research article production, purification, and...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 728735 8 pageshttpdxdoiorg1011552013728735

Research ArticleProduction Purification and Characterization ofa Major Penicillium glabrum Xylanase Using Brewerrsquos SpentGrain as Substrate

Adriana Knob12 Susan Michelz Beitel1 Diana Fortkamp1

Ceacutesar Rafael Fanchini Terrasan3 and Alex Fernando de Almeida4

1 Department of Biological Sciences Midwest State University Camargo Varela de Sa Street 03 85040-080 Guarapuava PR Brazil2 Department of Biology Midwest State University Camargo Varela de Sa Street 03 85040-080 Guarapuava PR Brazil3 Department of Chemical Engineering Federal University of Sao Carlos Rodovia Washington Luıskm 235 SP-310 13565-905 Sao Carlos SP Brazil

4Department of Biochemistry and Microbiology Sao Paulo State University 24-A Avenue 1515 13506-900 Rio Claro SP Brazil

Correspondence should be addressed to Adriana Knob adriknobgmailcom

Received 3 April 2013 Accepted 20 April 2013

Academic Editor Arzu Coleri Cihan

Copyright copy 2013 Adriana Knob 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

In recent decades xylanases have been used in many processing industries This study describes the xylanase production byPenicillium glabrum using brewerrsquos spent grain as substrate Additionally this is the first work that reports the purification andcharacterization of a xylanase using this agroindustrial waste Optimal production was obtained when P glabrum was grown inliquidmedium in pH55 at 25 ∘C under stationary condition for six daysThexylanase fromP glabrumwas purified to homogeneityby a rapid and inexpensive procedure using ammonium sulfate fractionation andmolecular exclusion chromatography SDS-PAGEanalysis revealed one band with estimated molecular mass of 1836 kDa The optimum activity was observed at 60 ∘C in pH 30The enzyme was very stable at 50 ∘C and high pH stability was verified from pH 25 to 50 The ion Mn2+ and the reducing agents120573-mercaptoethanol and DTT enhanced xylanase activity while the ions Hg2+ Zn2+ and Cu2+ as well as the detergent SDS werestrong inhibitors of the enzyme The use of brewerrsquos spent grain as substrate for xylanase production cannot only add value anddecrease the amount of this waste but also reduce the xylanase production cost

1 Introduction

Xylan is a major structural polysaccharide of plant-cell wallsbeing the second most prevalent in nature after cellulose Itis a heterogeneous polymer constituted primarily by a linear120573-(14)-D-xylose backbone which is partially acetylated andsubstituted at different degrees by a variety of side chainsmainly 120572-D-glucuronosyl and 120572-L-arabinosyl units Due toits structural complexity several hydrolases are required forcomplete xylan degradation The key enzyme in this processis endo-120573-(14)-xylanase (EC 3218) which cleaves the xylanbackbone to xylooligosaccharides [1 2]

Interest in xylanolytic enzymes has increased in recentyears due to their potential application in biotechnologyXylanolytic enzymes have applications in conversion of lig-nocellulosic materials to chemicals and fuels animal feed

digestion food and textile industries and as bleaching agentsin the pulp and paper processing [3 4]

Industrial production of enzymes on large scale is asso-ciated mainly with the substrate The use of agroindustrialwastes as low-cost substrates for the industrial enzymes pro-duction is a significant way to reduce production cost Thuslignocellulosic substrates have now received considerableinterest because of their possible use in secondary fermenta-tion process [5] Various lignocellulosic substrates involvingagroindustrial wastematerials like cornmeal corn cob wheatbran wheat straw rice straw sugarcane bagasse and coffeeby-products are being used as substrates for the productionof fungal xylanases [6ndash8]

Brewerrsquos spent grain (BSG) the main residue of brewingindustry is rich in cellulosic and noncellulosic polysaccha-rides It is produced at large amounts during the year and

2 BioMed Research International

represents around 85 of the total by-products generated [9]In Brazil the worldrsquos fourth largest beer producer (85 billionlitresyear) approximately 17 million tonnes of BSG weregenerated per year Despite the large amounts produced BSGhas received little attention as a low-cost by-product and itsuse is still limited mainly as animal feed [10]

Penicillium glabrum is a filamentous fungus that is dis-tributed worldwide frequently involved in food contamina-tion Due to its ability to disperse a large number of spores inthe environment P glabrum is frequently found in the foodmanufacturing industry [11] Despite its large implicationin food contamination few studies have been conductedto investigate the enzyme production by this fungus Onlytannase production by P glabrum has been described inthe literature [12] Since many agroindustrial wastes are apotentially valuable resource for industrial exploitation thiswork aimed to evaluate the xylanase production byP glabrumusing different agroindustrial wastes establish the best fungalgrowing conditions for xylanase production with the bestsubstrate and biochemically characterize the major enzymepurified

2 Materials and Methods

21 Organism and Growth P glabrum used in the presentwork is available in the Culture Collection of Environmen-tal Studies CentermdashCEAUNESP SP Brazil Conidia wereobtained from cultures inVogel solidmedium [13] containing15 (wv) glucose and 15 (mv) agar at 25 ∘C for 7 daysLiquid cultures were prepared in the same medium with1 (wv) carbon source and the pH was adjusted to 65Erlenmeyer flasks (125mL) containing 25mL of mediumwere inoculated with 10mL of spore suspension containing5 times 107 sporesmL and incubated at 30 ∘C for 5 days instationary condition

22 Preparation of Agroindustrial Wastes The agroindustrialwastes were obtained locally The residues were prepared byexhaustive washing with distilled water dried at 80 ∘C for 24ndash48 h and milled (35 mesh)

23 Enzyme Preparations and Assays Cultures were har-vested by filtration The culture filtrate was assayed for extra-cellular activity and protein The mycelium was washed withdistilled and sterilized water frozen and ground with sandin 005M sodium phosphate buffer pH 65 The slurry wascentrifuged at 3900 g at 4 ∘C and the supernatant was usedas intracellular protein source

Xylanase activity was determined at 50 ∘C using 10(wv) birchwood xylan (Sigma st Louis MO USA) in McIl-vaine buffer pH 65 This buffer is prepared by a mixtureof 01M citric acid and 02M sodium monohydrogen phos-phate After 5 and 10min of incubation the reaction wasinterrupted by the addition of 35-dinitrosalicylic acid (DNS)and the reducing sugars released were quantified [14] usingxylose as standardOne unit of enzyme activitywas defined asthe amount of enzyme capable of releasing 1 120583mol of reducingsugar per min under assay conditions Specific activity wasexpressed as unit per milligram of protein All enzymatic

assays were developed in triplicate and the results arepresented through mean values

24 Protein Determination Total protein was determined bymodified Bradfordmethod [15] using bovine serum albumin(BSA) as standard

25 Culture Conditions for Xylanase Production

251 EnzymeProduction onDifferent Carbon Sources Vogelrsquosliquid medium was supplemented with various carbonsources at a concentration of 1 (wv) The inoculated flaskswere incubated at 28 ∘C under stationary condition for fivedays Xylanase activity was determined as described previ-ously

252 Effect of Incubation Period Initial pH and Temperatureon Xylanase Production The incubation periodrsquos influenceon xylanase production by P glabrum was studied understanding culture and under shaking culture (120 rpm) for 8days The effect of initial pH on the enzyme production wasassayed from 30 to 80 and the temperature influence wasverified from 15 to 40 ∘C The initial pH values were adjustedby the addition of 10M sodiumhydroxide or phosphoric acidsolutions

26 Purification of a Major P glabrum Xylanase

261 Ammonium Sulfate Fractionation The crude enzyme(50mL) was fractionated by ammonium sulfate precipitation(0ndash90 wv) The supernatant of 90 ammonium sulfatesaturation obtained after centrifugation (6000 g 20min4 ∘C) was extensively dialyzed against 005M ammoniumacetate buffer pH 68 before analyses

262 Exclusion Molecular Chromatography The proteinsample obtained in the step above was chromatographed onSephadex G-75 column (26 times 640 cm) equilibrated andeluted with 005M ammonium acetate buffer pH 68 flowingat 18mLh Fractions (3mL) whose protein content was esti-mated by reading absorbance at 280 nm and xylanase activ-ity assayed as described previously To determine xylanasemolecular mass through gel filtration chromatography thecolumnwas calibrated using blue dextran for the void volumedetermination and ribonuclease (154 kDa) chymotrypsin(250 kDa) ovalbumin (430 kDa) and bovine serum albumin(670 kDa) as standardsThemolecularweight of xylanasewasestimated from a regression curve (1198772 = 0993) by plottinglog of the molecular weights of the standards against theratio between elution volumes of the standards and the voidvolume of the column

263 Electrophoresis Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) was performedusing a gradient of 8ndash18 (wv) polyacrylamide accordingto Laemmli [16] The resolved protein bands were visu-alized after staining with 01 (wv) Coomassie BrilliantBlue R-250 in methanol acetic acid and distillated water(4 1 5 vvv) The proteins phosphorylase b bovine serum

BioMed Research International 3

albumin ovalbumin carbonic anhydrase trypsin inhibitorand 120572-lactalbumin (SDS-LMW markers-Sigma st LouisMO USA) were used to plot the standard curve log ofmolecular weight against relative mobility in the gel

27 Enzyme Characterization

271 Temperature and pH Optima Thermal and pH StabilityTo determine the optimum pH the purified xylanases wereassayed at 50 ∘C in different pH values using 005M glycine-HCl buffer from pH 16 to 25 and McIlvaine buffer from30 to 70 The optimum temperatures were determined byperforming the reaction at temperatures ranging from 15 ∘Cto 75 ∘C in McIlvaine buffer pH 30

For pH stability assays the purified enzymes were diluted(1 2 vv) in 005M glycine-HCl buffer from pH 16 to 25 andMcIlvaine buffer for pH range from 30 to 70 The sampleswere incubated at 4 ∘C for 24 h After this period the xylanaseactivity was assayed under optimal conditions To evaluatethe thermal stability the purified enzyme was incubated at50 ∘C 55 ∘C and 60 ∘C at the optimal pH determined abovefor different periods

272 Effect of Substances The effect of metallic ions andother compounds on the xylanase activity was evaluated atconcentrations of 2mM and 10mM The residual activitieswere measured in relation to the control without substancesby performing the enzyme assay at the optimal conditions

273 Substrate Specificity Specificity of xylanase againstbirchwood beechwood and oat spelt xylans carboxymethylcellulose (CM-cellulose) and Avicel were assayed Substratesolutions of 1 (wv) were prepared in a buffer of optimumpH activity for the enzyme

274 Kinetic Parameters Theenzymewas incubatedwith oatspelt beechwood and birchwood xylans at concentrationsbetween 40 and 30mgmL The Michaelis-Menten constant(119870119898) and maximum reaction velocity (119881max) were estimated

from the Lineweaver-Burk reciprocal plots using ldquoGraFitrdquo 50software

3 Results and Discussion

31 Influence of the Carbon Source on Xylanase ProductionDistinct substrates such as pure carbohydrates and someagroindustrial wastes were evaluated for xylanase production(Table 1) Among the pure carbohydrates highest valuesof xylanase activity were obtained with oat spelt xylancorresponding to 2544UmL and 6496Umg protein Ingeneral higher levels of xylanolytic enzymes can be achievedwith substrates derived from xylan According to Kulka-rni et al [1] xylanase activity is inducible and xylan-richsubstrates play an important role in xylanase inductionWhen P glabrum was cultivated in medium with lactosesucrose cellobiose Avicel andCM-cellulose enzyme activitywas not detected Conversely lactose and sucrose increasedxylanase production by Penicillium canescens 10-10c [17] Inthe presence of glucose xylose andmaltose only low levels of

Table 1 Influence of pure carbohydrates and agroindustrial wasteson xylanase production by P glabrum

Carbon source(1 wv)

Intracellular protein(mg)

Xylanase activity(UmL) (Umg protein)

Pure carbohydratesGlucose 175 004 007Xylose 104 025 060Maltose 089 006 014Lactose 034 ND NDSucrose 134 ND NDCellobiose 111 ND NDAvicel 025 ND NDCM-cellulose 034 ND NDOat spelt xylan 198 2544 6496

Agroindustrial wastesSugarcanebagasse 053 834 2514

Wheat bran 184 2054 4335Oat bran 114 1354 2831Rice straw 124 1765 3034Brewerrsquos spentgrain 210 3432 10265

Soybean meal 076 823 2043Corn cobs 083 732 1823Citrus pectin 034 010 034Orangebagasse 038 016 065

enzyme activity were verified when compared to the cultureswith oat spelt xylan

Among the agricultural and agroindustrial wastes high-est xylanase production was observed with brewerrsquos spentgrain (3432UmL and 10265Umg protein) These valueswere higher than those obtained with oat spelt xylan Theproduction of xylanolytic enzymes by Penicillium janczewskiiwith brewerrsquos spent grain as substrate was previouslydescribed by Terrasan et al [18] However lower levels ofxylanase activity were obtained when compared to this studycorresponding to 158UmL under optimized conditionsXylanase activity was also observed in the presence of wheatbran oat bran and rice straw however the values obtainedwere lower than that observed by brewerrsquos spent grain Onlylow levels of xylanase activity were verified with sugarcanebagasse soybean meal and corn cobs Orange bagasse andcitrus pectin provided minimal fungal growth with absentor no significant levels of xylanase produced The differentproduction levels observed among the lignocellulosic mate-rials are probably related on differences in composition andthe accessibility of the substrates to the fungi Consideringthe elevated xylanase production obtained with BSG thissubstrate was selected for the subsequent optimization exper-iments

32 Effects of Culture Conditions on Xylanase ProductionCultivation conditions are essential for the successful pro-duction of an enzyme and optimization of parameters such


1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25

Intr

acel

lula

r pro

tein

(mg)

(a)

1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25In

trac

ellu

lar p

rote

in (m

g)

(b)

Figure 1 Time course of xylanase production by P glabrum instationary (a) and shake culture at 120 revminminus1 (b) Culture con-ditions Vogel medium with 1 (wv) brewerrsquos spent grain pH 65 at28 ∘C (◼) Xylanase activity (UmL) (∙) intracellular protein (mg)

as period of cultivation pH and temperature is importantfor a process development In standing culture (Figure 1(a))with brewerrsquos spent grain the highest xylanase productionwas verified in 60 day-old cultures (4245UmL) In shak-ing condition (Figure 1(b)) maximal xylanase activity wasobserved on 35-day-old cultures corresponding to the valuesof 2585UmL The highest P glabrum growth measured bythe intracellular protein concentration occurred on the fifthday in standing culture and on the third day in shake cultureUnder shaking aswell as under standing condition xylanaseswere expressed during the stationary phase reaching thedecline phase (Figure 1)

Temperature and pH are important environmentalparameters that determine growth rates of microorganismsand significantly affect the level of xylanases produced Theinfluence of pH culture on xylanase production during Pglabrum cultivation is presented in Figure 2(a) Xylanase

activity was detected in all pH evaluated The highest activitywas observed at initial pH 55 corresponding to the valuesof 4854UmLWith rare exceptions xylanase production byfilamentous fungi occurs in cultures with an initial pH under70 Trichoderma harzianum [19] and Penicillium janczewskii[18] showed enhanced xylanase production at pH 50 and55 respectively P glabrum could grow in media with initialpH between 30 and 80 (Figure 2(a)) with maximal growthin the range of 45 to 65 This result clearly indicates theacidophilic nature of this fungus

The effect of temperature on xylanase production by Pglabrum is presented in Figure 2(b) The highest xylanaseactivity was verified at 25 ∘C corresponding to 5143UmLSimilarly maximum xylanase production by Trichodermaviridewas achieved at 25 ∘C [20]The optimal growth verifiedat 25 ∘C is in accordance with the literature that describesthis temperature as ideal for P glabrum [11] The maximaltemperature for this filamentous fungus was 35 ∘C which isin agreement with some data reporting the absence of growthabove 37 ∘C [21 22]

33 Purification of Xylanase The major P glabrum xylanasewas purified by protein precipitation with ammonium sulfateand molecular exclusion chromatography High enzyme pro-portion (about 84) was observed in the 90 ammoniumsulfate saturation supernatantThemolecular exclusion chro-matography elution profile resulted in one xylanase activitypeak (Figure 3) The fractions corresponding to this peakwere pooled and the sample was submitted to SDS-PAGEshowing electrophoretic homogeneity (Figure 4)

The electrophoretic analysis revealed that xylanase cor-responded to a single molecular mass band of 1836 kDaNative enzyme molecular mass of 213 kDa was estimated forP glabrum xylanase by molecular exclusion chromatographyshowing monomeric form The molecular mass of the Pglabrum xylanase is in agreement with those found for thecatalytic domain of low molecular mass xylanases belongingto family 11 [23] Sanghvi et al [7] partially purified a xylanasefrom T harzianum with 290 kDa molecular mass while270 kDa was the molecular mass estimated for the xylanasefrom Penicillium sp [8]

The summary of the P glabrum xylanase purification ispresented in Table 2 The procedure resulted in an overallyield of 7694 and the specific activity increased 510-foldThe purified xylanase exhibited high specific activity corre-sponding to 45789Umgminus1 protein

34 Properties of Purified Xylanase

341 Effects of pH and Temperature Thermal and pH Stabil-ities The effects of temperature and pH on the activity ofthe purified xylanase were investigatedThe xylanase showedoptimal activity at pH 30 (Figure 5(a)) Similarly Laetiporussulphureus xylanase exhibited optimum activity at pH 30[24] However most xylanases present optimal activity inpH between 50 and 70 [25] and among the acidophilicxylanases the majority of them showed high activity onlyunder slight acid conditions


3 4 5 6 7 80

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

Initial pH

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

(a)

0

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

Temperature (∘C)15 20 25 30 35 40

(b)

Figure 2 Effect of initial pH (a) and temperature (b) on xylanase production by P glabrumCulture conditions Vogel mediumwith 1 (wv)brewerrsquos spent grain under stationary condition for six days at 28 ∘C (a) and pH 55 (b) (◼) Xylanase activity (UmL) (∙) intracellular protein(mg)

0 10 20 30 40 50 60 70 80 90000002004006008010012014016018020

Fraction

0

25

50

75

100

125

150

175

200

Xyla

nase

activ

ity (U

mL)

119860280

Figure 3 Gel filtration on Sephadex G-75 of the xylanase fromP glabrum The column was equilibrated and eluted with 50mMammonium acetate buffer pH 68 The flow rate and fraction sizewere 18mLh and 30mL respectively (◼) 119860

280

and (∙) xylanaseactivity

The optimum temperature for xylanase activity was 60 ∘C(Figure 5(b)) Values of optimum temperature of xylanasehydrolysis vary according to the producing microorganismUsually xylanases from filamentous fungi show optimumtemperature between 40 ∘C and 55 ∘C [26ndash28] Neverthelessother fungal xylanases show optimum temperature at 60 ∘Cor above [24 29]

A pH stability study is an essential part of an enzymecharacterization before it can be exploited commercially Thexylanase produced by P glabrum was stable over a broad pHrange (Figure 6(a)) Xylanase activity was maintained over80 at pH from 25 to 50 Microbial xylanases are usuallystable over a wide pH range (3ndash10) [1] The optimum activityin very acidic conditions and pH stability exhibited by

9766

45

30

20

14

M1 (kDa)

Figure 4 SDS-PAGE (8ndash18) of the purified P glabrum xylanaseLane M low molecular weight standard proteins lane 1 purifiedenzyme

P glabrum xylanase make this enzyme attractive for someindustrial applications such as in feed and food industries

Thermal stability is an interesting enzyme property dueto the great industrial importance Then enzyme stabilityanalyses were carried out The purified xylanase from Pglabrum was incubated without substrate at 50 ∘C 55 ∘C and60 ∘C (Figure 6(b)) The estimated half-lives (119879

12) at 60 ∘C

and 55 ∘C were 15 and 32min respectively This enzymewas stable at 50 ∘C with 119879

12of 150min retaining 70 of

its activity over 60min at this temperature The P glabrumxylanase is more thermostable than many fungal xylanasessuch as those from Penicillium expansum [27] andAspergillusniger B03 [28] however less thermostable than the xylanasefrom thermophilic Talaromyces thermophilus [30]


Table 2 Purification of xylanase from P glabrum

Purification step Total activity(U)

Total protein(mg)

Specific activity(Umg protein)

Purification(fold)

Recovery()

Culture filtrate 257150 2861 8984 100 10000Supernatant 90 of (NH4)2SO4 216501 742 29178 325 8419Sephadex G-75 197810 432 45789 510 7692

342 Effect of Substances In order to verify the effect of sub-stances on xylanase activity the purified enzyme was incu-bated in the presence of several metallic ions sodium dode-cyl sulfate (SDS) tetrasodium ethylenediaminetetraacetate(EDTA) dithiothreitol (DTT) phenylmethylsulfonyl fluo-ride (PMSF) and 120573-mercaptoethanol at 2mM and 10mMconcentrations (Table 3) In general the xylanase activity wasenhanced with increased concentration of the substancesused Hg2+ Cu2+ and Zn2+ were strong inhibitors of thexylanase Likewise Penicillium sclerotiorum and Aspergillusficuum xylanases were inhibited by these ions [26 31] Theinhibition by Hg2+ seems to be a general property of xylanas-es indicating the presence of thiol groups of cysteine residuesin their active sites or around them [32] Xylanase activityremains unaltered in the presence of Na+ and Mg2+ Slightactivation was observed in the presence of Ca2+ Co2+ andBa2+ Additionally P glabrum xylanase was remarkably stim-ulated when incubated with Mn2+ as A niger B03 xylanase[28]

EDTA a metal chelator decreased xylanase activity indi-cating that the purified enzyme requires metal ions for theiractions Total loss of activity was observed in the presenceof SDS indicating that hydrophobic interactions must beimportant in maintaining xylanase structure The reducingagents 120573-mercaptoethanol and DTT stimulated xylanaseactivity The enzymatic activity stimulation in the presenceof these thiol group-protecting agents can be explained bypreventing the oxidation of sulfhydryl groups Dutta et al[33] and Cardoso and Filho [34] also related the involvementof cysteine residues in the maintenance of tertiary structureof the active site in Penicillium citrinum and Acrophialophoranainiana xylanases respectively

343 Substrate Specificity and Kinetic Studies Specificitystudies indicated that the P glabrum xylanase did nothydrolyze Avicel or CM-cellulose but acted only on xylansThe purified xylanase exhibited typical Michaelis-Mentenkinetics for oat spelt birchwood and beechwood xylansallowing the corresponding kinetic constants to be calcu-lated P glabrum xylanase showed 119870

119898values of 53 31

and 12mgmLminus1 and 119881max values of 21210 19421 and39317 120583molminminus1mgminus1 of protein for birchwood beech-wood and oat spelt xylans respectively The 119870

119898and 119881max

values exhibited by P glabrum xylanase are in agreement withthe values presented by other fungal xylanases which rangefrom 009 to 409mg mLminus1 for 119870

119898and from 0106 to 10000

for 119881max [4] The values of 119870119898for these substrates indicated

that this enzyme has higher affinity for oat spelt xylanSimilarly the xylanases from A nainiana [34] and Fusarium

1 2 3 4 5 6 70

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

pH

(a)

10 20 30 40 50 60 70 800

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Temperature (∘C)

(b)

Figure 5 Influence of pH (a) and temperature (b) on the purifiedP glabrum xylanase Assay conditions 005M glycine-HCl bufferfrom pH 16 to 25 and McIlvaine buffer from 30 to 70 50 ∘C (a)McIlvaine buffer pH 30 (b)

oxysporum [35] showed highest value of 119870119898

for oat speltxylan

4 Conclusions

In this study a P glabrum strain was able to produce highlevels of xylanase using brewerrsquos spent grain as substrateConventional purification methods were effective to purifythe major xylanase from P glabrum The purification proce-dure resulted in higher overall yields as compared to others


1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

Incubation time (min)

(b)

Figure 6 pH (a) and thermal (b) stabilities of purified P glabrum xylanaseThe enzymatic preparation was incubated without substrate withglycine-HCl buffer from pH 16 to 25 and McIlvaine buffer from pH 30 to 70 at 4 ∘C for 24 h (a) the enzymatic preparation was incubatedat (998771) 50 (∙) 55 and (◼) 60 ∘C without substrate (b) In both assays the residual xylanase activity was assayed with McIlvaine buffer pH 30at 60 ∘C

Table 3 Effect of different substances on relative activity () ofpurified xylanase from P glabrum

Relative activity ()

Substance Concentration2mM 10mM

Control 100 100CuCl2 697 plusmn 03 213 plusmn 03ZnSO4 844 plusmn 20 752 plusmn 11MnSO4 1789 plusmn 16 2067 plusmn 22BaCl2 1175 plusmn 15 1249 plusmn 17CaCl2 1220 plusmn 14 1265 plusmn 12NH4Cl 1020 plusmn 155 1343 plusmn 17NaCl 993 plusmn 13 992 plusmn 15SDS ND NDPMSF 920 plusmn 09 893 plusmn 05MgSO4 972 plusmn 17 970 plusmn 12DTT 1134 plusmn 12 1344 plusmn 20CoCl2 1172 plusmn 16 1222 plusmn 14HgCl2 471 plusmn 05 NDPb(CH3COO)2 687 plusmn 20 191 plusmn 06EDTA 890 plusmn 20 648 plusmn 10120573-mercaptoethanol 1240 plusmn 20 1360 plusmn 20

described in the literature [24 26 36] Furthermore thespecific activity of purified xylanase is higher than those ofpreviously purified xylanases [26 31 37] P glabrum xylanasewas active at very low pH with optimum at 30 and it wasstable in an acid pH range These characteristics make itpotentially useful in some biotechnological processes suchas for animal feed clarification and maceration of juicesand wines Additionally the use of brewerrsquos spent grain assubstrate for xylanase production can not only add value and

decrease the amount of this waste but also reduce xylanaseproduction cost

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors would like to thank CAPES (Coordination forthe Improvement of Higher Education Personnel) and CNPq(National Council of Technological and Scientific Develop-ment) for the financial support and the scholarship awardedto the authors

References

[1] N Kulkarni A Shendye and M Rao ldquoMolecular and biotech-nological aspects of xylanasesrdquo FEMS Microbiology Reviewsvol 23 no 4 pp 411ndash456 1999

[2] T Collins C Gerday and G Feller ldquoXylanases xylanase fami-lies and extremophilic xylanasesrdquo FEMS Microbiology Reviewsvol 29 no 1 pp 3ndash23 2005

[3] AMoure P Gullon H Domınguez and J C Parajo ldquoAdvancesin the manufacture purification and applications of xylo-oligosaccharides as food additives and nutraceuticalsrdquo ProcessBiochemistry vol 41 no 9 pp 1913ndash1923 2006

[4] A Knob C R F Terrasan and E C Carmona ldquo120573-Xylosidasesfrom filamentous fungi an overviewrdquoWorld Journal of Microbi-ology and Biotechnology vol 26 no 3 pp 389ndash407 2010

[5] S Murugan D Arnold U D Pongiya and P M NarayananldquoProduction of xylanase from Arthrobacter sp MTCC 6915using saw dust as substrate under solid state fermentationrdquoEnzyme Research vol 2011 Article ID 696942 7 pages 2011

[6] U A Okafor T N Emezue V I Okochi B M Onyegeme-Okerenta and S Nwodo-Chinedu ldquoXylanase production by


Penicillium chrysogenum (PCL501) fermented on cellulosicwastesrdquo African Journal of Biochemistry Research vol 1 no 4pp 48ndash53 2007

[7] G V Sanghvi R D Koyani and K S Rajput ldquoThermostablexylanase production and partial purification by solid-state fer-mentation using agricultural waste wheat strawrdquoMycology vol1 no 2 pp 106ndash112 2010

[8] P S Murthy and M M Naidu ldquoProduction and application ofxylanase from Penicillium sp utilizing coffee by-productsrdquo Foodand Bioprocess Technology vol 5 no 2 pp 657ndash664 2012

[9] S Aliyu and M Bala ldquoBrewerrsquos spent grain a review of itspotentials and applicationsrdquo African Journal of Biotechnologyvol 10 no 17 pp 324ndash331 2011

[10] S IMussatto and I C Roberto ldquoChemical characterization andliberation of pentose sugars from brewerrsquos spent grainrdquo Journalof Chemical Technology and Biotechnology vol 81 no 3 pp268ndash274 2006

[11] J I Pitt and A D Hocking Fungi and Food Spoilage BlackieAcademic and Professional London UK 1997

[12] J van de Lagemaat and D L Pyle ldquoModelling the uptake andgrowth kinetics of Penicillium glabrum in a tannic acid-contain-ing solid-state fermentation for tannase productionrdquo ProcessBiochemistry vol 40 no 5 pp 1773ndash1782 2005

[13] H J Vogel ldquoA convenient growth medium for Neurospora(Medium N)rdquo Microbial Genetics Bulletin vol 13 pp 42ndash431956

[14] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[15] J J Sedmak and S EGrossberg ldquoA rapid sensitive and versatileassay for protein using coomassie brilliant blue G250rdquo Analyti-cal Biochemistry vol 79 no 1-2 pp 544ndash552 1977

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

[17] A A Assamoi F Delvigne J M Aldric J Destain and PThonart ldquoImprovement of xylanase production by Penicilliumcanescens 10-10c in solid-state fermentationrdquo BiotechnologieAgronomie Societe et Environnement vol 12 no 2 pp 111ndash1182008

[18] C R F Terrasan B TemerM C T Duarte and E C CarmonaldquoProduction of xylanolytic enzymes by Penicillium janczewskiirdquoBioresource Technology vol 101 no 11 pp 4139ndash4143 2010

[19] S Isil and A Nilufer ldquoInvestigation of factors affecting xylanaseactivity from Trichoderma harzianum 1073 D3rdquo BrazilianArchives of Biology and Technology vol 48 no 2 pp 187ndash1932005

[20] M Goyal K L Kalra V K Sareen and G Soni ldquoXylanase pro-duction with xylan rich lignocellulosic wastes by a local soil iso-late of Trichoderma viriderdquo Brazilian Journal of Microbiologyvol 39 no 3 pp 535ndash541 2008

[21] M Sinigaglia M R Corbo and C Ciccarone ldquoInfluence oftemperature pH and water activity on ldquoin vitrordquo inhibition ofPenicillium glabrum (Wehmer) Westling by yeastsrdquo Microbio-logical Research vol 153 no 2 pp 137ndash143 1998

[22] L Nevarez V Vasseur A Le Madec et al ldquoPhysiological traitsofPenicillium glabrum strain LCP 085568 a filamentous fungusisolated from bottled aromatised mineral waterrdquo InternationalJournal of Food Microbiology vol 130 no 3 pp 166ndash171 2009

[23] A Torronem and J Rouvine ldquoStructural and functional prop-erties of low molecular weight endo-1 4-120573-xylanasesrdquo Journalof Biotechnology vol 57 no 1ndash3 pp 137ndash149 1997

[24] J W Lee J Y Park M Kwon and I G Choi ldquoPurification andcharacterization of a thermostable xylanase from the brown-rot fungus Laetiporus sulphureusrdquo Journal of Bioscience andBioengineering vol 107 no 1 pp 33ndash37 2009

[25] A M Madlala S Bissoon S Singh and L Christov ldquoXylanase-induced reduction of chlorine dioxide consumption duringelemental chlorine-free bleaching of different pulp typesrdquoBiotechnology Letters vol 23 no 5 pp 345ndash351 2001

[26] A Knob and E C Carmona ldquoPurification and characterizationof two extracellular xylanases from Penicillium sclerotiorum anovel acidophilic xylanaserdquo Applied Biochemistry and Biotech-nology vol 162 no 2 pp 429ndash443 2010

[27] A L de Souza Querido J L C Coelho E F de Araujo and VM Chaves-Alves ldquoPartial purification and characterization ofxylanase produced by Penicillium expansumrdquoBrazilian Archivesof Biology and Technology vol 49 no 3 pp 475ndash480 2006

[28] GDobrev andB Zhekova ldquoPurification and characterization ofendoxylanase Xln-2 fromAspergillus niger B03rdquoTurkish Journalof Biology vol 36 pp 7ndash13 2012

[29] E M Fawzi ldquoHighly thermostable purified xylanase from Rhi-zomucormieheiNRRL 3169rdquoAnnals ofMicrobiology vol 60 no2 pp 363ndash368 2010

[30] I Maalej I Belhaj N F Masmoudi and H Belghith ldquoHighlythermostable xylanase of the thermophilic fungus Talaromycesthermophilus purification and characterizationrdquo Applied Bio-chemistry and Biotechnology vol 158 no 1 pp 200ndash212 2009

[31] F Lu M Lu Z Lu X Bie H Zhao and Y Wang ldquoPurificationand characterization of xylanase from Aspergillus ficuum AF-98rdquo Bioresource Technology vol 99 no 13 pp 5938ndash5941 2008

[32] K B Bastawde ldquoXylan structure microbial xylanases and theirmode of actionrdquoWorld Journal ofMicrobiologyampBiotechnologyvol 8 no 4 pp 353ndash368 1992

[33] T Dutta R Sengupta R Sahoo S S Ray A Bhattacharjee andS Ghosh ldquoA novel cellulase free alkaliphilic xylanase fromalkali tolerant Penicillium citrinum production purificationand characterizationrdquo Letters in Applied Microbiology vol 44no 2 pp 206ndash211 2007

[34] O A V Cardoso and E X F Filho ldquoPurification and character-ization of a novel cellulase-free xylanase from Acrophialophoranainianardquo FEMS Microbiology Letters vol 223 no 2 pp 309ndash314 2003

[35] I Jorge O de la Rosa J A Navas-Cortes R M Jimenez-Dıazand M Tena ldquoExtracellular xylanases from two pathogenicraces ofFusariumoxysporum f sp ciceris enzymeproduction inculture and purification and characterization of amajor isoformas an alkaline endo-120573-(14)-xylanase of low molecular weightrdquoAntonie van Leeuwenhoek vol 88 no 1 pp 49ndash59 2005

[36] S Shrivastava P Shukla and K Mukhopadhyay ldquoPurificationand preliminary characterization of a xylanase from Ther-momyces lanuginosus strain SS-8rdquo 3 Biotech vol 1 no 4 pp255ndash259 2011

[37] RD Kamble andA R Jadhav ldquoIsolation purification and char-acterization of xylanase produced by a new species ofBacillus insolid state fermentationrdquo International Journal of Microbiologyvol 2012 Article ID 683193 8 pages 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


represents around 85 of the total by-products generated [9]In Brazil the worldrsquos fourth largest beer producer (85 billionlitresyear) approximately 17 million tonnes of BSG weregenerated per year Despite the large amounts produced BSGhas received little attention as a low-cost by-product and itsuse is still limited mainly as animal feed [10]

Penicillium glabrum is a filamentous fungus that is dis-tributed worldwide frequently involved in food contamina-tion Due to its ability to disperse a large number of spores inthe environment P glabrum is frequently found in the foodmanufacturing industry [11] Despite its large implicationin food contamination few studies have been conductedto investigate the enzyme production by this fungus Onlytannase production by P glabrum has been described inthe literature [12] Since many agroindustrial wastes are apotentially valuable resource for industrial exploitation thiswork aimed to evaluate the xylanase production byP glabrumusing different agroindustrial wastes establish the best fungalgrowing conditions for xylanase production with the bestsubstrate and biochemically characterize the major enzymepurified

2 Materials and Methods

21 Organism and Growth P glabrum used in the presentwork is available in the Culture Collection of Environmen-tal Studies CentermdashCEAUNESP SP Brazil Conidia wereobtained from cultures inVogel solidmedium [13] containing15 (wv) glucose and 15 (mv) agar at 25 ∘C for 7 daysLiquid cultures were prepared in the same medium with1 (wv) carbon source and the pH was adjusted to 65Erlenmeyer flasks (125mL) containing 25mL of mediumwere inoculated with 10mL of spore suspension containing5 times 107 sporesmL and incubated at 30 ∘C for 5 days instationary condition

22 Preparation of Agroindustrial Wastes The agroindustrialwastes were obtained locally The residues were prepared byexhaustive washing with distilled water dried at 80 ∘C for 24ndash48 h and milled (35 mesh)

23 Enzyme Preparations and Assays Cultures were har-vested by filtration The culture filtrate was assayed for extra-cellular activity and protein The mycelium was washed withdistilled and sterilized water frozen and ground with sandin 005M sodium phosphate buffer pH 65 The slurry wascentrifuged at 3900 g at 4 ∘C and the supernatant was usedas intracellular protein source

Xylanase activity was determined at 50 ∘C using 10(wv) birchwood xylan (Sigma st Louis MO USA) in McIl-vaine buffer pH 65 This buffer is prepared by a mixtureof 01M citric acid and 02M sodium monohydrogen phos-phate After 5 and 10min of incubation the reaction wasinterrupted by the addition of 35-dinitrosalicylic acid (DNS)and the reducing sugars released were quantified [14] usingxylose as standardOne unit of enzyme activitywas defined asthe amount of enzyme capable of releasing 1 120583mol of reducingsugar per min under assay conditions Specific activity wasexpressed as unit per milligram of protein All enzymatic

assays were developed in triplicate and the results arepresented through mean values

24 Protein Determination Total protein was determined bymodified Bradfordmethod [15] using bovine serum albumin(BSA) as standard

25 Culture Conditions for Xylanase Production

251 EnzymeProduction onDifferent Carbon Sources Vogelrsquosliquid medium was supplemented with various carbonsources at a concentration of 1 (wv) The inoculated flaskswere incubated at 28 ∘C under stationary condition for fivedays Xylanase activity was determined as described previ-ously

252 Effect of Incubation Period Initial pH and Temperatureon Xylanase Production The incubation periodrsquos influenceon xylanase production by P glabrum was studied understanding culture and under shaking culture (120 rpm) for 8days The effect of initial pH on the enzyme production wasassayed from 30 to 80 and the temperature influence wasverified from 15 to 40 ∘C The initial pH values were adjustedby the addition of 10M sodiumhydroxide or phosphoric acidsolutions

26 Purification of a Major P glabrum Xylanase

261 Ammonium Sulfate Fractionation The crude enzyme(50mL) was fractionated by ammonium sulfate precipitation(0ndash90 wv) The supernatant of 90 ammonium sulfatesaturation obtained after centrifugation (6000 g 20min4 ∘C) was extensively dialyzed against 005M ammoniumacetate buffer pH 68 before analyses

262 Exclusion Molecular Chromatography The proteinsample obtained in the step above was chromatographed onSephadex G-75 column (26 times 640 cm) equilibrated andeluted with 005M ammonium acetate buffer pH 68 flowingat 18mLh Fractions (3mL) whose protein content was esti-mated by reading absorbance at 280 nm and xylanase activ-ity assayed as described previously To determine xylanasemolecular mass through gel filtration chromatography thecolumnwas calibrated using blue dextran for the void volumedetermination and ribonuclease (154 kDa) chymotrypsin(250 kDa) ovalbumin (430 kDa) and bovine serum albumin(670 kDa) as standardsThemolecularweight of xylanasewasestimated from a regression curve (1198772 = 0993) by plottinglog of the molecular weights of the standards against theratio between elution volumes of the standards and the voidvolume of the column

263 Electrophoresis Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) was performedusing a gradient of 8ndash18 (wv) polyacrylamide accordingto Laemmli [16] The resolved protein bands were visu-alized after staining with 01 (wv) Coomassie BrilliantBlue R-250 in methanol acetic acid and distillated water(4 1 5 vvv) The proteins phosphorylase b bovine serum













Carbon source(1 wv)











1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25

Intr

acel

lula

r pro

tein

(mg)

(a)

1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25In

trac

ellu

lar p

rote

in (m

g)

(b)












3 4 5 6 7 80

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

Initial pH

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

(a)

0

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

Temperature (∘C)15 20 25 30 35 40

(b)


0 10 20 30 40 50 60 70 80 90000002004006008010012014016018020

Fraction

0

25

50

75

100

125

150

175

200

Xyla

nase

activ

ity (U

mL)

119860280


280




9766

45

30

20

14

M1 (kDa)




12) at 60 ∘C







Total protein(mg)


Purification(fold)

Recovery()





119898values of 53 31


119898and 119881max


119898and from 0106 to 10000



1 2 3 4 5 6 70

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

pH

(a)

10 20 30 40 50 60 70 800

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Temperature (∘C)

(b)



for oat speltxylan

4 Conclusions



1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)


(b)










Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Carbon source(1 wv)











1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25

Intr

acel

lula

r pro

tein

(mg)

(a)

1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25In

trac

ellu

lar p

rote

in (m

g)

(b)












3 4 5 6 7 80

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

Initial pH

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

(a)

0

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

Temperature (∘C)15 20 25 30 35 40

(b)


0 10 20 30 40 50 60 70 80 90000002004006008010012014016018020

Fraction

0

25

50

75

100

125

150

175

200

Xyla

nase

activ

ity (U

mL)

119860280


280




9766

45

30

20

14

M1 (kDa)




12) at 60 ∘C







Total protein(mg)


Purification(fold)

Recovery()





119898values of 53 31


119898and 119881max


119898and from 0106 to 10000



1 2 3 4 5 6 70

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

pH

(a)

10 20 30 40 50 60 70 800

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Temperature (∘C)

(b)



for oat speltxylan

4 Conclusions



1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)


(b)










Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25

Intr

acel

lula

r pro

tein

(mg)

(a)

1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

45

Xyla

nase

activ

ity (U

mL)

Culture age (days)

00

05

10

15

20

25In

trac

ellu

lar p

rote

in (m

g)

(b)












3 4 5 6 7 80

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

Initial pH

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

(a)

0

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

Temperature (∘C)15 20 25 30 35 40

(b)


0 10 20 30 40 50 60 70 80 90000002004006008010012014016018020

Fraction

0

25

50

75

100

125

150

175

200

Xyla

nase

activ

ity (U

mL)

119860280


280




9766

45

30

20

14

M1 (kDa)




12) at 60 ∘C







Total protein(mg)


Purification(fold)

Recovery()





119898values of 53 31


119898and 119881max


119898and from 0106 to 10000



1 2 3 4 5 6 70

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

pH

(a)

10 20 30 40 50 60 70 800

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Temperature (∘C)

(b)



for oat speltxylan

4 Conclusions



1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)


(b)










Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


3 4 5 6 7 80

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

Initial pH

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

(a)

0

10

20

30

40

50

60

Xyla

nase

activ

ity (U

mL)

00

05

10

15

20

25

30

Intr

acel

lula

r pro

tein

(mg)

Temperature (∘C)15 20 25 30 35 40

(b)


0 10 20 30 40 50 60 70 80 90000002004006008010012014016018020

Fraction

0

25

50

75

100

125

150

175

200

Xyla

nase

activ

ity (U

mL)

119860280


280




9766

45

30

20

14

M1 (kDa)




12) at 60 ∘C







Total protein(mg)


Purification(fold)

Recovery()





119898values of 53 31


119898and 119881max


119898and from 0106 to 10000



1 2 3 4 5 6 70

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

pH

(a)

10 20 30 40 50 60 70 800

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Temperature (∘C)

(b)



for oat speltxylan

4 Conclusions



1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)


(b)










Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Total protein(mg)


Purification(fold)

Recovery()





119898values of 53 31


119898and 119881max


119898and from 0106 to 10000



1 2 3 4 5 6 70

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

pH

(a)

10 20 30 40 50 60 70 800

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Temperature (∘C)

(b)



for oat speltxylan

4 Conclusions



1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)


(b)










Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 80

20

40

60

80

100

Resid

ual a

ctiv

ity (

)

pH

(a)

0 20 40 60 80 100 120 140 160 180 200 2200

20

40

60

80

100

Resid

ual a

ctiv

ity (

)


(b)










Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article production, purification, and...

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