research article purification and characterization of a

Research ArticlePurification and Characterization of a Thermostable120573-Mannanase from Bacillus subtilis BE-91 PotentialApplication in Inflammatory Diseases

Lifeng Cheng Shengwen Duan Xiangyuan Feng Ke Zheng Qi Yang and Zhengchu Liu

Institute of Bast Fiber Crops Chinese Academy of Agricultural Sciences Changsha Hunan 410205 China

Correspondence should be addressed to Zhengchu Liu liuzhengchucaascn

Received 30 July 2016 Revised 14 September 2016 Accepted 19 September 2016

Academic Editor Kai Wang

Copyright copy 2016 Lifeng Cheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

120573-mannanase has shown compelling biological functions because of its regulatory roles in metabolism inflammation andoxidation This study separated and purified the 120573-mannanase from Bacillus subtilis BE-91 which is a powerful hemicellulose-degrading bacterium using a ldquotwo-steprdquo method comprising ultrafiltration and gel chromatography The purified 120573-mannanase(about 282 kDa) showed high specific activity (79 8592 IUmg)The optimum temperature and pHwere 65∘C and 60 respectivelyMoreover the enzyme was highly stable at temperatures up to 70∘C and pH 45ndash70 The 120573-mannanase activity was significantlyenhanced in the presence ofMn2+ Cu2+ Zn2+ Ca2+Mg2+ andAl3+ and strongly inhibited by Ba2+ and Pb2+119870

119898

and119881max values forlocust bean gum were 714mgmL and 1075 120583molminmL versus 1749mgmL and 3345 120583molminmL for Konjac glucomannanrespectively Therefore 120573-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutralenvironments Based on such data the 120573-mannanase will have potential applications as a dietary supplement in treatment ofinflammatory processes

1 Introduction

Mannan consists of a series of complex polysaccharideswhich are found in the cell wall of marine algae [1] Thebackbone is comprised of 120573-14-linked mannose residuesKonjac glucomannan is a randomly arranged polymer of 120573-14-linked glucose and mannose residues at ratio of 10 16Both the backbones of mannan and Konjac are modified by120572-16-linked galactosyl residues to form galactomannan andgalactoglucomannan respectively [2]120573-mannanase (EC 32178) is a hemicellulase that attacks

the internal glycosidic bonds of mannan backbone to re-lease the condensed 120573-14-manno-oligosaccharides [3] 120573-mannanases are widely applied in pulp and paper processing[4] feed [5] food [6] pharmaceutical [7] oil and textile in-dustries [8] to randomly hydrolyze the 120573-14 mannopyrano-side linkage in mannan galactomannan glucomannan andgalactoglucomannan120573-mannanase is widely produced by bacteria [9 10] acti-

nomycetes [11] fungi [12] plants and animals [13] Among

them 120573-mannanase from bacteria is wildely used because ofnumerous advantages including extracellular secretion eco-nomic production and purification and novel characteristicssuch as tolerance to heat and alkaline conditions [14]

Although multiple 120573-mannanase-producing bacteriahave been reported [15 16] they are far from the diverse in-dustry needs Currently acidic and alkaline 120573-mannanasehas been proposed to meet the industrial demands [17]However the requirements of high energy in productionand the environmental impact limit their developmentNeutral and weakly acidic 120573-mannanase with lower energyfor production has attracted considerable interest over thepast few years however it has rarely been characterizedIt is clarified that 120573-mannanase with high activity in shortfermentation time confers lower costs during the productionprocedures Therefore the exploitation of strains producinghigh 120573-mannanases activity is valuable and profitable Incurrent study we isolated and preserved a powerful hemi-cellulose-degrading bacterium (BE-91) Then we explored

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6380147 7 pageshttpdxdoiorg10115520166380147

2 BioMed Research International

the efficient purification process and characterized the en-zymatic properties of its 120573-mannanase

2 Materials and Methods

21 Microorganism Media and Fermentation Conditions Bsubtilis BE-91 a strain used for herbaceous fiber extractionwas identified and preserved by the Institute of Bast FiberCrops Chinese Academy of Agricultural Science (ChangshaHunan China) B subtilis BE-91 was cultured in Petri dishcontaining 05 yeast extract 1 NaCl 05 Konjac glu-comannan 1 bacto tryptone 005 trypan blue and 15agarThe seedmediumwasmainly composed of 01 glucose04 Konjac glucomannan 03 beef extract 02 yeastextract 05 peptone and 05 NaCl The fermentationmedium primarily consisted of 02 yeast extract 07Konjac glucomannan 05 peptone 03 beef extract and05 NaCl B subtilis BE-91 was first activated in the seedmedium at 35 plusmn 1∘C for 55 h Subsequently the suspensionwas serially diluted spread onto Petri dishes and incubatedat 35 plusmn 1∘C for 18 h The single colony exhibiting the largesthydrolytic halo was transferred into an Erlenmeyer flask withthe seed medium and cultured at 35 plusmn 1∘C for 6 h at 180 rpmConsequently 2 culture was inoculated in the fermentationmedium and cultured for 6 h at 35 plusmn 1∘C at 180 rpm [18]

22 Classification of Strain BE-91 The 16S rDNA of strainBE-91 was PCR amplified from genomic DNA using theBacterial Identification PCR Kit (TaKaRa Japan) The ob-tained 16S rDNA was sequenced by the ABI 3730XL 96-capillary DNA analyzer The primers were as follows P151015840-AGAGTTTGATCMTGGCTCAG-31015840 and P2 51015840-TACGGY-TACCTTGTTACGACTT-31015840 The resulting sequence alignedclosely with the related standard sequences of other bacteriaretrieved from GenBank Neighbor-joining clusters wereconstructed by Mega 60 [19]

23 Enzymatic Assays 120573-mannanase activity was estimatedby initiating the reaction at 65∘C for 10min in 005molL cit-ric acid01molL Na

2HPO4buffer (pH 60) with 02 (wv)

Konjac glucomannan as substrate The amounts of reducingsugar in the reaction were quantified based on a standardcurve generated with mannose using the 35-dinitrosalicylicacid (DNS) method One unit (IU) of 120573-mannanase activitywas defined as the amount of protein producing 1 120583molL ofreducing sugar per minute (eg mannose) under standardconditions [20]

24 Purification of 120573-Mannanase The bacterial 120573-mannan-asewas purified using a two-step process involving ultrafiltra-tion (Sartorius Germany) and gel filtrationThe fermentationliquidwas fractionated orderly by 100 kDa 50 kDa and 5 kDamembrane thresholdsThe solution filteredwith 5 kDaltMWlt 50 kDa was further purified on a Sephadex G-100 gel col-umn (B16 cmtimes 100 cm Pharmacia)The eluate was obtainedat a rate of 05mLmin and collected in 5mL fractions 120573-mannanase activity was determined by the DNS methodwhereas the proteinwas quantified by the Coomassie brilliant

blue staining against bovine serum albumin (BSA) standard[21]

25 The Determination of Apparent Molecular Weight Themolecular mass of the 120573-mannanase was determined bySDS-PAGE (Bio-Rad USA) with 3 stacking gel and 12separating gel [22] The protein bands were stained with001 Coomassie brilliant blue R-250 and destained witha water-methanol-acetic acid (9 9 2) solvent Zymogramanalysis was performed by the method of Chanhan [17]The molecular weight of 120573-mannanase was derived fromthe relative mobility of molecular weight markers resolvedsimultaneously

26 The Effect of Temperature on the Activity and Stability of120573-Mannanase The activity of 120573-mannanase was assayed at arange of temperatures between 50 and 70∘C in 005molL cit-ric acid-01molL Na

2HPO4buffer at pH 60The thermosta-

bility was assessed by preincubating the enzyme without asubstrate at different temperatures varying over 20ndash80∘C for30min The residual activity was promptly measured by theDNS method The 120573-mannanase activity was considered tobe 100 when preincubated at 4∘C

27 The Effect of pH on the Activity and Stability of 120573-Mannanase 120573-mannanase activity was evaluated by incu-bating the purified enzyme at different pH conditions rangingfrom 40 to 80 in 005molL citric acid-01molL Na

2HPO4

buffer at 4∘C The stability at a particular pH was tested bypreincubating the purified enzyme without a substrate for30min in various 005molL citric acid-01molL Na

2HPO4

buffers at pH 30ndash85 at 4∘C The residual 120573-mannanaseactivity was immediately measured after treatment by theDNS procedure

28 The Effect of Metal Ions on the Activity of 120573-MannanaseIn order to examine the effects of metal ions on the activity of120573-mannanase the enzyme was incubated for 30min at 4∘C inthe presence of various 10mmolL metal ions CaCl

2sdot2H2O

ZnCl2 FeCl

3 PbCl

2sdot2H2OMnCl

2sdot4H2OMgCl

2sdot6H2O KCl

CuCl2sdot2H2O AlCl

3 BaCl

2 and NH

4Cl The residual 120573-

mannanase activity was measured at a specific condition andthat of the treatment in the absence of additives as a control

29 Substrate Specificity and Kinetic Parameters Various gly-cans such as Konjac glucomannan [23] locust bean gumfrom Ceatonia siliqua seeds (Sigma G0753) carob galac-tomannan (Megazyme P-GALML) guar galactomannan(Megazyme P-GGMMV) ivory nut mannan (MegazymeP-MANIV) 14-beta-D-mannan (Megazyme P-MANCB)wheat arabinoxylan (Megazyme P-120601a) beechwoodxylan (Megazyme P-141101a) and carboxymethyl cellulose(Megazyme P-CMC4M) were examined In brief 02 (wv)glycans were incubated with 120573-mannanase at 65∘C for 10minin 005molL citric acid-01molL Na

2HPO4buffer at pH

60 and the reducing sugars were measured by DNS TheMichaelis-Menten kinetic parameters119881max and119870119898 were cal-culated for 120573-mannanase The assays of the purified enzymewere carried out by the standardDNSprocedure as described

BioMed Research International 3

Table 1 120573-mannanase activities of five bacteria

Bacterium number Activity (IUmL)a Specific activity (IUmg)a

BE-23 0 0BE-78 1915 plusmn 45 8798 plusmn 132BE-46 832 plusmn 21 3116 plusmn 94BE-83 707 plusmn 16 1197 plusmn 255BE-91 2737 plusmn 65 23192 plusmn 263aData are mean plusmn SD 119899 = 3

above using 1ndash5mgmL locust bean gum and 05ndash25mgmLKonjac glucomannan as substrates The kinetic constantswere determined from the Michaelis-Menten equation bydirectly inputting the initial rates fromLineweaver-Burk plotsor the nonlinear regression [24]

210 Statistical Analysis Each 120573-mannanase activity experi-ment was performed in triplicate and expressed asmeanplusmn SD(standard deviation) The statistical analyses were performedwith SPSS 150 (SPSS Inc Chicago IL USA) One-way ortwo-way analysis of variance (ANOVA) was used to comparevarious treatment groups

3 Results and Discussion

31 Screening of the High 120573-Mannanase Activity ProducingStrain Four bacteria were stochastically selected for the120573-mannanase activity assay Figure 1 exhibited the halosproduced on the screening plate Table 1 summarized the120573-mannanase activity of the four bacteria (strain BE-23without120573-mannanase activitywas used as a negative control)Strain BE-91 fermented for 9 h exhibited the highest activityup to 2737 IUmL Wild-type B subtilis MA139 yielded amaximum 120573-mannanase activity of 170 IUmL after 3 daysof fermentation and the maximum enzyme activity of Bsubtilis TJ-102 was 2053 IUmL at 38 h [25 26] Notably BE-91 secreted 120573-mannanase with higher activity in shorter time

32 Classification of B subtilis BE-91 The 1508 bp sequenceof 16S rDNA of strain BE-91 was analyzed by a phylogenictree (Figure 2) The homology between BE-91 16S rDNA (gi260159552) and B subtilis 16S rDNA (gi 530330588 and gi341831474) was 99 It was confirmed that the similarity of Bsubtilis type strains about 16S rRNA gene sequence is higherthan 98 [27 28] We also obtained ge98 similarity to 16SrRNA gene sequences of B subtilis isolates

33 Isolation and Purification of 120573-Mannanase 2000mL offermentation liquor was purified by ultrafiltration and chro-matography Specific activity recovery andmultiple purifica-tions at each step were summarized in Table 2 The recoveryof 120573-mannanase in B subtilis BE-91 exceeded 660multiplepurifications achieved 329-fold pure 120573-mannanase activityand the specific activity of the purified enzyme reached798592 IUmg The purified 120573-mannanase was shown tobe homogeneous judged by SDS-PAGE analysis (Figure 3)Compared with the previous separation and purification

1

2

3

4 5

Figure 1 Clear halos produced by control and active colonies with120573-mannanase activity 1 BE-78 2 BE-46 3 BE-83 4 BE-23 (negativecontrol) 5 BE-91

methods [29 30] the two-step method has the advantages ofhigh efficiency high yield and easy operation

34 Apparent Molecular Weight of 120573-Mannanase The ap-parent molecular weight of 120573-mannanase was 282 kDa(Figure 3) lower than those of the most known 120573-mannan-ases from Bacillus spp (Bacillus licheniformis THCM 3140 kDa B subtilis WY34 396 kDa B subtilis Z-2 38 kDaBacillus circulans CGMCC1554 32 kDa) [28 31ndash34] Simi-larly the molecular weights of 120573-mannanases from Penicil-lium occitanis Po16 and Bacillus halodurans PPKS-2 were22 and 18 kDa respectively [30 31] Due to low molecularweights these enzymes may rapidly penetrate the lignocellu-lose systems and depolymerize the mannans more efficiently[35]

35 Optimal Temperature and Thermostability of 120573-Man-nanase The purified 120573-mannanase was maximally active at65∘C (Figure 4) and remained more than 80 active at70∘C (Figure 5) Compared with the optimal temperaturesobtained for other 120573-mannanases (40∘C for Penicillium occ-itanis Pol6 50∘C for both Bacillus circulans TN-31 and Bsubtilis B36 60∘C for Paenibacillus sp DZ3) [29 31 36] 120573-mannanase of BE-91 showed a pronounced activity at highertemperatures As compared to the thermostability of the 120573-mannanase from wild-type B subtilis BCC41051 (60∘C for30min) [37] this 120573-mannanase retains 80 residual activityafter incubation at 20ndash70∘C for 30min indicating enhancedthermostability

36 Optimal pH and Stability of 120573-Mannanase The optimalpH and the stability of BE-91 120573-mannanase were measured atvarious pHs The optimum enzyme activity was obtained atpH 60 (Figure 6) and more than 80 maximal activity wasretained at pH 45ndash70 (Figure 7) Interestingly the optimalpH of BE-91 120573-mannanase was the same as that of B subtilisMA139 (pH 60) an enzyme that can potentially be used


Table 2 Purification of 120573-mannanase by ultrafiltration and gel chromatography

Purification step Total activity (IU) Total protein (mg) Specific activity (IUmg) Recovery () Purification multiple (fold)Fermentation liquor 4296508 1767 24314 100 1Ultrafiltration 3283174 86 380702 764 156Gel chromatography 2835002 36 798592 660 329

Bacillus deserti KCTC 13246 GQ465041Bacillus eiseniae KCCM 90092 HM035089Bacillus horneckiae MTCC 9535 EU861362Bacillus crescens KCTC 33627 LN625239Bacillus acidicola DSM 14745 AF547209Bacillus filamentosus DSM 27955 KF265351Bacillus cibi DSM 16189 AY550276Bacillus idriensis KCCM 90024 AY904033Bacillus aerophilus MTCC 7304 KM055067

Bacillus galliciensis DSM 21539 FM162181Bacillus subtilis DSM 10 AJ276351Strain BE-91

Bacillus abyssalis DSM 25875 JX232168Bacillus fengqiuensis DSM 26745 KC291653Bacillus berkeleyi KCTC 12718Bacillus hemicellulosilyticus

JN187498DSM 16731 AB043846

Bacillus daliensis CGMCC 110369Bacillus gibsonii DSM 8722 X76446Bacillus cecembensis MTCC9127 AM773821Bacillus cereus DSM 31 AE016877Bacillus thuringiensis DSM 2046 D16281Bacillus beijingensis DSM 19037 EF371374Bacillus endoradicis CCBAU05776 GU434676Bacillus kokeshiiformis KCTC 33163 JX848633Paenibacillus amylolyticus DSM 11730 D85396Paenibacillus alvei DSM 29 AJ320491Paenibacillus kobensis DSM 10249 AB073363

Bacillus circulans DSM 11 AY724690

GU583651

Figure 2 Phylogenetic tree based on 16S rDNA sequences of strain BE-91 and other bacteria by Mega 60 using neighbor-joining analysiswith 1000 bootstrap replicates

as a feed additive for monogastric animals [25] At pH lt40 the 120573-mannanase activity was negligible retaining lt80of its maximal value obtained after incubation at pH gt75 4∘C for 30min A relatively broad zone of optimumactivity was observed Therefore BE-91 120573-mannanase canbe considered a weakly acidic and neutral enzyme therebyrendering suitability for animal feed industry [38]

37 The Effect of Metal Ions on 120573-Mannanase Stability Theeffect of a variety of metal ions on 120573-mannanase activity wasmeasured (Table 3)The highest induction was achieved withMn2+ which showed 168 baseline activity followed byAl3+Ca2+ Cu2+ Zn2+ Mg2+ and NH

4

+ respectively K+ and Fe3+had no obvious effects on 120573-mannanase activity in these con-ditions Ba2+ and Pb2+ greatly inhibited the enzyme activityto a final rate of 83 and 74 respectively This suggests thatBE-91 120573-mannanase should not be contaminated by Ba2+ andPb2+

Table 3 Effects of different metal ions (1mmolL) on 120573-mannanaseactivity

Metal ions Relative activity ()a

Blank 100K+ 99 plusmn 32NH4

+ 103 plusmn 27Ca2+ 117 plusmn 36Zn2+ 115 plusmn 29Mn2+ 168 plusmn 45Cu2+ 116 plusmn 21Mg2+ 107 plusmn 28Ba2+ 83 plusmn 31Pb2+ 74 plusmn 29Fe3+ 99 plusmn 36Al3+ 121 plusmn 43aData are mean plusmn SD 119899 = 3


170130

95

72

55

43

34

26

17

(kDa) M 1 2 3

Figure 3 SDS-PAGE analysis of 120573-mannanase Lane M proteinmolecular weight standard Lane 1 culture broth Lane 2 purified120573-mannanase Lane 3 zymogram of purified 120573-mannanase

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

50 55 60 65 70 7545Temperature (∘C)

Figure 4 Optimum temperature curve of 120573-mannanase

38 Kinetic Parameters The purified enzyme hydrolyzedKonjac glucomannan but only slightly hydrolyzed ivorynut mannan guar galactomannan and 14-beta-D-mannanWheat arabinoxylan beechwood xylan and CMC werebarely hydrolyzed as shown in Table 4 This 120573-mannanaseexhibited the highest activity with Konjac glucomannanenriched in glucose units This finding suggests that 120573-mannanase of BE-91 preferentially hydrolyzes the 120573-14-linkage of the glucosylated mannan backbone

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

20 25 30 35 40 45 50 55 60 65 70 75 80 8515Temperature (∘C)

Figure 5 Thermal stability curve of 120573-mannanase

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

45 5 55 6 65 7 75 84pH

Figure 6 Optimum pH curve of 120573-mannanase

Table 4 Hydrolytic activity of the purified enzyme on differentpolysaccharides

Substrate (05 wv) Relative activity ()a

Konjac glucomannan 100Locust bean gum 8815 plusmn 18Carob galactomannan 9185 plusmn 17Guar galactomannan 3570 plusmn 06Ivory nut mannan 3274 plusmn 0314-Beta-D-mannan 4622 plusmn 04Wheat arabinoxylan 0Beechwood xylan 0Carboxymethyl cellulose 0Assays were carried out at 65∘C at pH 60 for 10min in 005molL citric acid-01molL Na

2HPO4buffer

aData are mean plusmn SD 119899 = 3

119870119898and 119881max values of this 120573-mannanase estimated by

the Lineweaver-Burk plot were 714mgmL and 1075 120583molminmL respectively for locust bean gum versus 1749mgmL and 3345 120583molminmL for Konjac glucomannan


3 35 4 45 5 55 6 65 7 75 8 8525pH

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

Figure 7 pH stability curve of 120573-mannanase

respectively These results displayed higher affinity of 120573-mannanase towards natural Konjac glucomannan (119881max119870119898191 120583molminmg) than the locust bean gum (119881max119870119898150 120583molminmg) similar to the values obtained for Peni-cillium pinophilum C1 and Penicillium freii F63 hence con-stituting it as an adequate candidate in food industry for theproduction of oligosaccharides [17 18 39]

4 Conclusion

B subtilis bacteria are abundantmoderately stable andmost-ly nonpathogenic microorganisms Our results indicated thatB subtilis BE-91 could be considered a prominent candidatefor the production of extracellular 120573-mannanase In additionthis study developed an advanced purification approachldquotwo-step methodrdquo with high efficiency high yield and easyoperation Furthermore the 120573-mannanase purified from BE-91 was extremely stable at relatively high temperatures andvarious weak acidic or neutral pHs Finally the enzymeshowed a higher affinity towards natural Konjac glucoman-nan a major functional food material Therefore this 120573-mannanase purified and characterized from B subtilis BE-91for the first time is suitable for inflammatory diseases

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was funded by the Natural Science Foundation ofHunan Province (no 2016jj3126) National Innovation Engi-neering of China (no ASTIP-IBFC08) and the EarmarkedFund for China Modern Agriculture Research System (noCARS-19)

References

[1] D R Georgianna M J Hannon M Marcuschi et al ldquoPro-duction of recombinant enzymes in the marine alga Dunaliellatertiolectardquo Algal Research vol 2 no 1 pp 2ndash9 2013

[2] G Gubitz A Sachslehner and D Haltrich ldquoMicrobial man-nanases substrates production and applicationsrdquo in GlycosylHydrolases for Biomass Conversion chapter 14 pp 236ndash2622001

[3] P S Chauhan N Puri P Sharma and N Gupta ldquoMannan-ases microbial sources production properties and poten-tial biotechnological applicationsrdquo Applied Microbiology andBiotechnology vol 93 no 5 pp 1817ndash1830 2012

[4] X Pan J Zhou A Tian et al ldquoHigh level expression of a trun-cated 120573-mannanase from alkaliphilic Bacillus sp N16-5 inKluyveromyces cicerisporusrdquo Biotechnology Letters vol 33 no3 pp 565ndash570 2011

[5] C Kong J H Lee and O Adeola ldquoSupplementation of 120573-mannanase to starter and grower diets for broilersrdquo CanadianJournal of Animal Science vol 91 no 3 pp 389ndash397 2011

[6] A Adiguzel H Nadaroglu and G Adiguzel ldquoPurification andcharacterization of 120573-mannanase from Bacillus pumilus (M27)and its applications in some fruit juicesrdquo Journal of Food Scienceand Technology vol 52 no 8 pp 5292ndash5298 2015

[7] WHVanZyl S H Rose K Trollope and J F Gorgens ldquoFungal120573-mannanases mannan hydrolysis heterologous productionand biotechnological applicationsrdquo Process Biochemistry vol45 no 8 pp 1203ndash1213 2010

[8] A C Sehgal and R M Kelly ldquoStrategic selection of hyper-thermophilic esterases for resolution of 2-arylpropionic estersrdquoBiotechnology Progress vol 19 no 5 pp 1410ndash1416 2003

[9] S Titapoka S Keawsompong D Haltrich and S Nitis-inprasert ldquoSelection and characterization of mannanase-producing bacteria useful for the formation of prebioticmanno-oligosaccharides from copra mealrdquoWorld Journal of Microbiol-ogy and Biotechnology vol 24 no 8 pp 1425ndash1433 2008

[10] GAdiguzel Z SonmezAAdiguzel andHNadaroglu ldquoPurifi-cation and characterization of a thermostable endo-beta-14mannanase from Weissellaviridescens LB37 and its applicationin fruit juice clarificationrdquo European Food Research and Tech-nology vol 242 no 5 pp 769ndash776 2016

[11] P Bhoria G Singh and G S Hoondal ldquoOptimization of man-nanase production from Streptomyces sp PG-08-03 in sub-merged fermentationrdquo BioResources vol 4 no 3 pp 1130ndash11382009

[12] M Siti Norita M Rosfarizan and A B Ariff ldquoEvaluation of theactivities of concentrated crude mannan-degrading enzymesproduced by Aspergillus nigerrdquoMalaysian Journal of Microbiol-ogy vol 6 no 2 pp 171ndash180 2010

[13] R Prakash S L Johnston H L Boldingh et al ldquoMannans intomato fruit are not depolymerized during ripening despite thepresence of endo-120573-mannanaserdquo Journal of Plant Physiologyvol 169 no 12 pp 1125ndash1133 2012

[14] H X Liu J S Gong H Li et al ldquoBiochemical characterizationand cloning of an endo-14-120573-mannanase from Bacillus subtilisYH12 with unusually broad substrate profilerdquo Process Biochem-istry vol 50 no 5 pp 712ndash721 2015

[15] H Mou F Zhou X Jiang and Z Liu ldquoProduction purificationand properties of 120573-mannanase from soil Bacterium bacilluscirculans m-21rdquo Journal of Food Biochemistry vol 35 no 5 pp1451ndash1460 2011

[16] YHakamada YOhkubo and SOhashi ldquoPurification and char-acterization of 120573-mannanase from Reinekea sp KIT-YO10with transglycosylation activityrdquo Bioscience Biotechnology andBiochemistry vol 78 no 4 pp 722ndash728 2014

[17] P S Chauhan P Sharma N Puri and N Gupta ldquoPurificationand characterization of an alkali-thermostable 120573-mannanase


from Bacillus nealsonii PN-11 and its application in man-nooligosaccharides preparation having prebiotic potentialrdquoEuropean Food Research and Technology vol 238 no 6 pp 927ndash936 2014

[18] MWang S You S Zhang et al ldquoPurification characterizationand production of 120573-mannanase from Bacillus subtilis TJ-102and its application in gluco-mannooligosaccharides prepara-tionrdquo European Food Research and Technology vol 237 no 3pp 399ndash408 2013

[19] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[20] T Akino N Nakamura and K Horikoshi ldquoProduction of 120573-mannosidase and 120573-mannanase by an alkalophilic BacillussprdquoApplied Microbiology and Biotechnology vol 26 no 4 pp 323ndash327 1987

[21] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[22] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[23] M Chua K Chan T J Hocking P AWilliams C J Perry andT C Baldwin ldquoMethodologies for the extraction and analysis ofkonjac glucomannan from corms of Amorphophallus konjac KKochrdquo Carbohydrate Polymers vol 87 no 3 pp 2202ndash22102012

[24] H Lineweaver and D Burk ldquoThe determination of enzyme dis-sociation constantsrdquo Journal of the American Chemical Societyvol 56 no 3 pp 658ndash666 1934

[25] J Qiao Z Rao B Dong and Y Cao ldquoExpression of BacillussubtilisMA139 120573-mannanase in Pichia pastoris and the enzymecharacterizationrdquo Applied Biochemistry and Biotechnology vol160 no 5 pp 1362ndash1370 2010

[26] Y Wang P Shi H Luo et al ldquoCloning over-expression andcharacterization of an alkali-tolerant endo-120573-14-mannanasefrom penicillium freii F63rdquo Journal of Bioscience and Bioengi-neering vol 113 no 6 pp 710ndash714 2012

[27] BKDashMMRahman andPK Sarker ldquoMolecular identifi-cation of a newly isolated Bacillus subtilis BI19 and optimizationof production conditions for enhanced production of extracel-lular amylaserdquo BioMed Research International vol 2015 ArticleID 859805 9 pages 2015

[28] L Yang X Quan B Xue et al ldquoIsolation and identificationof Bacillus subtilis strain YB-05 and its antifungal substancesshowing antagonism against Gaeumannomyces graminis vartriticirdquo Biological Control vol 85 pp 52ndash58 2015

[29] M R S Chandra Y S Lee I H Park Y Zhou K K Kim andYL Choi ldquoIsolation purification and characterization of a ther-mostable 120573-mannanase from Paenibacillus sp DZ3rdquo Journal ofApplied Biological Chemistry vol 54 no 3 pp 325ndash331 2011

[30] S Vijayalaxmi P Prakash S K Jayalakshmi V H Mulimaniand K Sreeramulu ldquoProduction of extremely alkaliphilichalotolerent detergent and thermostable mannanase by thefree and immobilized cells of Bacillus halodurans PPKS-2Purification and characterizationrdquo Applied Biochemistry andBiotechnology vol 171 no 2 pp 382ndash395 2013

[31] M Blibech R E Ghorbel I Fakhfakh et al ldquoPurification andcharacterization of a low molecular weight of 120573-mannanase

from Penicillium occitanis Pol6rdquo Applied Biochemistry and Bio-technology vol 160 no 4 pp 1227ndash1240 2010

[32] F Kanjanavas P Khawsak A Pakpitcharoen et al ldquoOver-expression and characterization of the alkalophilic organicsolvent-tolerant and thermotolerant endo-l4-120573-mannanasefrom Bacillus licheniformis isolate THCM 31rdquo ScienceAsia vol35 no 1 pp 17ndash23 2009

[33] Z Jiang YWei D Li L Li P Chai and I Kusakabe ldquoHigh-levelproduction purification and characterization of a thermostable120573-mannanase from the newly isolated Bacillus subtilis WY34rdquoCarbohydrate Polymers vol 66 no 1 pp 88ndash96 2006

[34] Q Zhang X Yan L Zhang and W Tang ldquoCloning sequenceanalysis and heterologous expression of a beta-mannanase genefrom Bacillus subtilis Z-2rdquoMolekuliarnaia Biologiia vol 40 no3 pp 418ndash424 2006

[35] R Martınez-Palou and R Luque ldquoApplications of ionic liquidsin the removal of contaminants from refinery feedstocks anindustrial perspectiverdquo Energy amp Environmental Science vol 7no 8 pp 2414ndash2447 2014

[36] Y Maruyama and T Nakajima ldquoThe aman6 gene encodinga yeast mannan backbone degrading 16-120572-D-mannanase inBacillus circulans cloning sequence analysis and expressionrdquoBioscience Biotechnology and Biochemistry vol 64 no 9 pp2018ndash2020 2000

[37] P Summpunn S Chaijan D Isarangkul S Wiyakrutta and VMeevootisom ldquoCharacterization gene cloning and heterolo-gous expression of 120573-mannanase from a thermophilic Bacillussubtilisrdquo Journal of Microbiology vol 49 no 1 pp 86ndash93 2011

[38] D Y Kim S J Ham H J Lee et al ldquoCloning and characteri-zation of a modular GH5 120573-14-mannanase with high specificactivity from the fibrolytic bacterium Cellulosimicrobium spstrain HY-13rdquo Bioresource Technology vol 102 no 19 pp 9185ndash9192 2011

[39] H Cai P Shi H Luo et al ldquoAcidic 120573-mannanase from Penicil-lium pinophilum C1 Cloning characterization and assessmentof its potential for animal feed applicationrdquo Journal of Bioscienceand Bioengineering vol 112 no 6 pp 551ndash557 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the efficient purification process and characterized the en-zymatic properties of its 120573-mannanase

2 Materials and Methods

21 Microorganism Media and Fermentation Conditions Bsubtilis BE-91 a strain used for herbaceous fiber extractionwas identified and preserved by the Institute of Bast FiberCrops Chinese Academy of Agricultural Science (ChangshaHunan China) B subtilis BE-91 was cultured in Petri dishcontaining 05 yeast extract 1 NaCl 05 Konjac glu-comannan 1 bacto tryptone 005 trypan blue and 15agarThe seedmediumwasmainly composed of 01 glucose04 Konjac glucomannan 03 beef extract 02 yeastextract 05 peptone and 05 NaCl The fermentationmedium primarily consisted of 02 yeast extract 07Konjac glucomannan 05 peptone 03 beef extract and05 NaCl B subtilis BE-91 was first activated in the seedmedium at 35 plusmn 1∘C for 55 h Subsequently the suspensionwas serially diluted spread onto Petri dishes and incubatedat 35 plusmn 1∘C for 18 h The single colony exhibiting the largesthydrolytic halo was transferred into an Erlenmeyer flask withthe seed medium and cultured at 35 plusmn 1∘C for 6 h at 180 rpmConsequently 2 culture was inoculated in the fermentationmedium and cultured for 6 h at 35 plusmn 1∘C at 180 rpm [18]

22 Classification of Strain BE-91 The 16S rDNA of strainBE-91 was PCR amplified from genomic DNA using theBacterial Identification PCR Kit (TaKaRa Japan) The ob-tained 16S rDNA was sequenced by the ABI 3730XL 96-capillary DNA analyzer The primers were as follows P151015840-AGAGTTTGATCMTGGCTCAG-31015840 and P2 51015840-TACGGY-TACCTTGTTACGACTT-31015840 The resulting sequence alignedclosely with the related standard sequences of other bacteriaretrieved from GenBank Neighbor-joining clusters wereconstructed by Mega 60 [19]

23 Enzymatic Assays 120573-mannanase activity was estimatedby initiating the reaction at 65∘C for 10min in 005molL cit-ric acid01molL Na

2HPO4buffer (pH 60) with 02 (wv)

Konjac glucomannan as substrate The amounts of reducingsugar in the reaction were quantified based on a standardcurve generated with mannose using the 35-dinitrosalicylicacid (DNS) method One unit (IU) of 120573-mannanase activitywas defined as the amount of protein producing 1 120583molL ofreducing sugar per minute (eg mannose) under standardconditions [20]

24 Purification of 120573-Mannanase The bacterial 120573-mannan-asewas purified using a two-step process involving ultrafiltra-tion (Sartorius Germany) and gel filtrationThe fermentationliquidwas fractionated orderly by 100 kDa 50 kDa and 5 kDamembrane thresholdsThe solution filteredwith 5 kDaltMWlt 50 kDa was further purified on a Sephadex G-100 gel col-umn (B16 cmtimes 100 cm Pharmacia)The eluate was obtainedat a rate of 05mLmin and collected in 5mL fractions 120573-mannanase activity was determined by the DNS methodwhereas the proteinwas quantified by the Coomassie brilliant

blue staining against bovine serum albumin (BSA) standard[21]

25 The Determination of Apparent Molecular Weight Themolecular mass of the 120573-mannanase was determined bySDS-PAGE (Bio-Rad USA) with 3 stacking gel and 12separating gel [22] The protein bands were stained with001 Coomassie brilliant blue R-250 and destained witha water-methanol-acetic acid (9 9 2) solvent Zymogramanalysis was performed by the method of Chanhan [17]The molecular weight of 120573-mannanase was derived fromthe relative mobility of molecular weight markers resolvedsimultaneously

26 The Effect of Temperature on the Activity and Stability of120573-Mannanase The activity of 120573-mannanase was assayed at arange of temperatures between 50 and 70∘C in 005molL cit-ric acid-01molL Na

2HPO4buffer at pH 60The thermosta-

bility was assessed by preincubating the enzyme without asubstrate at different temperatures varying over 20ndash80∘C for30min The residual activity was promptly measured by theDNS method The 120573-mannanase activity was considered tobe 100 when preincubated at 4∘C

27 The Effect of pH on the Activity and Stability of 120573-Mannanase 120573-mannanase activity was evaluated by incu-bating the purified enzyme at different pH conditions rangingfrom 40 to 80 in 005molL citric acid-01molL Na

2HPO4

buffer at 4∘C The stability at a particular pH was tested bypreincubating the purified enzyme without a substrate for30min in various 005molL citric acid-01molL Na

2HPO4

buffers at pH 30ndash85 at 4∘C The residual 120573-mannanaseactivity was immediately measured after treatment by theDNS procedure

28 The Effect of Metal Ions on the Activity of 120573-MannanaseIn order to examine the effects of metal ions on the activity of120573-mannanase the enzyme was incubated for 30min at 4∘C inthe presence of various 10mmolL metal ions CaCl

2sdot2H2O

ZnCl2 FeCl

3 PbCl

2sdot2H2OMnCl

2sdot4H2OMgCl

2sdot6H2O KCl

CuCl2sdot2H2O AlCl

3 BaCl

2 and NH

4Cl The residual 120573-

mannanase activity was measured at a specific condition andthat of the treatment in the absence of additives as a control

29 Substrate Specificity and Kinetic Parameters Various gly-cans such as Konjac glucomannan [23] locust bean gumfrom Ceatonia siliqua seeds (Sigma G0753) carob galac-tomannan (Megazyme P-GALML) guar galactomannan(Megazyme P-GGMMV) ivory nut mannan (MegazymeP-MANIV) 14-beta-D-mannan (Megazyme P-MANCB)wheat arabinoxylan (Megazyme P-120601a) beechwoodxylan (Megazyme P-141101a) and carboxymethyl cellulose(Megazyme P-CMC4M) were examined In brief 02 (wv)glycans were incubated with 120573-mannanase at 65∘C for 10minin 005molL citric acid-01molL Na

2HPO4buffer at pH

60 and the reducing sugars were measured by DNS TheMichaelis-Menten kinetic parameters119881max and119870119898 were cal-culated for 120573-mannanase The assays of the purified enzymewere carried out by the standardDNSprocedure as described











1

2

3

4 5












JN187498DSM 16731 AB043846



GU583651




4







170130

95

72

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(kDa) M 1 2 3


0

20

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Rela

tive a

ctiv

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50 55 60 65 70 7545Temperature (∘C)



00

200

400

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1200

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tive a

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20 25 30 35 40 45 50 55 60 65 70 75 80 8515Temperature (∘C)


00

200

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Rela

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)

45 5 55 6 65 7 75 84pH





2HPO4buffer





3 35 4 45 5 55 6 65 7 75 8 8525pH

00

200

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1000

1200

Rela

tive a

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4 Conclusion


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























1

2

3

4 5












JN187498DSM 16731 AB043846



GU583651




4







170130

95

72

55

43

34

26

17

(kDa) M 1 2 3


0

20

40

60

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100

120

Rela

tive a

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50 55 60 65 70 7545Temperature (∘C)



00

200

400

600

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1000

1200

Rela

tive a

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20 25 30 35 40 45 50 55 60 65 70 75 80 8515Temperature (∘C)


00

200

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600

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1000

1200

Rela

tive a

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45 5 55 6 65 7 75 84pH





2HPO4buffer





3 35 4 45 5 55 6 65 7 75 8 8525pH

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)



4 Conclusion


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















JN187498DSM 16731 AB043846



GU583651




4







170130

95

72

55

43

34

26

17

(kDa) M 1 2 3


0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

50 55 60 65 70 7545Temperature (∘C)



00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

20 25 30 35 40 45 50 55 60 65 70 75 80 8515Temperature (∘C)


00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

45 5 55 6 65 7 75 84pH





2HPO4buffer





3 35 4 45 5 55 6 65 7 75 8 8525pH

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)



4 Conclusion


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















170130

95

72

55

43

34

26

17

(kDa) M 1 2 3


0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

50 55 60 65 70 7545Temperature (∘C)



00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

20 25 30 35 40 45 50 55 60 65 70 75 80 8515Temperature (∘C)


00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)

45 5 55 6 65 7 75 84pH





2HPO4buffer





3 35 4 45 5 55 6 65 7 75 8 8525pH

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)



4 Conclusion


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3 35 4 45 5 55 6 65 7 75 8 8525pH

00

200

400

600

800

1000

1200

Rela

tive a

ctiv

ity (

)



4 Conclusion


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article purification and characterization of a

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