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Eur Food Res TechnolDOI 10.1007/s00217-014-2170-7

ORIgInal PaPER

Purification and characterization of an alkali‑thermostable β‑mannanase from Bacillus nealsonii PN‑11 and its application in mannooligosaccharides preparation having prebiotic potential

Prakram Singh Chauhan · Prince Sharma · Neena Puri · Naveen Gupta

Received: 21 September 2013 / Revised: 9 January 2014 / accepted: 16 January 2014 © Springer-Verlag Berlin Heidelberg 2014

the purified β-mannanase from B. nealsonii Pn-11 make this enzyme attractive for biotechnological applications.

Keywords Bacillus nealsonii · alkali-thermostable mannanase · Endo-1,4-β-d-mannanase · Purification · Mannooligosaccharides (MOS) · Food industry

Introduction

Hemicellulose is the second most abundant heteropoly-mer present in nature, usually associated with cellulose and lignin in plant cell walls. The two most important and representative hemicelluloses are hetero-1,4-β-d-xylans and the hetero-1,4-β-d-mannans [1]. Mannan is the pre-dominant hemicellulosic polysaccharide in softwoods from gymnosperms. β-Mannanase (endo-1,4-β-d-mannanase, EC 3.2.1.78) is a hydrolase that catalyzes the random hydrolysis of β-1,4-mannosidic linkages in the main chain of mannans and releases linear/branched oligosaccharides of various lengths [2].

Various mannanases from fungi, yeasts and bacteria as well as from germinating seeds of terrestrial plants have been produced and characterized [1, 2]. Microbial man-nanases are mainly extracellular and can act in a wide range of pH and temperature because of which they have found various applications in pharmaceutical, food and feed technology, coffee extraction, bioethanol production, oil and textile industries and as slime control agents [1–4].

In the pulp and paper industry, alkali-thermostable man-nanase can act synergistically with xylanase as a biological prebleaching agent, allowing a significant reduction in the use of bleaching chemicals like chlorine and thereby lower-ing the levels of hazardous halogenated organic compounds released into the environment [5, 6]. Mannanase can also be

Abstract an alkali-thermostable β-mannanase from Bacillus nealsonii Pn-11 was purified 38.96-fold to homo-geneity with specific activity of 2,288.90 ± 27.80 U mg−1 protein and final recovery of 8.92 ± 0.09 %. The purified β-mannanase was an extracellular monomeric protein with a molecular mass of 50 kDa on SDS–PagE. The first 20 n-terminal amino acid sequence of mannanase enzyme was MVVKKlSSFIlIlllVTSal. The optimal tempera-ture and pH for enzyme were 65 °C and 8.8, respectively. It was completely stable at 60 °C for 3 h and retained >50 ± 1.0 % activity at 70 °C up to 3 h. The β-mannanase was highly stable between pH 5–10 and retained >85 % of the initial activity for 3 h. The metal ions ni+2, Co+2, Zn+2 and Mg+2 enhanced the enzyme activity. The enzyme remained stable after 3 h of preincubation with most of the tested organic solvents. according to substrate specificity study, the purified mannanase had high specificity to locust bean gum which was degraded mainly to mannooligosac-charides (MOS) like mannotriose, mannotetraose and man-nopentose. These MOS enhanced the growth of Lactobacil-lus casei but inhibited the growth of Salmonella enterica indicating potential prebiotic properties. The properties of

Electronic supplementary material The online version of this article (doi:10.1007/s00217-014-2170-7) contains supplementary material, which is available to authorized users.

P. S. Chauhan · P. Sharma · n. gupta (*) Department of Microbiology, BMS Block, Panjab University, Sector-14, Chandigarh 160014, Indiae-mail: ngmicro12@gmail.com

n. Puri Department of Microbiology, guru nanak Khalsa College, City Centre Road, Yamunanagar 135001, Haryana, India

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used for the production of mannooligosaccharides (MOS) from cheap agricultural by-products such as copra mannan as well as locust bean gum. These MOS are non-digestible oligosaccharides possessing important physiological prop-erties to behave as dietary fiber and prebiotics. They have beneficial effect on the growth of intestinal microorgan-isms, decreasing enteric pathogenic bacteria and improving the integrity of intestinal mucosa, thereby improving the human health [1, 2].

Bacillus nealsonii-Pn11 isolated in our laboratory pro-duces a highly alkali-thermostable β-mannanase. There are only few reports on mannanase active at alkaline pH as well as at high temperature [7]. Moreover, ManPn11 is not only active at high pH but also highly stable at alkaline pH which makes its more useful for degradation of hemicel-luloses which are more soluble in alkaline range and for application in industrial processes which are carried out at high pH and temperature.

Owing to the increasing biotechnological importance of alkali-thermostable mannanases, the present study was undertaken to purify and characterize a highly alkali-ther-mostable β-mannanase from B. nealsonii Pn-11 as well as to explore the role of this enzyme in food industry for the production of mannooligosaccharides having potential prebiotic property.

Materials and methods

locust bean gum (lBg), guar gum (gg), Sephadex-150 and diethylaminoethyl cellulose (DEaE-cellulose) were purchased from Sigma-aldrich, USa. all other chemicals were of analytical grades.

Microorganisms and culture conditions

The bacterial strain B. nealsonii Pn-11 capable of produc-ing extracellular β-mannanase (ManPn11) was isolated in our laboratory and used for the production of mannanase (genBank accession no. Jn624311, MTCC no. 11401). It was maintained on minimal medium lBg (0.5 %) slant and stored at 4 °C. The minimal medium (pH 8.0) used for mannanase production in this study contained FeSO4·7H20 (0.01 gl−1), CaCl2·2H2O (0.05 gl−1), MgSO4·7H2O (0.2 gl−1), KH2PO4 (2.32 gl−1), K2HPO4 (7.5 gl−1), nH4nO3 (0.3 gl−1) and locust bean gum (8.0 gl−1) [4]. The minimal medium was inoculated with 0.1 % inocu-lum of 18-h seed culture and incubated at 37 °C for 96 h. The cells were separated by centrifugation at 7,826×g for 10 min at 4 °C, and the cell-free supernatant was used as enzyme. Lactobacillus casei (MTCC no. 1423) was grown in de Man–Rogosa–Sharpe (MRS) broth at 37 °C for 12–15 h, while Salmonella enterica (MTCC no. 3219) was

cultivated in nutrient broth (nB) under the same conditions with shaking (150 rpm) at 37 °C.

assay for mannanase activity

Mannanase activity was assayed by measuring the amount of reducing sugars released by the enzyme using dinitro-salicylic acid (DnSa) method [8]. The mannanase assay mixture contained 0.5 ml of 1.0 % (w/v) locust bean gum (substrate) prepared in 100 mM glycine–naOH buffer, pH 8.8 and 0.5 ml of appropriately diluted enzyme. The reac-tion mixture was maintained at 65 °C for 5 min, and then, 3 ml of DnSa reagent was added and boiled for 10 min. The optical density was measured at 560 nm. One unit of mannanase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar as a d-mannose standard per minute by one ml of enzyme.

Protein assay

Protein concentration was determined by the method of lowry et al. [9]. Bovine serum albumin was used as the standard. The protein eluted with column chromatography was monitored by taking absorbance at 280 nm.

Protein purification and molecular weight determination

The crude mannanase (ManPn11) was purified to homo-geneity by using combination of ammonium sulfate pre-cipitation (60–80 %), gel filtration (Sephadex g-150) and ion-exchange chromatography (DEaE-Cellulose). all the purification steps were performed at 4 °C. Precipitation was done by first excluding the undesired protein with 40–60 % ammonium sulfate concentration and then salting out the proteins containing desired mannanase with 60–80 % ammonium sulfate. The precipitated proteins were col-lected by centrifugation (7,826×g, 10 min, 4 °C), dissolved in 100 mM glycine–naOH (pH 8.8) buffer and dialyzed against the same buffer. Further purification was done by gel filtration followed by ion-exchange chromatography. Sample from ammonium sulfate precipitation was loaded on a Sephadex-150 column (40 × 1.5 cm2) and equilibrated with 100 mM glycine–naOH (pH 8.8), and the protein was eluted at a flow rate of 1.0 ml min−1. The active frac-tions were pooled and concentrated by polyethylene gly-col (PEg) and then applied to a DEaE-cellulose column (15 × 1.2 cm2) equilibrated with 50 mM phosphate buffer, pH 7.0. The bound mannanase was eluted with 0–1.0 M naCl gradient at a flow rate of 0.8 ml min−1. The active fractions were pooled, concentrated by PEg and dialyzed against the same buffer. With the purified enzyme, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PagE) was performed using 12.5 % (w/v) gel, by the

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method of laemmli [10]. gel was stained with Coomassie Brilliant Blue R-250 and destained with methanol:acetic acid:water (4:1:5). The molecular weight of the purified mannanase enzyme was determined by SDS–PagE using commercial protein marker (Chromous Biotech PMn01). Zymogram was obtained by co-polymerizing 1.0 % (w/v) lBg with 12.5 % (w/v) polyacrylamide gel. after electro-phoresis, the gel was soaked in 25 % (v/v) isopropanol with gentle shaking to remove the SDS and renature the proteins in the gel. The gel was then washed four times at 4 °C in 100 mM glycine–naOH buffer (pH 8.8) for 30 min. after a further incubation for 45 min at 50 °C, zymogram was stained for residual carbohydrates with Congo red solution (0.1 %, w/v), destained with 1 M naCl [11]. The activity bands were observed as clear colorless areas.

n-terminal amino acid sequence

The purified mannanase was hydrolyzed in a PicoTag work-station using HCl vapor (6 M) at 100–110 °C for 24 h in the presence of 0.01 % phenol and 0.003 % β-mercaptoethanol. amino acids were analyzed using a HP 1100 amino acid analyzer. The n-terminal amino acid sequence was deter-mined by automated Edman degradation using PROCISE 491 HT protein sequencer (applied Biosystems, USa). n-terminal sequence homology was analyzed using Basic local alignment Search Tool (BlaST) database.

Effect of temperature on the activity and stability of β-mannanase

The optimal temperature for mannanase activity was deter-mined by performing enzyme assays in the temperature ranges of 40–85 °C. Thermostability of the purified man-nanase was analyzed by incubating the suitably diluted purified enzyme in 100 mM glycine–naOH buffer (pH 8.8) at temperatures between 60 and 80 °C up to 3 h. Samples were withdrawn sequentially at different time intervals, and residual activity was measured under standard assay conditions.

Effect of pH on the activity and stability of β-mannanase

The optimal pH of the enzyme was determined by prepar-ing the substrate (1.0 % lBg) as well as suitable enzyme dilutions in the buffers of different pH values, i.e., pH 7.2 (phosphate buffer), pH 7.6 (Tris–HCl buffer), pH 8.0 (Tris–HCl buffer), pH 8.4 (Tris–HCl buffer), pH 8.8 (glycine–naOH Buffer) and pH 9.2 (glycine–naOH Buffer) and performing the assay under standard conditions. The pH stability of purified mannanase was determined by measur-ing the residual enzyme activity under the standard condi-tions after preincubation of the enzyme in various buffers

(100 mM each) of pH 3.0–12.0 at 50 °C up to 3 h without the substrate.

Effect of organic solvents on the stability of enzyme

To determine the effect of organic solvents (dimethyl sul-foxide, methanol, ethanol, acetone, formaldehyde, ethyl acetate, chloroform, benzene, xylene, hexane, petroleum ether) on the stability of mannanase, 1.0 ml of suitably diluted purified enzyme was mixed with 1 ml of different organic solvents and then incubated at 37 °C for 3 h with constant shaking (150 rpm). The residual activity was measured using the standard assay method.

Effect of detergents, chelating agents, oxidizing agents, reducing agents and metal ions on enzyme activity

To study the effects of detergents (sodium dodecyl sul-fate, cetyl trimethyl ammonium bromide, polyethylene glycol, Tween-20, Tween-80), chelating agents (sodium citrate, ethylene diamine tetraacetic acid, sodium azide), oxidizing agents (hydrogen peroxide, ammonium persul-fate, potassium iodide), reducing agents (ascorbic acid, β-mercaptoethanol) at 1 % concentration and metal ions (na+, Ca+2, ni+2, Mg+2, li+, Zn+2, K+, Co+2, Hg+2, Fe+2, Cu+2, Mn+2, Fe+3) at 1 and 5 mM concentrations on the enzyme activity, suitably diluted purified enzyme was pre-incubated with reagents and metal ions for 1 h at 37 °C with constant shaking (150 rpm) and then enzyme assay was performed under standard conditions.

Substrate specificity and kinetic parameters

The substrate specificity of the enzyme was determined by measuring the enzyme activity after incubation of the enzyme in 100 mM glycine–naOH buffer (pH 8.8) con-taining 1.0 % of lBg, guar gum and copra mannan at 65 °C for 5 min. amount of the reducing sugars produced was estimated using DnSa method described above.

For the kinetic (Km, Vmax and Vmax/Km) experiments, sub-strates (i.e., locust bean gum, guar gum and copra mannan) were used at concentrations between 5 and 15 mg ml−1 and enzyme assay was done under standard conditions. The data were analyzed using the nonlinear regression com-puter program graphPad Prism.

analysis of the hydrolysis products

For analyzing the product of hydrolysis, enzyme was incu-bated with 1.0 % (w/v) lBg at 55 °C for 6 h. The excess enzyme was removed by ultrafiltration using a 10 kDa cutoff membrane. The hydrolysis products were analyzed by using high-pressure anion-exchange chromatography

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(HPaEC) with a model 2500 HPaEC 2500 system (Dionex, USa). The amount of MOS was estimated indirectly by estimating the amount of reducing groups released by the hydrolysis reaction.

Evaluation of prebiotics properties

Mannooligosaccharides (MOS) obtained by the hydrolysis of lBg by ManPn11 were used to determine the poten-tial prebiotic properties. L. casei and S. enterica were used as the target strains to determine the possible growth effect of MOS. Optical density of the culture solution of target strains was adjusted to 0.25 at 600 nm. Culture solution of each target strain (1 %) and different amount of MOS, equivalent to 1–3 mg ml−1 reducing sugars and con-trol (without MOS), were transferred into 10 ml of MRS medium for L. casei and nB medium for S. enterica. The mixture was incubated at 37 °C for 6 h, and then, colony-forming unit (cfu) was determined by the standard plate count assay using either de Man Rogosa Sharpe (MRS) agar or nutrient agar. The prebiotic properties of MOS were defined as growth-enhancing activity of L. casei and inhibition activity of S. enterica according to the following equations:

where M and C were the colony-forming unit (cfu) obtained in the growth experiments containing MOS and the cell number in control experiments (without MOS), respectively (log cfu ml−1).

Statistical analysis

all the experiments were carried out in triplicates, and the mean ± SD has been plotted. Data were analyzed using analysis of variance (anOVa) by Sigma Stat version 2.03, and values which were statistically significant (P value ≤0.05) were taken.

Results and discussion

Protein purification and molecular weight determination

The results of the purification procedure are summarized in Table 1. It was observed that after the final purification step, the enzyme was purified to 38.96 ± 0.32-fold with a recov-ery of 8.92 ± 0.09 %. The specific activity determined using locust bean gum as substrate was 2,288.90 ± 27.80 U mg−1 protein. SDS–PagE analysis of purified enzyme showed a major band of 50 kDa (Fig. 1a) which also coincided with active band on zymograph (Fig. 1b). This was similar to

Enhancing activity = M − C;

Inhibition activity = C − M;

Tabl

e 1

Sum

mar

y of

the

puri

ficat

ion

proc

edur

e fo

r β

-man

nana

se f

rom

B. n

eals

onii

Pn

-11

Puri

ficat

ion

step

sV

olum

e

(ml)

act

ivity

(U

ml−

1 )Pr

otei

n co

nc.

(mg

ml−

1 )To

tal a

ctiv

ity (

U)

Tota

l pro

tein

(m

g)Sp

ecifi

c ac

tivity

(U

mg−

1 )Pu

rific

atio

n fo

ldY

ield

(%

)

Cru

de s

uper

nata

nt20

070

.50

± 1

.30

1.20

± 0

.10

14,1

00 ±

75.

0024

0 ±

12.

0058

.75

± 1

.20

1.00

± 0

.010

0 ±

0.0

60–8

0 %

(n

H4)

2SO

4 pr

ecip

itatio

n7.

075

2.60

± 2

2.43

8.45

± 0

.98

5,26

8.20

± 2

9.34

59.1

5 ±

1.2

989

.06

± 1

.90

1.51

± 0

.00

37.3

6 ±

0.0

1

Seph

adex

g-1

503.

01,

036.

90 ±

31.

334.

20 ±

1.1

53,

110.

70 ±

54.

0612

.60

± 3

.44

246.

88 ±

2.5

04.

20 ±

0.0

522

.06

± 0

.25

DE

aE

-cel

lulo

se1.

01,

258.

70 ±

28.

500.

55 ±

0.0

11,

258.

90 ±

43.

220.

55 ±

0.0

72,

288.

90 ±

27.

8038

.96

± 0

.32

8.92

± 0

.09

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those reported for enzyme from Bacillus sp. JaMB-602 [12] and Chaetomium sp. CQ31 [13], but lower than that from Bacillus sp. JaMB-750 that had a molecular weight of 130 kDa [14]. The elution pattern of β-mannanase using salt gradient has been shown in Fig. 2.

The β-mannanase sequence was determined by Edman degradation. The first 20 n-terminal amino acid sequence of β-mannanase from B. nealsonii Pn-11 was found to

be M-V-V-K-K-l-S-S-F-I-l-I-l-l-l-V-T-S-a-l. This sequence showed 50 % homology with the enzyme from alkalophilic Bacillus sp. JaMB-602 [12] which belongs to glycoside hydrolase (gH) family 5. This suggests that Pn-11 mannanase might also belong to gH family 5.

Effect of temperature on the activity and stability of β-mannanase

The purified β-mannanase showed maximum activity at 65 °C, and >60 ± 1.5 % of the activity could be detected at temperatures between 55 °C and 70 °C (Fig. 3a). β-Mannanase reported from different species of Bacillus has been reported to be in the range of 50–70 °C. Bacil-lus sp. n16-5, Bacillus sp. aM-001, Bacillus sp. JaMB-602, Bacillus subtilis WY-34, Bacillus sp. JaMB-750 and Bacillus sp. MSJ-5 had an optimum temperature of 70, 65, 65, 65, 55 and 50 °C, respectively [7, 11, 12, 14–16].

Fig. 1 Purification of ManPn11: a SDS–PagE profile—Lane 1 marker, Lane 2 crude enzyme, Lane 3 enzyme purified after 60–80 % ammonium sulfate precipitation, Lane 4 active fraction of Sepha-dex-150 column, Lane 5 active fraction of DEaE-cellulose anion-exchange column, b zymogram of purified enzyme

Fraction number 0 5 10 15 20 25 30 35 40 45 50 55 60 65

Pro

tein

abs

orba

nce

at a

t 280

nm

0.0

0.5

1.0

1.5

2.0

Man

nana

se a

ctiv

ity (

U/m

l)

0

20

40

60

80

Con

cent

ratio

n of

NaC

l (M

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2Absorbance (280 nm)

Mannanase activity(U/ml)

NaCl Gradient

Fig. 2 Elution profile of DEaE-cellulose chromatography of ManPn11: The column (15 × 1.2 cm2) was washed with 100 mM phosphate buffer, pH 7.0, at a flow rate of 0.8 ml min−1 and then eluted with a linear gradient of 0–1 M naCl

40 45 50 55 60 65 70 75 80 85

20

40

60

80

100

Temperature (°C)

Rel

ativ

e ac

tici

ty (

%)

00

20

40

60

80

100

60oC 65oC

5 10 20 30 45 60 120 180

70oC 75oC 80oC

Time (min.)

Res

idua

l act

ivit

y (%

)(A)

(B)

Fig. 3 Optimum temperature and stability of ManPn11: a Tempera-ture activity profile was obtained by measuring mannanase activity at pH 8.8 (100 mM glycine–naOH buffer) in the temperatures ranging from 40 to 85 °C. b Thermal stability of the ManPn11 was deter-mined after preincubating the enzyme at 60, 65, 70, 75 and 80 °C in 100 mM glycine–naOH buffer (pH 8.8), aliquots were withdrawn at different time intervals and residual activity was measured under standard conditions

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Thermostability profile of ManPn11 showed that enzyme was completely stable up to 3 h (60 °C) (Fig. 3b). The half-life of ManPn11 at 70 °C was found to 3 h. The ther-mostability of ManPn11 is higher than other reported mannanase, but mannanase from Bacillus subtilis Wl-3 retained >70 % of its activity at 60 °C after 8 h of incuba-tion [17] and alkalophilic Bacillus sp. n16-5 retained 62 % activity after incubation at 70 °C for 2 h [18]. The thermo-stability of ManPn11 is significantly higher than those of all other reported mannanase [7, 11, 12, 19–22]. a higher temperature optimum and thermostability of mannanase from B. nealsonii Pn-11 make it a more suitable catalyst for possible application in biobleaching of pulp, where pulping is carried out at high temperature.

Effect of pH on the β-mannanase activity and stability

The optimum pH for activity of ManPn11 was found to be 8.8, and 80 ± 2.0 % activity was retained at pH 8.4 (Fig. 4a). The ManPn11 was highly stable between pH

5–10, retained >85 % of the activity up to 3 h. Even at pH 11, it retained 35 ± 2.5 % of activity (Fig. 4b). Some mannanases have been reported to be active at alkaline pH [12, 14, 15, 18]. However, most of them have not found to be stable at high pH, and only mannanase from Bacillus licheniformis THCM 3.1 retains 77 % activity at pH 9 after incubation for 48 h [23].

Effect of organic solvents on stability of purified mannanase

In the present study, ManPn11 remained stable after 3 h of preincubation with most of the tested organic solvents. It retained >80 % activity in acetone, ethyl acetate, chloro-form, benzene, xylene, hexane and petroleum ether (Fig. 5). activity was retained in the presence of organic solvents with log P values almost equal to or less than 4.0 (benzene 2.0, xylene 3.1, hexane 3.5 and petroleum ether 4.0). This could be due to well-known fact that hydrophilic solvents are usually superior to hydrophobic solvents for the better enzyme activity, as the earlier have a greater tendency to bind water tightly, which is essential for catalytic activity. Kanjanvas et al. [23] reported that mannanase from Bacil-lus licheniformis THCM 3.1 showed 15–25 % reduction in activity after incubation with acetone, toluene, benzene, dimethyl sulphoxide, 2-propanol, acetonitrile or cyclohex-ane. Similar results were observed with mannanase from B. subtilis g1 which showed 11–53 % reduction in enzyme activity by the addition of organic solvents [24].

Effect of detergents, oxidizing and reducing agents, and chelating agents on mannanase activity

The ManPn11 was found to be stable with most of the tested agents (Table 2). In the presence of non-ionic deter-gents (1 %v/v) viz; PEg, Tween-20, Tween-80, it retained >80 % residual activity. However, in the presence of ionic detergents sodium dodecyl sulfate (SDS) (1 %w/v) and cetyl trimethylammonium bromide (CTaB) (1 %w/v), enzyme activity decreased to 64 ± 1.2 and 10 ± 0.5 %, respectively. Similar to our results, Jiang et al. [11] reported that the mannanase produced by Bacillus subtilis WY34 retained 55 % of its original activity in the presence of SDS (1 mM). In contrast, the mannanase produced from Bacillus sp. strain JaMB-750 and Bacillus sp. n16-5 showed 100 and 88 % resistance against SDS [7, 25]. The strong inhibi-tory effect of CTaB, a potent cation surfactant, could be due to the destruction of the conformation of mannanase. In the current study, ManPn11 retained 88 ± 1.4 % activity in the presence of 1 % potassium iodide and 58 ± 0.3 % activity in presence of 1 % hydrogen peroxide. The reduc-ing agent β-mercaptoethanol had a little inhibitory effect on enzyme activity (retaining 89 ± 1.4 % activity). However,

20

40

60

80

100

7.2 7.6 8.0 8.4 8.8 9.2

pH

Rel

ativ

e ac

tici

ty (

%)

3 4 5 6 7 8 9 10 11 120

20

40

60

80

100

pH

Res

idua

l act

ivit

y (%

)

(A)

(B)

Fig. 4 Optimum pH and stability of ManPn11: a pH activity profile was obtained by measuring mannanase activity at 65 °C in the buffers ranging from pH 7.2–9.2. b pH stability of ManPn11 was determined after preincubating the enzyme at 50 °C for 3 h in buffers ranging from pH 3–12, and the residual activity was measured under standard conditions

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akino et al. [15] reported three β-mannanases from alka-lophilic Bacillus sp. aM-001 which retained about 100 % activity with 1 mM β-mercaptoethanol. ManPn11 retained

89 ± 2.3 % activity, in the presence of ethylene diamine tetraacetic acid (EDTa), similar to Bacillus sp n16-5 (88 %) [15] and Cellulosimicrobium sp. strain HY-13 (95.9 %) [26].

Effect of metal ions on mannanase activity

The activity of ManPn11 was enhanced by 10 ± 1.9, 18 ± 3.0 and 12 ± 3.0 % in the presence of 1 mM ni+2, Co+2 and Mg+2, respectively. However, activity was reduced in the presence of 1 mM of Fe+2, li+, Mn+2, Hg+2 and Fe+3 (Fig. 6). In agreement with our results, enhance-ment of mannanase activity has been reported in the pres-ence of ni+2, Co+2, Zn+2 and Mg+2 in case of Cellulosimi-crobium sp. strain HY-13 [26] and Pantoea agglomerans [27] and inhibition of mannanase activity by Cd+2, Mn+2, Hg+2 and ag+ in case of Bacillus sp n16-5 [20]. among the metal ions tested on β-mannanase activity in the pre-sent study, mercuric (Hg+2) ions and ferric ions (Fe+3) sig-nificantly inhibited enzyme activity. Inhibition in enzyme activity by Hg+2 suggests that enzyme contains an essential sulfhydryl group [28].

Substrate specificity and kinetic parameters

The activity of ManPn11 toward various substrates was determined. It exhibited high activity toward lBg (100 %) followed by guar gum (78.55 ± 1.6 %) but consider-ably weaker activity on copra mannan (34.80 ± 1.2 %). (Table 3).

The Michaelis–Menten constants were determined for locust bean gum, guar gum and copra mannan (Table 3). The results revealed that β-mannanase had a higher affinity

0

20

40

60

80

100

120

Contr

ol

Dimet

hyl s

ulph

oxid

eM

etha

nol

Ethan

olAce

tone

Form

alde

hyde

Ethyl

acet

ate

Chlor

ofor

mBen

zene

Xylen

eH

exan

e

Petro

leum

ethe

r

Solvents

Rel

ativ

e ac

tivi

ty (

%)

Fig. 5 Effect of different organic solvents on mannanase stability: ManPn11 was preincubated in the presence of 50 % (v/v) various organic solvents at 37 °C for 3 h. The activity after incubation was measured under standard conditions. The 100 % activity represented the control enzyme activity without organic solvents

Table 2 Effect of detergents, chelating agents, oxidizing and reduc-ing agents on the activity of purified mannanase

ManPn11 was incubated at 37 °C for 1 h, and activity after incuba-tion was measured under standard conditions. The 100 % activity rep-resented the control enzyme activity without any agents

Reagent (1 % v/v or w/v) Relative activity (%)

Detergents

Control 100 ± 2.0

Sodium dodecyl sulfate 64 ± 1.2

Cetyl trimethyl ammonium bromide 10 ± 0.5

Polyethylene glycol 85 ± 1.1

Tween-20 80 ± 1.5

Tween-80 87 ± 1.9

Chelating agents

Sodium citrate 84 ± 1.0

Ethylene diamine tetraacetic acid 89 ± 2.3

Sodium azide 78 ± 1.2

Oxidizing agents

Hydrogen peroxide 58 ± 0.3

ammonium persulfate 65 ± 2.6

Potassium iodide 88 ± 1.4

Reducing agents

ascorbic acid 42 ± 2.0

β-Mercaptoethanol 89 ± 1.4

Contro

l +

Na +2

Ca+2

Ni +2

Mg

+Li +2

Zn+K

+2

Co+2

Hg+2

Fe+2

Cu+2

Mn

+3

Fe

0

20

40

60

80

100

120

1401 mM 5 mM

Metal Ions

Rel

ativ

e ac

tivi

ty (

%)

Fig. 6 Effect of metal ions on mannanase activity: ManPn11 was preincubated with various metal ions at 1 and 5 mM concentration at 37 °C for 1 h. The activity after incubation was measured under standard conditions. The 100 % activity represented the control enzyme activity without metal ions

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toward lBg (Vmax/Km, 104.2 ± 1.80 μmol min−1 mg−1) than that of other mannans like guar gum and copra man-nan. The determined Vmax for lBg is 750.41 ± 54.63 μ mol ml−1 min−1 which is lower than that mannanase of B. subtilis WY34 (970.3 ± 10.3 μmol ml−1 mg−1) and equal to mannanase of Paenibacillus sp. DZ3 (714 U mg−1) [11, 29]. Kinetic studies revealed that the ManPn11 had more affinity toward natural mannans, and hence it is applicable in the food industry for the production of oligosaccharides.

analysis of the hydrolysis products

The hydrolysis products of lBg were analyzed by HPaEC using mannooligosaccharides standards (Fig. 1S). as shown in Fig. 7, the main products were oligosaccharides, including 11 % mannobiose, 23 % mannotriose, 20 % mannotetrose, 18 % mannopentose and some other oli-gosaccharides that were detected but could not be identi-fied because no standards were available for comparison. This type of hydrolysis pattern indicates that the ManPn11 is an endomannanase, and therefore, extensive hydroly-sis of cheap and commercial available locust bean gum with ManPn11 will result in the production of a mixture

of mannooligosaccharides (MOS). The use of alkali-ther-mostable endomannanase will further improve the yield of MOS due to high solubility of mannan in alkaline media and reduction in viscosity of the reaction mixture [30]. MOS produced by acid hydrolysis of hemicellulosics are neither environment friendly nor cost-effective [31].

Potential of mannooligosaccharides (MOS) as a prebiotic agent

The MOS formed by the hydrolysis of lBg by ManPn11 were tested for their potential prebiotic properties, i.e., their effect on growth of L. casei as a positive prebiotic strain, as well as on growth of S. enterica as an example of potentially pathogenic strains. The different amounts of MOS, equivalent to 1.0, 2.0 and 3.0 mg ml−1 reduc-ing sugars, were tested for their prebiotic properties (Table 4). When varying amounts of MOS were added into the growth medium, the growth-enhancing activity of L. casei was in the range of 1.38 ± 0.11 to 3.37 ± 0.27 log cfu ml−1 and inhibition activity of S. enterica was in range of 0.52 ± 0.14 to 2.08 ± 0.12 log cfu ml−1. The experi-ment containing 3.0 mg ml−1 of MOS showed the high-est enhancing and inhibition activity of 3.37 ± 0.27 log cfu ml−1 and 2.08 ± 0.12 log cfu ml−1 against L. casei and S. enterica, respectively (Fig. 8). These results indicate that lBg-hydrolysate contains MOS which have a potential prebiotic property of enhancing growth of L. casei while apparently inhibiting growth of S. enterica. MOS that are already available in the market are carbohydrates extracted from the outer wall of yeast cells. They are used because of their ability to reduce growth of pathogenic bacteria, such as S. enterica and Escherichia coli in the digestive tract

Table 3 Kinetic parameters for the purified mannanase

Substrates Relative activity (%) Km (mg ml−1) Vmax (μmol ml−1 min−1) Vmax/Km (μmol min−1 mg−1)

locust bean gum 100.0 ± 2.0 7.22 ± 0.29 750.41 ± 54.63 104.22 ± 1.80

guar gum 78.55 ± 1.6 11.59 ± 0.16 344.68 ± 39.26 29.73 ± 1.10

Copra mannan 34.80 ± 1.2 18.73 ± 0.40 256.89 ± 26.47 13.71 ± 0.12

Fig. 7 HPaEC profile of the degradation of lBg by ManPn11: The hydrolysis products of lBg released by ManPn11 analyzed on HPaEC model-Dionex 2500, column type-CarboPac Pa100 col-umn, eluent-0.01 M naOH and 0.05 M sodium acetate, flow rate-1.0 ml min−1, injection volume-20 μl

Table 4 Effect of mannooligosaccharides on the growth of L. casei and S. enterica

* MOS equivalent to reducing groups

*MOS treatment Survival cell (log cfu ml−1)

Lactobacillus casei Salmonella enterica

Control (without MOS) 9.52 ± 0.11 10.33 ± 0.37

1.0 mg ml−1 10.90 ± 0.22 9.80 ± 0.23

2.0 mg ml−1 11.78 ± 0.27 8.88 ± 0.30

3.0 mg ml−1 12.89 ± 0.38 8.25 ± 0.25

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of animals. However, an ideal prebiotic should not only reduce growth of pathogens but must also promote growth of desirable and beneficial bacteria such as Lactobacillus and Bifidobacteria [32].

Conclusion

In conclusion, this study reports the purification and char-acterization of an alkali-thermostable, organic solvent tol-erant β-mannanase from B. nealsonii Pn-11. The purified β-mannanase was an extracellular monomeric protein with a molecular mass of 50 kDa on SDS–PagE. ManPn11 was highly stable between pH 5–10 and at temperature 60 °C which makes this enzyme suitable for industrial applica-tions. The purified enzyme showed higher affinity toward lBg (Vmax/Km, 104.2 ± 1.80 μmol min−1 mg−1) than that of other mannans like guar gum and copra mannan. It hydrolyzes locust bean gum to give mainly mannotetra-ose, mannotriose and mannobiose. The mannooligosaccha-rides (MOS) generated by ManPn11 from lBg show the potential prebiotic properties, viz., promote the growth of L. casei and inhibit the growth of S. enterica. Further study on cloning of mannanase gene is pursuing in our laboratory to elucidate the reaction mechanism of this enzyme and to determine structural functional relationship.

Acknowledgments Prakram Singh Chauhan is thankful to Council of Scientific and Industrial Research (CSIR), new Delhi, India, for providing a Senior Research Fellowship.

Conflict of interest none.

Compliance with Ethics Requirements all ethical considerations were complied with while working with bacterial strains.

References

1. Chauhan PS, Puri n, Sharma P, gupta n (2012) Mannanases: microbial sources, production, properties and potential biotech-nological applications. appl Microbiol Biotechnol 93:1817–1830

2. VanZyl WH, Rose SH, Trollope K, gorgens JF (2010) Fungal β-mannanases: mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45:203–1213

3. Chauhan PS, george n, Sondhi S, Puri n, gupta n (2014) an overview of purification strategies for microbial mannanases. Int J Pharm Bio Sci 5:176–192

4. Chauhan PS, Sharma P, Puri n, gupta n (2013) a pro-cess for reduction in viscosity of coffee extract by enzymatic hydrolysis of mannan. Bioprocess Biosyst Eng. doi:10.1007/s00449-013-1118-9

5. Kansoh al, nagieb Za (2004) Xylanase and mannanase enzymes from Streptomyces galbus nR and their use in biobleaching of softwood kraft pulp. anton Van leeuwonhoek 85:103–114

6. Bhoria P, Singh g, Sharma JR, Hoodal gS (2012) Biobleaching of wheat straw-rich-soda pulp by the application of alkalophilic and thermophilic mannanase from Streptomyces sp. Pg-08-3. afr J Biotechnol 11:6111–6116

7. Ma Y, Xue Y, Dou Y, Xu Z, Tao W, Zhou P (2004) Characteriza-tion and gene cloning of a novel β-mannanase from alkalophilic Bacillus sp. n16-5. Extremophiles 8:447–454

8. Miller gl (1959) The use of dinitrosalicylic acid reagents for the determination of reducing sugars. anal Chem 31:426–428

9. lowry OH, Rosebrough nJ, Farr al, Randall RJ (1951) Pro-tein measurement with folin phenol reagent. J Biol Chem 193:265–275

10. laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. nature 277:680–682

11. Jiang ZQ, Wei Y, li D, li l, Chai P, Kusakabe I (2006) High-level production, purification and characterization of a thermosta-ble β-mannanase from the newly isolated Bacillus subtilis WY34. Carbohydr Polym 66:88–96

12. Takeda n, Hirasawa K, Uchimura K, nogi Y, Hatada Y, akita M (2004) alkaline mannanase from a novel species of alkaliphilic Bacillus. J appl glycosci 51:229–236

13. Katrolia P, Qiaojuan Y, Zang P, Zhou P, Yang S, Jiang Z (2013) gene cloning and enzymatic characterization of an alkali-tolerant endo-1,4-β-mannanase from Rhizomucor miehei. J agric Food Chem 61:394–401

14. Takeda n, Hirasawa K, Uchimura K, nogi Y, Hatada Y, akita M (2004) Purification and enzymatic properties of a highly alkaline mannanase from alkaliphilic Bacillus sp. strain JaMB-750. J Biol Macromol 4:67–74

15. akino T, Kato C, Horikoshi K (1989) The cloned β–mannanase gene from alkalophilic Bacillus sp. aM-001 produces two β-mannanases in E. coli. arch Microbiol 152:10–15

16. Zhang M, Chen Xl, Zhang ZH, Sun CY, Chen ll, He Hl, Zhou BC, Zhang YZ (2009) Purification and functional characteriza-tion of endo-β-mannanase Man5 and its application in oligosac-charides production from konjac flour. appl Microbiol Biotech-nol 83:865–873

17. Yoon KH, Chung S, lim B (2008) Characterization of the Bacil-lus subtilis Wl-3 mannanase from a recombinant Escherichia coli. Microbiology 46:344–349

A B C A B C

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Fig. 8 Effect of mannooligosaccharides on growth of bacteria: growth enhancement of L. casei (filled bar); growth inhibition of S. enterica (checked bar) (A MOS equivalent to 1 mg ml−1 reducing groups, B MOS equivalent to 2 mg ml−1 reducing groups, C MOS equivalent to 3 mg ml−1 reducing groups)

Eur Food Res Technol

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18. He X, liu n, Zhang Z, Zhang B, Ma Y (2008) Inducible and constitutive expression of a novel thermo stable alkaline β-mannanase from alkalophilic Bacillus sp. n16-5 in Pichia pas-toris and characterization of the recombinant enzyme. Enzyme Microbiol Technol 43:13–18

19. Meenakshi Singh g, Bhalla a, Hoondal gS (2010) Solid state fermentation and characterization of partially purified ther-mostable mannanase from Bacillus sp. Mg-33. Bioresources 5:1689–1701

20. Yang P, li Y, Wang Y, Meng K, luo H, Yuan Y, Bai Y, Zhan Z, Yao B (2009) a novel β-mannanase with high specific activity from Bacillus circulans CgMCC1554: gene cloning, expres-sion and enzymatic characterization. appl Biochem Biotechnol 159:85–94

21. Yanan li, Yang P, Meng K, Wang Y, luo H, Wu n, Fan Y, Yao B (2008) gene cloning, expression and characterization of a novel β-mannanase from Bacillus circulans CgMCC 1416. J Microbiol Biotechnol 18:160–166

22. Summpunn P, Chaijan S, Isarangkul D, Wiyakrutta S, Meevooti-som V (2011) Characterization, gene cloning and heterologous expression of β-Mannanase from a thermophilic Bacillus subtilis. J Microbiol 49:86–93

23. Kanjanvas P, Khawsak P, Pakpitcharoen a, areekit S, Sriyaphai T, Pothivejkul K, Santiwatanakul S, Matsui K, Kajiwara T, Chan-siri K (2009) Over-expression and characterization of the alka-lophilc, organic solvent-tolerant, and thermotolerant endo-1,4-β-mannanase from Bacillus licheniformis isolate THCM 3.1. Sci asia 35:17–23

24. Vu TT, Quyen DT, Dao TT, nguyen SlT (2012) Cloning, high-level expression, purification, and properties of a novel endo-β-1,4-mannanase from Bacillus subtilis g1 in Pichia pastoris. J Microbiol Biotechnol 22:331–338

25. Hatada Y, Takeda n, Hirasawa K, Ohta Y, Usami R, Yoshida Y, Ito S, Horikoshi K (2005) Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JaMB-750, its expression in B. subtilis and characterization of the recombinant enzyme. Extremophiles 9:497–500

26. Kim DY, Ham SJ, lee HJ, Kim YJ, Shin DH, Rhee YH, Son KH, Park HY (2011) a highly active endo-1,4-β-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bac-terium in the gut of Eisenia fetida. Enzyme Microbiol Technol 48:365–370

27. Wang J, Shao Z, Hong Y, li C, Fu X, liu Z (2010) a novel β- mannanase from Pantoea agglomerans a021: gene cloning, expression, purification and characterization. World J Microbiol Biotechnol 26:1777–1784

28. naganagouda K, Salimath PV, Mulimani VH (2009) Purification and characterization of endo-β-1,4 mannanase from Aspergillus niger gr for application in food processing industry. J Microbiol Biotechnol 19:1184–1190

29. Chandra MRS, lee YS, Park IH, Zhou Y, Kim KK, Choi Yl (2011) Isolation, purification and characterization of a thermosta-ble β-mannanase from Paenibacillus sp. DZ3. J Korean Soc appl Bio Chem 54:325–331

30. Mamo g, Hatti-Kaul R, Mattiasson B (2006) a thermostable alkaline active endo-β-1,4-xylanase from Bacillus halodurans S7: purification and characterization. Enzyme Microbiol Technol 39:1492–1498

31. Vazquez MJ, alonso Jl, Dominguez H, Parajo JC (2012) Xylo-oligosaccharides: manufacture and applications. Tre Food Sci Technol 11:387–393

32. ghazzewi FH, Tester RF (2012) Efficacy of cellulase and man-nanase hydrolysates of konjac glucomannan to promote the growth of lactic acid bacteria. J Sci Food agric 92:2394–2396