mannanase can randomly cleave bonds within mannan chain while β-mannosidase enzyme is capable of removing one or more mannose units from the ends of chains. Mannanase can hydrolyze copra meal into mannooligosaccharides. The effects of copra mannan hydrolysates in gastrointestinal tracts are to stimulate the growth of intestinal microflora and limit pathogenic bacteria. (Titapoka et al. 2008). Mannanases are produced by various microorganisms, including bacteria, yeast, and fungi. In particular, mannanase from Bacillus circulans NT 6.7 can hydrolyze mannan into mannooligosaccharides. Hydrolysates can inhibit the growth of pathogens (Salmonella serovar Enteritidis S003 and E. coli E010) and can promote the growth of probiotic bacteria (Lactobacillus reuteri KUB-AC5) (Phothichitto et al. 2006). This result suggests that mannanase from B. circulans NT 6.7 is suitable for the preparation of mannooligosaccharides. Therefore, this research aimed to characterize the purified mannanase from B. circulans NT 6.7 and its application in the preparation of mannooligosaccharides as prebiotics.
Results and discussion
Production and purification of mannanase
Purification table of mannanase from B. circulans NT 6.7
Total volumn (mL)
Total enzyme activity
Total protein (mg)
Specific activity (unit/mg protein)
Characterization of the purified mannanase
These results were similar to the results of Phothichitto et al. (2006), who reported the optimum pH and temperature of the crude enzyme from B. circulans NT 6.7 were pH 6.0 and 50 °C, respectively. Piwpankaew et al. (2014) also reported the optimum pH and temperature of recombinant β-mannanase of B. circulans NT 6.7 were pH 6.0 and 50 °C, respectively. The optimum pH and temperature of the purified M1 were similar to those of other bacterial mannanases. Mohammad et al. (1996) showed the optimum temperature of purified mannanase from Bacillus sp. KK01 was 60 °C. The purified mannanase from Bacillus licheniformis had optimum temperature and pH at 60 °C and pH 7.0 (Zhang et al. 2000). Therefore, the mannanase was optimum temperature at high temperature, making the enzyme more attractive for industrial applications.
Substrate specificity of purified M1
Relative activity (%)
Locust bean gum
Defatted copra meal
Xylan from birchwood
Xylan from oat spelt
Effect of metal ions and EDTA on purified M1 activity
Relative activity of M1 (%)
Hydrolysis Product of mannan
Hydrolysis product of mannan hydrolysed by the purified M1
Locust bean gum
Defatted copra meal
The purified M1 could hydrolyze konjac mannan, locust bean gum and defatted copra meal into mannooligosaccharides. However, the ratio of mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose (M2–M6) depended on the substrates. Moreover, the purified M1 tended to hydrolyze galactomannan (locust bean gum and defatted copra meal) into mannooligosaccharides more often than glucomannan (konjac mannan). Therefore, purified M1 could be galacto-mannanase, which would be suitable for mannooligosaccharide preparation from copra meal (galactomannan).
Enhancement/inhibition properties of defatted copra meal-hydrolysate (DCM-hydrolysate)
Defatted copra meal can be used as a substrate for mannanase production and mannooligosaccharide preparation. The purified M1 from B. circulans NT 6.7 was a dimeric protein and the pH and temperature optimum were 6.0 and 60 °C, respectively. The purified M1 was specified to mannan substrates; konjac mannan, locust bean gum, ivory nut, guar gum and defatted copra meal. The M1 was activated by Mn2+, Mg2+, and Cu2+ and inhibited by EDTA. The purified M1 hydrolyzed konjac mannan, locust bean gum and defatted copra meal into mannooligosaccharides. The ratio of mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose (M2–M6) was dependent on the substrates and tended to hydrolyze galactomannan (locust bean gum and defatted copra meal) into mannooligosaccharides rather than glucomannan (konjac). Therefore, purified M1 could be galacto-mannanase, which is suitable for mannooligosaccharide preparation from copra meal. The defatted copra meal hydrolysate could effectively promote the growth of beneficial bacteria and inhibit pathogenic bacteria more than commercial mannooligosaccharides, prepared from yeast cell wall. These results thus provide preliminary data regarding the potential of the defatted copra meal hydrolysate as a novel candidate for a prebiotic.
Microorganism and enzyme production
Bacillus circulans NT. 6.7 (Phothichitto et al. 2006) was cultivated in a producing medium: 1 % defatted copra meal, 3 % peptone, 1.5 %, KH2PO4, 0.06 %, MgSO4·7H2O, and 2.5 % corn steep liquor at pH 7.0 in a 5-l fermenter (Biostat-B). The cultivation was conducted at agitation speeds of 600 rpm, aeration 0.75 vvm (Feng et al. 2003) at 45 °C (Phothichitto et al. 2006) for 6 h. Cells were removed by centrifugation at 8000g for 15 min at 4 °C, and the supernatant was assayed for mannanase activity.
The β-mannanase was assayed by incubating a reaction mixture of 0.5 mL of sample and 0.5 mL of 50 mM potassium phosphate buffer at pH 7.0 with 1 % (w/v) locust bean gum at 60 °C for 60 min. The amount of reducing sugar released was determined by 3, 5-dinitrosalicylic acid (DNS) method using d-mannose as the standard (Miller 1959).
One unit of enzyme activity was defined as the amount of enzyme that produced 1 μm of reducing sugar per minute under the experimental conditions.
The protein concentration was determined by the method of Lowry et al. (1951). Bovine serum albumin was used as a standard.
Purification of Mannanase
The crude enzyme was concentrated 10× by ultrafiltration using a 10 kDa Mw cut-off membrane (Minimate TFF System, PALL, USA). Two mL of concentrated enzyme were applied to 1.6 × 20 cm Q-Sepharose column (Titapoka et al. 2008) and equilibrated with 20 mM Tris–HCl buffer (buffer A) at pH 8.5, with a flow rate of 3 mL/min. The column was washed with buffer A until there was no eluting protein. Then, the protein was eluted with 0–1 M sodium chloride in 20 mM Tris–HCl buffer (buffer B) at pH 8.5, eluting protein was detected by absorbance measurements at 280 nm. The fractions were collected using a fraction collector (ÄKTA explorer, GE Healthcare Life Sciences, Sweden). All fractions were measured for mannanase activity.
Determination of Molecular weight
The molecular weight of purified protein was determined by gel filtration (Sephacryl S-100, (GE healthcare). The maker of gel filtration calibration kit LMW (low molecular weight) use composed of aprotinin (6500 kDa), ribonuclease (13,700 kDa), carbonic anhydrase (29,000 kDa), ovalbumin (43,000), conalbumin (75,000 kDa) and Blue Dextran 2000 (GE healthcare).
The molecular weight and purity of the purified enzyme were also determined by SDS-PAGE according to Laemnli (1970) using 10 % acrylamide separating gel and stained with silver stain plus kit (Bio-Rad). The pre-stained marker (Bio-Rad) composed of myosin (195,755), β-galactosidase (107,181), bovine serum albumin (59,299), ovalbumin (41,220), carbonic anhydrase (27,578), soybean trypsin inhibitor (20,514), lysozyme (15,189) and aprotinin (6458) was used.
To confirm the activity of the purified proteins, native protein samples were separated on a 10 % acrylamide separating gel. Then, the native gel was incubated at 50 °C for 1–2 h. Congo red 0.1 % was used for staining. Congo red was washed by 1 M NaCl and the clear zone activity was fixed with 5 % acetic acid. Enzyme activity on the substrate gel was visualized as a clear zone against a blue background.
Determination of optimum pH and optimum temperature
The optimum pH and temperature of enzyme activity were determined at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 at 30, 40, 50, 60 and 70 °C, respectively. The following 50 mM buffer solutions were used: citrate (pH 3.0–6.0), phosphate (pH 6.0–8.0) and glycine-NaOH (pH 8.0–10.0).
Determination of pH stability and temperature stability
The effect of pH on enzyme stability was determined at pH 3.0–10.0 in 50 mM buffer: citrate (pH 3.0–6.0), phosphate (pH 6.0–8.0) and glycine-NaOH (pH 8.0–10.0). The enzyme solution was incubated in the buffer system at 60 °C for 60 min. The remaining enzyme activity was measured at temperature optimum for 60 min. Thermal stability of the enzyme was determined at 30, 40, 50, 60, 70, and 80 °C in 50 mM buffer at pH 6.0. After 60 min, the remaining enzyme activity was measured.
Effect of metal ions on enzyme activity
The effects of metal ions Li+, Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Zn2+, Ni2+, Co2+, urea, SDS, EDTA and β-mercaptoethanol on the enzyme activity were determined in the presence of 1 mM of each ion under optimum conditions.
Determination of substrate specificity
The activity of the purified enzyme was determined as previously described under optimum conditions on 1.0 % (w/v) substrates: locust bean gum (Sigma), alpha-mannan (Megazyme), ivory nut mannan (Megazyme), konjac mannan (Megazyme), guar gum (Megazyme), xylan (from oat spelts) (Sigma), xylan (from birchwood) (Sigma), carboxymethylcellulose (CMC) (Fluka), avicel (Megazyme), copra meal and defatted copra meal.
Determination of hydrolysis product
The 1 % substrates of konjac mannan, locust bean gum and defatted copra meal were hydrolyzed by the purified M1 at pH 6.0 at 60 °C for 120 min. The hydrolysis products were analyzed by HPLC under the following conditions: column, Aminex-HPX42C; mobile phase, DI water; column temperature, 75 °C; flow rate, 0.4 mL/min; and refractive index detector. The mannose (Fluka), mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose (Megazyme) were used as standards.
Enhancement/inhibition properties of defatted copra meal hydrolysate on beneficial bacteria/pathogenic bacteria
SB is the optical density of cell in medium with DCM-hydrolysate product/commercial mannooligosaccharides.
CB is the optical density of cell in medium without DCM-hydrolysate product/commercial mannooligosaccharides.
PP drafted the manuscript. All authors participated, contributed to design of research. All authors read and approved the final manuscript.
This research was financially supported by the following organizations, all of which are in Thailand: the Office of the Higher Education Commission; the Ministry of Education,Thailand; Valaya Alongkorn Rajabhat University under the Royal Patronage Pathumthani; Kasetsart University Research and Development Institute (KURDI); and the Center for Advanced Studies in Agriculture and Food, Institute for Advanced Studies, Kasetsart University Bangkok. We also thank the Center for Agricultural Biotechnology (CAB), Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom for supporting this research by allowing the use of their purification system.
The authors declare that they have no competing interests.
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