Characterization of mannanase from Bacillus circulans NT 6.7 and its application in mannooligosaccharides preparation as prebiotic

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.

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 3.2.1.78) and β-mannosidase (EC 3.2.1.25). 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.

Production and purification of mannanase
Defatted copra meal was used for mannannase production in a producing medium in 5-l fermenter cultivation: 600 rpm, 0.75 vvm and 45 °C. B. circulans NT 6.7 exhibited the highest cell growth and mannanase activity with 2.43 × 10 9 CFU/mL and 27.70 units/ mL respectively at 6 h. The crude enzyme was concentrated 10× by ultrafiltration and applied to anion-exchange chromatography. The result showed that the purified mannanase from Q-Sepharose chromatography had 628-folds of purification and specific activity of 295 units/mg protein (Table 1). The purified protein was named M1.
The molecular weight of purified M1 were 39.80 and 75.85 kDa, by gel filtration and the zymogram of purified M1 also showed two bands in active gel (Fig. 1a). N-terminal sequences of these two fractions (39.80 and 75.85 kDa) were the same and the  Fig. 1b. Therefore, the purified M1 could be a dimeric protein containing two subunits (40 kDa). The result was consisted with Piwpankaew et al. (2014) reported that the mannanase gene of B. circulans NT 6.7 consisted of 1083 nucleotides encoding 360 amino acid residues with theoretical molecular weight 40.29 kDa. The purified M1 also had the same molecular weight as other Bacillus species; Zakaria et al. (1998) reported that the mannanase from Bacillus subtilis KU-1 had a molecular weight of 39 kDa. The purified MAN 5 had a single band on SDS-PAGE at 40.5 kDa (Zhang et al. 2009).

Characterization of the purified mannanase
The effects of temperature on enzyme activity were determined for the purified M1. The optimum temperature was measured using the standard assay while varying the temperature from 30 to 70 °C. The optimum temperature was 60 °C for the 60 min assay (Fig. 2a). The effects of the pH value on β-mannanase activity were determined for purified enzyme using the standard assay and varying the pH from 3.0 to 10.0. The optimum pH β-mannanase activity was 6.0 (Fig. 2b). 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.
The effect of various metal ions and EDTA on the purified M1 activity was tested by adding 1 mM of the selected metal ions or EDTA to the enzyme reactions. The mannanase activity (M1) was activated by Mn 2+ (123.53 %), Cu 2+ (121.53 %) and Mg 2+ (116.26 %). While, the purified M1 was inhibited 45-65 % by EDTA, which indicated that the purified M1 could be a metalloenzyme (Table 3). The magnesium ion is the most widespread metal present in enzymes. Manganese ions function as enzyme activators and components of metalloenzymes (enzymes that contain a metal ion in their structure), and Cu 2+ is a metal ion that functions well as a redox center. Magnesium plays a central role as the essential partner of phosphate-containing substrates, including ATP, which is largely present in cells as Mg 2+ complex (Luthi et al. 1999).

Enhancement/inhibition properties of defatted copra meal-hydrolysate (DCM-hydrolysate)
The results demonstrated that DCM-hydrolysate and commercial mannooligo-saccharide from yeast cell walls could support the growth of beneficial bacteria. It was shown that probiotic bacteria could use the oligosaccharides in DCM-hydrolysate and commercial mannooligosaccharide to support their growth. DCM-hydrolysate could promote the growth of Lactobacillus sp. (8 strains) (Fig. 3) and inhibit the growth of pathogenic bacteria (5 strains) as shown in Fig. 4. This result was similar to the report by Phothichitto et al. (2006), in which the culture filtrate of B. circulans NT. 6.7 grown on copra meal could promote the growth of L. reuteri AC-5 up to 49.593 %, but could not inhibit S. serovar Enteritidis S003 or E. coli E010. Titapoka et al. (2008) reported that the copra meal hydrolysate by mannanase from Klebsiella oxytoca KUB-CW2-3 showed higher enhanced activity for the growth of L. reuteri KUB-AC5. The commercial mannooligosaccharides from yeast cell walls could stimulate the growth of beneficial bacteria and inhibit the growth of three strains of pathogenic bacteria (Shigella dysenteria DMST 1511, Staphylococcus aureus TISTR 029, and Salmonella enterica serovar Enteritidis DMST 17368). This result was similar to that of Line et al. (1997), who reported that yeast cell wall carbohydrates, especially mannose residue, were effective in preventing Salmonella sp. colonization. Newman (1994) reported that MOS derived from the outer cell wall of selected S. cerevisiae strains had the ability to adhere to pathogenic bacteria, such as Salmonella or E. coli. These results suggest that defatted copra meal-hydrolysate from purified M1 has the ability to promote beneficial bacteria and inhibit pathogenic bacteria. Therefore, defatted copra meal-hydrolysate from purified M1 could be a novel candidate for a prebiotic.

Conclusions
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 Mn 2+ , Mg 2+ , and Cu 2+ 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.

Enzyme assay
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.

Protein assay
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.

Zymogram analysis
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 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 + , Ca 2+ , Cu 2+ , Fe 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Ni 2+ , Co 2+ , 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 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
Defatted copra meal hydrolysate was prepared under optimal conditions three trials. 12 strains of beneficial bacteria and five strains of pathogenic bacteria were cultivated in 5 mL of media with 1 % DCM-hydrolysate or commercial mannooligosaccharides (yeast cell wall). Lactobacillus and Pediococcus were cultivated in 5 mL of MRS broth and BHI broth (Agnes et al. 2005) respectively, at 37 °C for 4 h under anaerobic conditions. Enterococcus and pathogenic bacteria were cultivated in 5 mL of BHI broth and NB medium, respectively, at 37 °C under aerobic conditions for 4 h. Cell growth was determined by measuring optical density at 600 nm. The enhancement and inhibition activities were calculated by the following equations (Phothichitto et al. 2006): 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.