A Novel β-1,4-mannanase Isolated from Paenibacillus polymyxa KT551

Abstract

A β-1,4-mannanase producing bacterium was isolated from soil collected in Akita Prefecture, Japan. The bacterium was identified as Paenibacillus polymyxa KT551 and was shown to produce a novel β-1,4-mannanase. The novelty of the enzyme was established by its N-terminal amino acid sequence, molecular weight and isoelectric point. The isolated β-1,4-mannanase showed activity against mannotetraose, mannopentaose and mannohexaose to produce mannobiose, mannotriose and mannotetraose. However, the enzyme exhibited no activity against mannobiose and mannotriose. Moreover, the crude enzyme preparation of the bacterium had no or minimal β-mannosidase or α-galactosidase activity. Therefore, the enzyme preparation from P. polymyxa KT551 holds potential for the efficient production of mannooligosaccharides from natural resources of galactomannans.

Introduction

Waste disposal in the food processing industry is a major problem to be solved from the standpoints of environmental burden and economics. In Akita Prefecture, Japan, the waste disposal of soybean coat (episperm) from crushed natto production and byproduct generated in the processing of Japanese yam (Dioscorea japonica, yamaimo) are typical examples of the problem (Aspinall et al., 1967).

These wastes are known to contain large amounts of β-1,4-mannans with galactose branches; these galactomannans have been suggested as utilizable sources of galactomannooligosaccharide. A mannooligosaccharides mixture prepared from the residue of brewed coffee beans was registered as a “Food for Specified Health Use”, and is known as a prebiotic to improve the intestinal microflora (Asano et al., 2003). For the industrial production of mannooligosaccharides, application of commercially available β-1,4-mannanase preparations are proposed for the hydrolysis of natural resources having a β-1,4-mannan skeleton, such as konjac mannan, guar gum, tara gum and locust bean gum. However, almost all commercially available β-1,4-mannanase preparations show additional β-mannosidase activity, and large amounts of mannose and/or mannobiose are produced along with mannooligosaccharides (Elbein et al., 1977). Therefore, the production of β-1,4-mannanase without β-mannosidase or α-galactosidase activity is needed for the efficient production of galactomannooligosaccharides as prebiotics.

Our survey on β-1,4-mannanase producing bacteria in soil samples collected in Akita Prefecture, Japan, resulted in the isolation of a bacterium producing β-1,4-mannanase. In this paper, we report a novel β-1,4-mannanase isolated from the bacterium.

Materials and Methods

General    High-performance anion-exchange chromatography (HPAEC) was performed with a Dionex DX-500 system (Dionex, Sunnyvale, CA, USA) with a pulsed amperometric detector. A CarboPac PA-1 column (4 mm i.d. x 250 mm) with a PA-1 guard column was used. The mannooligosaccharides, D-mannose and D-galactose, were analyzed using the following gradient elution condition: 0.1 M NaOH (eluent A) and 0.1 M NaOH containing 0.5 M sodium acetate (eluent B) at a flow rate of 1 mL/min; 0 – 1 min, 100% eluent A; 1 – 25 min, a linear gradient of eluent B from 0% to 5% (v/v); 25 – 30 min, a linear gradient of eluent B from 5% to 100%.

TOYOPEARL Phenyl-650M (Tosoh, Tokyo, Japan), EAH Sepharose 4B (GE Healthcare Bio-Science, Pittsburgh, PA, USA), Bio-gel P-2 (Bio-Rad, Hercules, CA, USA) were used for column chromatography.

The molecular mass of the protein was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) using the Voyager System 4347 (Applied Biosystems, Foster City, CA, USA). Sinapinic acid was dissolved in 30% acetonitrile containing 0.1% trifluoroacetic acid (TFA), and used as the matrix.

Materials    Glucomannan of Amorphophallus konjac was obtained from Wako Pure Chemical Industries (Osaka, Japan). Galactomannan of guar gum, locust bean gum and yeast mannan from Saccharomyces cerevisiae was obtained from Sigma-Aldrich (St. Louis, MO, USA), and tara gum from Sansho (Osaka, Japan). Chitosan (low molecular weight) was obtained from Fulka Chemie (Buchs, Switzerland). RBB-Galactomannan, mannobiose (M2), mannotriose (M3), mannotetraose (M4), mannopentaose (M5) and mannohexaose (M6) were obtained from Megazyme International Ireland (Wicklow, Ireland). Copra meal was obtained from Fuji Oil Co., Ltd. (Tokyo, Japan). Sodium carboxymethylcellulose (CMC), pullulan and soluble starch and other chemicals were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan).

Isolation of β-1,4-mannanase producing bacterium    The screening of β-1,4-mannanase-producing bacteria was performed using a basal medium contained the following components in 1 L of distilled water (pH 6.5): KH₂PO₄ (2 g), NH₄NO₃ (2 g), MgSO₄·7H₂O (0.2 g), yeast extract (Difco, 2.5 g) and RBB-galactomannan (2 g). Utilization and production of gases from carbohydrates were determined with an API 50CHB (bioMérieux, Lyon, France).

16S rDNA sequencing    Total DNA was extracted from 2-day cultured cells. Purified genomic DNA was used for the amplification of 16S rRNA genes using the PCR method with PrimeSTAR^® HS DNA polymerase (Takara Bio, Inc. Tokyo, Japan) with the forward primer 9F and the reverse primer 1541R (Nakazawa et al., 2010). Amplification was carried out using a DNA thermal cycler (GeneAmp PCR System 9700; Applied Biosystems) as follows: 95°C for 3 min, followed by 30 cycles of denaturation (95°C, 30 s), primer annealing (55°C, 15 s), and primer extension (72°C, 1 min). At the end of the cycle, the reaction mixture was kept at 72°C for 5 min and then cooled to 4°C. The amplified 16S rDNA fragment was purified with Microcon-100 concentrators (Merck, Damstadt, Germany) and directly sequenced using an Applied Biosystems 373A DNA sequencer and the manufacturer's protocol for Taq cyclesequencing with fluorescent dye-labeled dideoxynucleotides (Perkin-Elmer, Waltham, MA, USA).

Assay for β-1,4-mannanase activity    The reaction mixture (1 mL), consisting of konjac glucomannan (1% (w/v)), 0.1 M of potassium phosphate buffer (KPB, pH 7.0) and the enzyme mixture, was incubated at 30°C for 30 min. The amount of reducing sugar formed by the reaction was measured by the Somogyi-Nelson method using D-mannose as the standard. One unit of enzyme activity was defined as the amount of the enzyme that released 1 µmol of reducing sugar per min. The amount of protein was determined by Lowry's method, using BSA as the standard.

Purification of β-1,4-mannanase Paenibacillus polymyxa    KT551 was cultured in a 2-L flask in medium consisting of copra meal (20 g), polypeptone (4.8 g), yeast extract (2.0 g), KH₂PO₄ (4.0 g), MgSO₄·7H₂O (0.2 g), and 400 mL of water, at 30°C for 72 h under shaking by a rotary-shaker (200 rpm). The culture broth was then centrifuged for 20 min at 10,000 x g, and the supernatant was used for the purification of β-1,4-mannanase.

The supernatant was dialyzed against water, and ammonium sulfate (13.2 g) was added to the dialyzed solution. Then, the solution was mounted on a column (1.5 cm i.d. x 16.5 cm) of TOYOPEARL Phenyl-650M equilibrated with 50 mM KPB (pH 7.0) in 1 M (NH₄)₂SO₄. The column was washed with 50 mM KPB (pH 7.0) in 1 M (NH₄)₂SO₄ at a flow rate of 500 mL/4.5 h, and then eluted with a linear gradient of 1 M to 0.6 M (NH₄)₂SO₄ in 50 mM PBS buffer (pH 7.0) at flow rate of 710 mL/14 h. Individual 10 mL fractions were collected and assayed for β-1,4-mannanase activity and protein amount.

The fractions showing β-1,4-mannanase activity were collected, and the buffer of the collected fractions was exchanged to 5 mM Tris-HCl buffer (pH 8.0) by an Amicon unit fitted with an Amicon PM3 ultrafiltration membrane (3 kDa cut-off), and was applied to a column (1.8 cm i.d. x 14 cm) of EAH Sepharose 4B equilibrated with 5 mM Tris-HCl buffer (pH 8.0). The column was washed with the same buffer at a flow rate of 400 mL/3.5 h, and then eluted with a linear gradient of 0.0 M to 0.8 M NaCl in 5 mM Tris-HCl buffer (pH 8.0) at flow rate of 640 mL/12 h. The amount of protein and β-1,4-mannanase activity of the factions (10 mL) were assayed. The fractions with β-1,4-mannanase activity were collected, and the combined solution was concentrated with an Amicon PM10 membrane (10 kDa cut-off).

Characterization of the isolated β-1,4-mannanase    SDS-PAGE was performed on a 5 – 20% gradient polyacrylamide gel (Atto, Tokyo, Japan) with Laemmli's buffer (Laemmli, 1970) containing 0.1% SDS. Molecular weight under denaturing conditions was determined using a Low Molecular Weight (LMW) Calibration Kit (GE Healthcare Bio-Science). Isoelectric focusing (IEF) was conducted as previously reported (Toeda et al., 2012). Optimum pH, temperature, and metal and substrate specificity of the isolated enzyme were determined using the assay method for β-1,4-mannanase activity mentioned above.

Chromatographic identification of hydrolysis products from mannooligosaccharide    The oligomers of D-mannose (dimer - hexamer, 5.0 mg each) were dissolved in water (1.0 mL), and the solutions (0.1 mL) were added to 0.9 mL of isolated enzyme (1 U/mL) in 10 mM KPB (pH 7.0). The mixtures were kept at 30°C for 4 h, and the hydrolysis products were quantitatively determined by HPAEC.

Results and Discussion

Isolation and identification of bacteria    Bacteria producing β-1,4-mannanase were screened using RBB-galactomannan as the carbon source, and the activities of β-1,4-mannanase were detected as a clear zone in the blue background. The most promising bacterial strain, KT551, was obtained from soil collected in Taiyu-mura, Akita, Japan. KT551 was characterized as mesophilic, fermentative, motile, spore-forming, rod-shaped (1.0 − 1.2 × 2.5 − 4.0 µm) and Gram-positive.

The sequences of strain KT551 showed 99% homology to Paenibacillus jamilae and P. polymyxa. Although P. polymyxa showed 98% homology to P. jamilae in 16sRNA, both strains showed 15% homology in genome DNA (Aguilera et al., 2001). Differences in characteristics of the strains included acid formation from methyl β-xyloside and gas formation from carbohydrate. KT551 was positive on acid formation from methyl β-xyloside and gas formation from glucose. KT551 was identified as P. polymyxa and was named as P. polymyxa KT551.

Purification of β-1,4-mannanase    The purification process of the β-1,4-mannanase produced by P. polymyxa KT-551 is summarized in Table 1. The dialyzed culture supernatant was purified by TOYOPEARL Phenyl column chromatography. The specific activity of the enzyme increased about 17-fold to 8.57 U/mg protein, and the yield of enzymatic activity was 55.1%. This fraction was further purified by EAH Sepharose 4B column chromatography. The specific activity of the enzyme increased about 28.4-fold to 14.3 U/mg protein, and the yield of enzymatic activity was 18.2%. Electrophoretic analysis of the purified β-1,4-mannanase on SDS-PAGE showed a single band around 34 kDa (Fig. 1). Furthermore, the precise molecular mass of the KT551 mannanase was determined as 32.5 kDa (data not shown) by MALDI-TOF-MS. The molecular mass of 32.5 kDa corresponds to the 34-kDa protein found by SDS-PAGE. The enzyme also showed a single band on native PAGE, and the isoelectric point (pI) was determined as 8.6.

Table 1. Purification of β-1,4-mannnanase from Paenibacillus polymyxa KT551

	Total activity (U)	Total Protein (mg)	Specific Activity (U/mg)	Purification (fold)	Yield (%)
Dialysis	2363	4691	0.504	1.00	100
TOYOPEARL Phenyl	1303	152	8.57	17.0	55.1
EAH-Sepharose 4B	430	30	14.3	28.4	18.2

Fig. 1.

SDS-PAGE of β-1,4-mannanase

The optimum temperature and pH of the isolated β-1,4-mannanase were determined as 30 – 40°C and 7.0 – 7.5, respectively. The enzyme activity was inhibited by Ag⁺, Cu²⁺, Mn²⁺, Fe²⁺, SDS and N-bromosuccinimide (Table 2). These results indicated that the tryptophan residue may correspond to the enzyme activity. The sequence of the N-terminal amino acid of the purified β-1,4-mannanase was determined as ASGFTVSGTKLTDSTGLPFV.

Table 2. Effect of various compounds on the activity of β-1,4-mannanase from Paenibacillus polymyxa KT551

Compounds	Relative activity(%)	Compounds	Relative activity(%)
Control	100	Mn2+	42
Ag+	27	Sn2+	88
Cu2+	31	Fe2+	25
Ba2+	85	Co2+	98
Ca2+	81	SDS	46
Al3+	58	EDTA	52
Pb2+	84	ICH2COOH	64
Zn2+	88	N-bromosuccinimide	16
Mg2+	84	N-ethylmaleimide	103
Cd2+	79

Many have reported on the isolation of β-1,4-mannanase from bacteria such as Bacillus subtilis (Emi et al., 1972), an alkalophilic Bacillus sp., (Akino et al., 1988), Bacillus circulans K-1 (Yosida et al., 1997) and Paenibacillus cookie (Li et al., 2012). Among them, the sequence of N-terminal amino acids of the β-1,4-mannanase isolated from B. circulans K-1 has high homology (85 %) to that isolated from P. polymyxa KT551. However, the molecular weight (62 kDa) and isoelectric point (pI = 5.4 – 6.2) of the enzyme isolated from B. circulans K-1 differed from those from P. polymyxa KT551. Therefore, the enzyme isolated from P. polymyxa KT551 was proved to be a novel β-1,4-mannanase.

Substrate specificity    The substrate specificity of the novel β-1,4-mannanase from P. polymyxa KT551 was determined, and the results are shown in Table 3. Konjac glucomannan and tara gum were shown to be good substrates, followed by locust bean gum. While guar gum also has a β-1,4-mannan structure, it was a poor substrate for the enzyme. The β-1,4-mannanase from P. polymyxa KT551 showed no hydrolytic activities against CMC, chitosan, pullulan, soluble starch and yeast mannan (α-1,6-mannan). Thus, the enzyme exhibits specific activity against β-1,4-mannosidic linkages of mannans. Moreover, the enzyme has no α-galactosidase activity; no D-galactose was detected as a hydrolysis product of tara gum and locust bean gum.

Table 3. Substrate specificity of β-1,4-mannanase isolated from Paenibacillus polymyxa KT551

Polysaccharides	Relative activity(%)	Main chain(sugar)
Konjac glucomannan	100	β-1,4(Man,Glc)
Tara gum	98	β-1,4(Man)
Locust bean gum	68	β-1,4(Man)
Guar gum	17	β-1,4(Man)
CMC	ND	β-1,4(Glc)
Chitosan	ND	β-1,4(GlcN)
Pullulan	ND	α-1,3(Glc)
Soluble starch	ND	α-1,4(Glc)
Yeast mannan	ND	α-1,6(Man)

In order to clarify the activities against oligomers, β-1,4-linked mannobiose - mannohexaose were enzymatically hydrolyzed. As shown in Table 4, mannobiose and mannotriose remained intact. Mannotetraose was almost intact; however, a small amount of mannotriose was detected. Mannopentaose and mannohexaose gave mannotetraose, mannotriose and mannobiose as main products, but mannose was not detected. In the case of mannopentaose, mannotetraose was obtained as one of the main products but the corresponding mannose was not detected. A reasonable explanation for this phenomenon remains to be clarified, and hypothetical glycosidation routes such as the formation of mannotetraose from mannobiose may be predicted. However, these results indicated that the enzyme obtained from P. polymyxa KT511 was shown to have no β-mannosidase activity. Therefore, P. polymyxa KT551 must be a potent commercial source of β-1,4-mannanase without β-mannosidase activity.

Table 4. Activity of β-1,4-mannanase isolated from Paenibacillus polymyxa KT551 against mannooligosaccharides

Substrates	Amount formed (%)
	M2	M3	M4	M5	M6
M2	100
M3		100
M4		5	95
M5	32	40	28	0
M6	18	46	36	0	0

In conclusion, the enzyme preparation from P. polymyxa KT551 was proved to have β-1,4-mannanase activity with no or minimal β-mannosidase or α-galactosidase activity. Therefore, the crude β-1,4-mannanase preparation prepared from P. polymyxa KT551 represents a potent tool for the efficient production of mannooligosaccharide, indicating its utility in resolving the problem of waste disposal.

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