research article characterization of a new providencia...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 386769 10 pageshttpdxdoiorg1011552013386769

Research ArticleCharacterization of a New Providencia sp Strain X1 ProducingMultiple Xylanases on Wheat Bran

Abhay Raj1 Sharad Kumar1 Sudheer Kumar Singh2 and Mahadeo Kumar3

1 Environmental Microbiology Section CSIR-Indian Institute of Toxicology Research MG Marg Post Box No 80Lucknow Uttar Pradesh 226 001 India

2Microbiology Division CSIR-Central Drug Research Institute Jankipuram Extension Sector 10 Sitapur RoadLucknow Uttar Pradesh 226 003 India

3 Animal Facility CSIR-Indian Institute of Toxicology Research MG Marg Post Box No 80 Lucknow Uttar Pradesh 226 001 India

Correspondence should be addressed to Abhay Raj arajiitrresin

Received 4 August 2013 Accepted 26 September 2013

Academic Editors H Kallel K Ohmiya and M Petruccioli

Copyright copy 2013 Abhay Raj 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

Providencia sp strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposingsite Strain X1 produced cellulase-free extracellular xylanase which was higher in wheat branmedium than in xylanmedium whencultivated at pH 80 and 35∘C Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran andbirchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33 35 40 48 6075 and 95 kDa and 33 and 44 kDa respectively The crude xylanases were produced on wheat bran medium and showed optimumactivity at pH 90 and 60∘CThe thermotolerance studies showed activity retention of 100 and 85 at 40∘C and 60∘C after 30 minpreincubation at pH 90 It was tolerant to lignin ferulic acid syringic acid and guaiacol and retained 90 activity after ethanoltreatmentThe enzyme preparationwas also tolerant tomethanol and acetone and showed good activity retention in the presence ofmetal ions such as Fe2+ Mg2+ Zn2+ and Ca2+ The crude enzyme preparation was classified as endoxylanase based on the productpattern of xylan hydrolysis Pretreatment of kraft pulp with crude xylanases for 3 h at 60∘C led to a decrease in kappa number by285 The properties of present xylanases make them potentially useful for industrial applications

1 Introduction

Xylan is the major component of the hemicellulosic com-plex found in the plant cell wall Complete degradation ofxylan requires a coordinated action of several hydrolyticenzymes among which xylanases (EC 3218) are sufficientto hydrolyze 120573-14-D-xylosidic linkages in xylanThe xylan isthe most abundant renewable polysaccharide after celluloseand its hydrolysis by xylanases into simple compoundsmainly xylose is important for several biotechnologicalapplications The global demand of xylanase has increasedover the years due to its potential application in variousindustrial processes such as in animal feed food processingtextiles pharmacy and pulp and paper industry [1] Xylanaseis produced by a variety of microorganisms including bacte-ria fungi and actinomycetes [2ndash4] However compared tofungi major attention has been given to bacteria specially

Bacillus spp due to their ability to produce cellulase-freethermostable and alkaline xylanase [1 5]

Cellulase-free xylanases functioning at high temperatureand pH are required for application in pulp and paperindustry where they reduce toxic wastes making the bleach-ing process environmentally friendly However relativelyhigh cost of enzyme production has hindered the industrialapplication of the enzymatic process In microbial-basedenzyme production processes approximately 30ndash40 ofproduction of many industries is attributed to the cost of thegrowth substrates [6] Xylanases are generally producedwhenmicroorganisms are grown in culturemedia containing xylanand xylan hydrolysate as carbon sources However the com-mercially available xylan is too expensive for use at industrialscale productionTherefore agroresidues such as wheat branrice bran rice straw corn stalk corn cobs and sugarcanebagasse have been explored as substrates for the production

2 The Scientific World Journal

of xylanaseThewheat bran has been found to be the best sub-strate for higher xylanase production by Bacillus spp [2 7 8]

Although a highly purified enzyme is a prerequisite forcharacterization and structural studies however from appli-cation perspective using purified enzyme is not economicalfor applications like biobleaching [9] Also it has beenalready demonstrated that hydrolysis product profile of crudeand purified enzyme preparations are similar [10] Thususing agroresidues as substrate for microbial productionof xylanase with a combination of all characters that iscellulase-free functions at high pH and temperature couldbe one of the ways towards substantially reducing the enzymecost as verified by recent publications [7 11] For this reasonthere is still a need of continued search for more efficientxylanolytic bacterial strains that can produce enzymes suit-able for pulp biobleaching In the present study we screeneda new Providencia sp strain X1 from saw-dust decomposingsite It produces multiple xylanases when grown on wheatbran mediumThe crude enzyme preparation was alkali- andthermotolerant in nature

2 Materials and Methods

21 Chemicals Chemicals like birchwood xylan 35-dinitro-salicylic acid (DNSA) carboxymethyl cellulose (CMC)Congo red and D-xylose were purchased from Sigma (StLouis MO USA) Other reagents used in this work were ofanalytical grade

22 Isolation of Xylanolytic Bacteria Serial dilutions of 10(wv) soil sample collected from saw-dust decomposing sitelocated around a saw mill in Lucknow Uttar Pradesh Indiawere used for screening of the bacteria Agar platesweremadeby using modified Bergrsquos mineral salts (BMSs) medium thatcontained (gL) NaNO

330 K

2HPO405 MgSO

4sdot7H2O 02

MnSO4sdotH2O 002 FeSO

4sdotH2O 002 and CaCl

2sdot2H2O 002

agar powder 160 and yeast extract 50 [12] adjusted to pH70 by using 20 Na

2CO3 and 10 birchwood xylan (wv)

as a source of carbon After incubation at 35∘C for 48 h sixbacterial colonies of distinct morphology (named as X1-X6)were isolated and their purity was verified by microscopicobservation after Gram-staining

23 Screening of Xylanase Producing Isolates All isolatedxylanolytic bacteria were further tested for their xylanaseactivity by growing them on agar plate and in broth mediumAll six isolates were grown on BMS agar plate which con-tained birchwood xylan (10 wv) After incubation for 48 hat 35∘C xylan hydrolysis zone was measured by Congo redstainingmethod [13]These bacterial isolates were rescreenedby growing in BMS broth that contained birchwood xylan(10 wv) at 35∘C for 48 h at 120 rpm The xylanase activityin culture supernatant was measured by using the DNSAmethod [14]

24 Characterization of Bacterium The selected bacteriumwas characterized by conventional and molecularapproaches Morphological and biochemical tests were

performed according to standard method [15] Physiologicaltests namely thermotolerance and halotolerance assayswere carried out on Petri dishes For thermotolerance assay100 120583L culture broths were added to 90mL distilled waterand the dilution tubes were heated at 80 90 and 100∘C inwater bath for 30min Samples (50 120583L) were spread ontonutrient agar plates and incubated at 35∘C for 48 h Similarlyhalotolerance was assayed by adding 80 NaCl (wv) to themedia Molecular characterisation of bacterium was doneby 16S rRNA gene sequencing Total DNA was isolated frombacterium using kit (UltraClean Microbial DNA isolationKit MO BIO USA) The 16S rRNA gene of DNA samplewas PCR amplified using 16S rRNA universal primers27F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and 1492R(51015840-TACGGTTACCTTGTTACGACTT-31015840) at annealingtemperature of 56∘C (35 cycles) The PCR product waspurified by gel extraction (Gel extraction Kit Qiagen) andwas sequenced in an ABI 3130 genetic analyzer using BigDye Terminator version 31 cycle sequencing kit The taskof sequencing was outsourced to Ms Eurofins Genomica professional company in Bangalore India The 16S rRNAgene sequences were compared with available sequencesusing NCBI-BLAST A phylogenetic tree was constructedusing Bio Informatics Bacterial Identification (BIBI) software(httpumr5558-sud-str1univ-lyon1frlebibilebibicgi)

25 Xylanase Production by Bacterium Xylanase was pro-duced by selected bacterium in liquid culture condition usingBMS broth containing wheat bran and birchwood xylan asproduction substrate Erlenmeyer conical flasks (250mL)containing 100mL of BMS broth (pH 80) supplemented with10 (wv) of wheat bran or xylan were autoclaved for 20minat 121∘C The pH of the medium was adjusted using 20Na2CO3 Flasks were inoculated with 10mL of overnight

grown pre-culture with an inoculum size of 1 times 106 CFUmLonnutrient agar plate and incubated at 35∘C 120 rpm for 120 h(Innova New Brunswick USA) The culture broths werewithdrawn every 24 h and measured for change of bacterialgrowth and enzyme activity

26 Measurement of Bacterial Growth Culture broth (2mL)waswithdrawn tomeasure bacterial growth by taking absorb-ance at 620 nm in a glass cuvette with 10 cm path length (UV-visible 2300 spectrophotometer Techcomp Korea)

27 Estimation of Enzyme Activity The culture broth wascentrifuged at 10000timesg for 30min and xylanase activityin culture supernatant was determined by measuring thereleased reducing sugars formed by enzymatic hydrolysis ofbirchwood xylan Briefly 05mL of supernatant and 15mL of10 (wv) birchwood xylan prepared in 100mM phosphatebuffer pH 80 was incubated at 50∘C for 15minThe reactionwas terminated by adding 20mL of 35-dinitrosalicylic acid(DNS) reagent The color development due to formation ofreducing sugars wasmonitored at 540 nm [14] and quantifiedby comparisonwith the standard graph of D-xylose One unit(IU) of xylanase activity was defined as the amount of enzymethat liberated 1 120583Mof reducing sugars equivalent to D-xylose

The Scientific World Journal 3

per minute under the assay conditions Cellulase activitywas assayed by the same method except that low viscositycarboxymethyl cellulose (CMC) was used as substrate Oneunit of cellulase activity was defined as the quantity ofenzyme releasing 1 120583M reducing sugar equivalent to glucoseper minute under the assay conditions

28 Zymogram Analysis Crude enzyme preparationsobtained after ammonium sulfate precipitation (80saturation) and ultrafiltration (Amicon Ultra-15 10 kDaMillipore) were used for zymogram analysis [16] Samples(25ndash30 120583g protein) were subjected to SDS-PAGE [17] using10 polyacrylamide in gel Electrophoresis was carried outusing Mini-Gel Electrophoresis unit (Microkin TechnoSource Mumbai India) The samples were loaded induplicate without the addition of 120573-mercaptoethanol orany other reducing agents and without boiling to ensurethat all active isoforms present there may be detected Afterelectrophoresis the gel was cut in two parts One part wasstained with Coomassie brilliant blue R-250 staining and theother portion was used for zymogram analysis The gel forzymogram analysis was washed two times for 30min at 4∘Cin 100mM sodium phosphate buffer (pH 70) containing 25isopropanol to remove SDS and renature the protein Thegel was then incubated in the same buffer containing 10birchwood xylan solution at 37∘C for 30min to allow theenzyme to digest xylan Zymogramwas developed by stainingthe gels in Congo red solution (01 wv) for 15min The gelwas then destained with NaCl solution (1M) until zones ofclearance and then immersed in 05 (wv) acetic acid tostop the reaction Presence of halos around the proteinbands was considered as enzyme activity The molecularweight of proteins was determined by comparing them withstandard protein marker (GenScript USA) Total proteinconcentration in crude xylanase preparations was measuredby the method of Bradford [18] using the Genei ProteinEstimation Kit (Bangalore Genei India)

29 Partial Characterization of Crude Xylanase Preparation

291 Optimum pH and Stability of the Xylanase The opti-mum pH for xylanase activity was determined by incubatingthe crude enzyme preparation with 10 (wv) birchwoodxylan prepared in 100mM of citrate buffer (pH 40ndash60)phosphate buffer (pH 60ndash80) Tris-HCl buffer (pH 80ndash90)or glycine-NaOH buffer (pH 90ndash110) at 50∘C for 15minTo determine the stability of enzyme at different pH thecrude xylanase preparation was preincubated in the abovementioned buffers of pH 40ndash110 at 37∘C for 30min withoutsubstrate and the remaining activity was measured in phos-phate buffer (pH 80) at 50∘C

292 Optimum Temperature and Stability of the XylanaseThe optimum temperature for xylanase activity was deter-mined by incubating the crude enzyme preparationwith 10(wv) birchwood xylan prepared in Tris-HCl buffer (pH 90)for 15min at 30∘C 40∘C 50∘C 60∘C 70∘C 80∘C 90∘C and100∘C Thermostability of the xylanase was determined by

preincubating the crude enzyme preparation at the abovementioned temperatures in Tris-HCl buffer (pH 90) withoutsubstrate for 30min and then residual activity was measuredat 60∘C

293 Effect of Metals and Solvents on Xylanase Activity Theeffect ofmetal ions on crude xylanase activitywas determinedby addition of metal ions (FeSO

4 MnSO

4 MgCl

2 ZnSO

4

CuSO4 and CaCl

2) at 2mM final concentration in a 20mL

reaction mixture Similarly the effect of organic solvents onenzyme activity was determined using ethanol acetone andmethanol at 25 (vv) concentration in a 20mL reactionmixture The activity of enzyme without any metal andsolvent was considered as control and was taken as 100

294 Effect of Lignin and Its Degradation Product on XylanaseActivity To determine the effect of lignin and its degrada-tions products on crude xylanase activity the enzyme assayswere performed by the addition of lignin ferulic acid gallicacid syringic acid guaiacol and phenol The effect of ligninwas measured over a range of concentration from 0125 to15mgL and the effects of ferulic acid gallic acid syringicacid guaiacol andphenolwere investigated over a range from25 to 10mgmL The enzyme reaction mixture in absence oflignin ferulic acid gallic acid syringic acid guaiacol andphenol was treated as a control All the experiments were car-ried out in triplicate and the results presented are mean plusmn SD

295 Determination of ExoEndoxylanolytic Activity Theexoendoxylanolytic activity of the crude enzymepreparationof strain X1 was examined by carrying out thin layer chro-matography (TLC) on silica gel plates The crude xylanasepreparation control (standard xylan) and treated xylan(1mL of 1 xylan incubatedwith 05mL of crude xylanase for30min at 50∘C) were extracted with methanol [19] Aliquotsof 10 120583L for standard D-xylose (1mgmL) and methanolicextract of crude xylanase preparation control and treatedxylan were applied on TLC plate and developed in a solventmix consisting of n-butanol-acetic acid-water (6 4 3 vvv)The TLC plate was sprayed with 995 ethanol-concentratedsulfuric acid mix (1 1 vv) and visualized after heating at100∘C

296 Biobleaching Studies One gram unbleached hardwoodKraft pulp (Star Paper Mill Saharanpur Uttar PradeshIndia) was washed extensively and treated with 25mL ofcrude xylanase preparation (10 IUmL) at 60∘C (pH 90) for3 h at 100 rpm At the end of reaction control and enzymetreated pulp samples were filtered washed with tap waterand dried in an oven at 50∘C to a constant weight Reducingsugars released from untreated and enzyme-treated pulpwere measured according to Miller [14] Kappa number ameasurement of lignin content in pulp was determined byreaction of pulp with acidified potassium permanganate [20]All the experiments were carried out in triplicate and theresults presented are mean plusmn SD of the three values


Table 1 Screening test for selecting bacteria with high xylanase producing ability

Isolate no Observation of plate assaya Xylanase activityb (IUmL)Clearance zone diameter (cm) Colony diameter (cm) Diameter ratio

X1 25 14 18 174 plusmn 04

X2 13 13 10 96 plusmn 02

X3 25 25 10 64 plusmn 01

X4 24 13 18 94 plusmn 02

X5 14 14 10 46 plusmn 01

X6 23 21 11 72 plusmn 01aMedium used Bergrsquos mineral salts-xylan agar growth conditions pH 70 35∘C 48 hbMedium used Bergrsquos mineral salts-xylan broth growth conditions pH 80 35∘C 48 h 120 rpm

3 Results and Discussion

31 Isolation and Characterization of Bacterial Strain X1 Sixbacterial strains (X1-X6) which are morphologically distinctand formed clear zones on xylan-agar plate were isolatedfrom saw-dust decomposing soil Strain X1 which showed thehighest clearance was selected for further characterizationThe property was corroborated by measurement of xylanaseactivity which again was the highest (174 plusmn 04 IUmL) instrain X1 (Table 1) The extracellular enzyme matrix fromstrain X1 was free from cellulase contamination (data notshown) Strain X1 was Gram-positive and spore forming rodAlso it was catalase oxidase caseinase and tryptophanasepositive It was able to metabolize variety of sugars exceptfructose and sucrose (Table 2) The isolate was moderatelythermophilic and thermotolerant It grew at 50∘C and showedits tolerance up to 90∘C for 30min It was also tolerant toalkaline pH (pH 10) and salt stress (NaCl 80) This abilityto withstand temperature salt stress and pH stress maybe an intrinsic property of the strain [21] The nucleotideblast analysis of 16S rDNA sequence showed gt98 identitywith several Providencia sp however maximum homology(99) was observed with Providencia rettgeri (Figure 1) The16S rDNA sequence has been deposited in NCBI GenBank(Accession no JN384121)

32 Xylanase Production by Strain X1 The xylanase produc-tion studies suggested that wheat bran as substrate was suit-able for growth and enzyme production The bacterial strainwhen grown on wheat bran produced extracellular xylanase(363 plusmn 16 IUmL) after 48 h of incubation (Figure 2(a)) Theenzyme production was lower when grown on pure xylan(174 plusmn 08 IUmL) after 48 h of incubation and a decliningtrendwas observed afterwards (Figure 2(b)) Higher xylanaseactivity in wheat bran medium could be due to improvedbacterial growth (OD

62024) in this medium compared to

(OD620

13) in xylan medium at 48 h (Figures 2(a) and 2(b))High production of xylanase in wheat bran medium has alsobeen reported earlier during submerged fermentation (SmF)of Geobacillus thermoleovorans and Bacillus pumilus strainsMK001 [22 23] The suitability of wheat bran as substratecould be due to the presence of 45hemicellulose whichmayfulfill the role of inducer and 23 organic nitrogen which isessential for protein synthesis [24]

Table 2Morphological and biochemical characteristics of strainX1

Test ResultsWhole colony White circularEdge EntireSurface SmoothElevation RaisedCell shape RodSpore position SubcentralGram-reaction +Growth at 50∘C +Growth at 80 NaCl +Motility +Catalase +Oxidase +Caseinase +Urease minusGelatinase minusTryptophanase +Sugar utilization

Glucose +Fructose minusLactose +Maltose +Xylose +Sucrose minusArabinose +Mannose +

+ positive minus negative

33 Zymogram Analysis Zymogram technique wasemployed to observe the presence of multiple forms ofxylanase expressed in presence of wheat bran and birchwoodxylan The zymogram analysis of crude xylanase preparationof organism grown on wheat bran contained multiplexylanases as indicated by the presence of halos around sevendistinct proteins bands corresponded to estimated molecularweight of 33 35 40 48 60 75 and 95 kDa (Figure 3) Incontrast crude xylanase preparation of organism grown onbirchwood xylan contained only two xylanases of 33 kDaand 44 kDa (Figure 4) This suggested differential expressionof xylanases in Providencia sp strain X1 as has also beenreported earlier fromMyceliophthora sp [25] Multiplicity ofxylanases has been documented in various microorganismswith evidence for the occurrence of 3 to 30 xylanases inbacteria and fungi [6] possibly as a result of differential


Providencia rettgeri~v~T~DQ885263

Providencia rettgeri~v~N~GU193984

Providencia rettgeri~v~N~HQ654052

Providencia rettgeri~v~N~HM590704

Providencia rettgeri~v~N~EU660367


Providencia rettgeri~v~N~GQ923882



Providencia rettgeri~v~T~AM040492=

Providencia stuartii~v~N~AB680338

Providencia rettgeri~v~N~AB680422


Providencia vermicola~v~N~EF192136

Providencia vermicola~v~N~HM582881

Providencia vermicola~v~T~AM040495=

Providencia vermicola~v~N~JN092794Providencia vermicola~v~N~JN092797

Providencia vermicola~v~N~JN092796

Strain X1 JN384121




Providencia rettgeri~v~N~JN644501


Providencia stuartii~v~N~AY803746



Providencia alcalifaciens~v~N~JF290496

Providencia alcalifaciens~v~N~AB480755001

Figure 1 Phylogenetic tree of the Providencia sp strain X1 and their related genera has been linked based on 16S rRNA sequence comparisons

mRNA processing and posttranslational modifications aswell as the product of multiple genes [26 27] To the bestof our knowledge this is the first evidence of large numberof xylanases induction in bacteria by wheat bran Thepresence of multiple xylanases in crude preparation fromone organism could synergistically function for superiorxylan hydrolysis [21]

34 Characterization of Crude Xylanase Preparation ofOrganism Grown on Wheat Bran

341 Effects of pH and Temperature on Activity and Stabilityof Xylanase The crude xylanase preparation was active inthe pH range of pH 40ndash110 (gt60 activity) with the highest

activity at pH 90 (Figure 5) Also the enzyme activity was76 and 61 of its optimum activity at pH 100 and pH 110respectively The stability studies at different pH suggestedthat crude enzyme preparation was stable over the pH rangesof 40ndash110 but retained 97ndash100 of its residual activity after30min preincubation at pH 60ndashpH 80 (Figure 5) Howevermore than 80 65 and 58 of its activity was retained bythe enzyme after preincubation at pH 90 pH 10 and pH 110respectively thus suggesting that crude enzyme preparationwas moderately stable at elevated pH Most of the xylanasesisolated from the Bacillus spp exhibited the highest activitywithin pH range of 60ndash80 [28ndash31] Although xylanasesisolated from Bacillus sp strain 41M-1 and Bacillus sp AR-009 [32 33] showed the highest activity at pH 90 but they


0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5

Xyla

nase

activ

ity (I

Um

L)

Incubation time (days)

Gro

wth

(A620

nm)

(a)

Xyla

nase

activ

ity (I

Um

L)

0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5Incubation time (days)

Gro

wth

(A620

nm)

(b)

Figure 2 Growth (◻) and xylanase () production by strain X1 in wheat bran (a) and xylan medium (b) at 35∘C pH 80 and shaking at120 rpm

30

200

150100

8060

40

(kDa) M 21

1 (95kDa)2 (75kDa)3 (60kDa)4 (48kDa)5 (40kDa)

6 (35kDa)7 (33kDa)

Figure 3 Zymogram analysis shows presence of seven distinctxylanases in crude enzyme preparation of strain X1 grown on wheatbran Lane 1 Coomassie brilliant blue stained gel Lane 2 Congo redfollowed by acetic acid stained gel and Lane M protein molecularweight marker

have lower stability at this pH values At pH 90 xylanaseisolated from Bacillus sp AR-009 retained only 54 of itsactivity after 1 h [33]

The activity of crude xylanase preparationwas the highestat 60∘C The enzyme retained more than 80 and 40of the maximum activity when assayed at 70∘C and 80∘Crespectively (Figure 6) The stability studies at different tem-peratures revealed that crude xylanase preparation did notlose significant activity up to 50∘Cafter 30min preincubationHowever it retained 85 and 45 of its original activityafter 30min incubation at 60∘C and 70∘C respectively Above70∘C the enzymersquos stability decreased gradually and only10 activity was retained at 80∘C (Figure 6) The significantenzyme stability at higher temperatures that is 50∘C and

44kDa

33kDa30

200150100

8060

40

(kDa) M 21

Figure 4 Zymogram analysis shows presence of two xylanases incrude enzyme preparation of strain X1 grown on birchwood xylanLane 1 Coomassie brilliant blue stained gel Lane 2 Congo redfollowed by acetic acid stained gel and Lane M protein molecularweight marker

60∘C would be important for its industrial application Mostof the xylanases isolated from Bacillus spp showed the high-est activity in the temperature range of 50ndash55∘C [28 30 34]Some xylanases of bacterial origin showed optimum activityat 60∘C but their pH optima were less than pH 90 [35 36]Differently in the present study the optimum pH and tem-perature for xylanase were 90 and 60∘C respectively therebysuggesting it to be an alkali and thermotolerant enzyme

342 Effect of Metal Ions and Solvents on Enzyme ActivityThe addition of Fe2+ Mg2+ Zn2+ and Ca2+ ions at finalconcentration of 2mMshowed a partial inhibition on activityof the crude xylanase (retaining 93ndash98 of activity) whereas


0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty

Figure 5 Effect of pH on activity and stability of crude xylanasepreparation of strain X1

30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty

Figure 6 Effect of temperature on activity and stability of crudexylanase preparation of strain X1

addition of 2mM of Mn2+ strongly inhibited the xylanaseactivity by 65 (Table 3) Mn2+ has earlier been reportedto strongly inhibit the activity of the xylanase of Bacillushalodurans S7 [37] In contrast to this study presence ofFe2+ Mg2+ Zn2+ and Ca2+ ions at the concentration of1mM increased 63 56 63 and 31 activity of xylanasefrom Arthrobacter sp MTCC 5214 receptively [38] Thestability studies with hydrophilic solvents suggested it tobe showing good solvent tolerance in 25 ethanol (91)methanol (82) and acetone (78) (Table 3) In contrastcomplete inhibition ofMacrotermes subhyalinus xylanase wasobserved at 30 and 60 vv of alcohols (methanol ethanolpropanol and butanol) [39] Impurities like metal ions and

Table 3 Effect of metal ions and solvents on activity of crudexylanase preparation from strain X1

Additive Relative xylanase activity ()None 100 plusmn 40

MetalCaCl2 98 plusmn 40

FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44

SolventEthanol 91 plusmn 43

Methanol 82 plusmn 38

Acetone 78 plusmn 50

solvents which can potentially inhibit the activity of xylanaseexist in rawpulpThus significant activity of xylanase isolatedfrom Providencia sp strain X1 in presence ofmetal ions (Fe2+Mg2+ Zn2+ and Ca2+) and hydrophilic solvents enhances itssuitability for paper pulping applications

343 Effect of Lignin and Phenolic Compounds on Activityof Xylanase The effect of soluble lignin on xylanase activitywas investigated at lignin concentration ranging from 0 to15mgmL The result showed that inhibition in xylanaseactivity was observed at low lignin concentration with a 10reduction in activity at 025mgmL of lignin However noincrease in inhibition was observed at higher concentrationsof lignin (Figure 7(a)) The inhibition in enzyme activitycan be correlated with either precipitation of protein outof the solution or by binding of the enzyme onto lignin[40] Morrison et al [41] have studied the impact of solublelignin and found a 25 inhibition in enzyme activity at lowconcentration of lignin (0075mgmL) In contrast Kaya etal [42] reported that the addition of lignin at increasingconcentrations (0ndash006) increased the hydrolysis rate ofxylanase by more than 20 Berlin et al [43] examinedthe inhibition of cellulase xylanase and 120573-glucosidase bylignin preparation Cellulase displayed varying levels of inhi-bition while xylanases displayed consistent inhibition and 120573-glucosidase was the least affected

The kraft pulp contains trace amount of low molecularweight phenolic compounds which may potentially affectthe activity of enzyme In the present study four phenoliccompounds namely ferulic acid syringic acid guaiacoland phenol were tested to see their effect on xylanaseactivity at concentrations ranging from 0 to 10mgmLThese compounds are either lignin degradation productsor naturally present in plant biomass Addition of ferulicacid syringic acid and guaiacol increased the activity ofxylanase by 91 21 and 26 respectively as comparedto control at concentration of 10mgmL (Figure 7(b)) Con-versely phenol had less inhibitory effect on xylanase activityat higher concentration (10mgmL) Morrison et al [41] haveexamined the impact of 120588-coumaric acid and gallic acid


0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)

Ferulic acid Syringic acid Guaiacol Phenol

0mgmL50mgmL

25mgmL100mgmL

(b)

Figure 7 Influence of lignin (a) and its monomeric compounds (b) on activity of crude xylanase preparation of strain X1

on xylanase activity and found 85 and 84 inhibition atlow concentration (01) of 120588-coumaric acid and gallic acidKaya et al [42] found that at low concentration (005) ofvanillin acid acetovanillone guaiacol and protocatechuicacid increased the rate of hydrolysis of xylan Other authorshave also investigated the effect of phenolic compounds andconcluded that the degree of inhibition varied depending onthe enzyme the microorganism it was derived from and thephenolic compound tested [44]

344 Endoxylanolytic Activity The thin layer chromatog-raphy (TLC) (Figure 8) study revealed absence of bandsin methanolic extract of control xylan and crude xylanase(Lanes 2 and 4)Themethanolic extract of treated xylan (Lane3) revealed three bands which were below the standard D-xylose (Lane 1) These results indicated that Providencia spstrain X1 xylanase released a range of xylooligosaccharidesfrom xylan suggesting it to be an endoxylanase Similarlyxylanase produced by Bacillus halodurans S7 was an endoxy-lanase [37]

345 Pulp Pretreatment Studies The biobleaching efficiencyof Providencia sp strain X1 xylanase was studied by treatmentof pulp with crude enzyme (10 IUmL) after incubation of 0to 3 h The results reveal gradual increase of reducing sugarswith time The amount of reducing sugars present in filtrateof untreated pulp was 59 120583gmL and it increased to 185455 and 460120583gmL in filtrate of treated pulp after 1 2 and3 h respectively (Figure 9) Kamble and Jadhav [31] reportedthat the amount of reducing sugars released from kraft pulpby the xylanase isolated from Cellulosimicrobium sp MTCC10645 was significantly greater with increasing time Kappanumber is the measurement of the amount of lignin presentin the pulp Kappa number of untreated pulp was 137 Aftertreating it with enzyme it decreased to 98 which indicatesthat the kappa number of pulp decreased by 285 after 3 hincubation Biobleaching of hardwood kraft pulp with crudexylanase dose of 20 IU gminus1 pulp fromBacillusmegaterium andB pumilusASHhas resulted in a decrease in kappa number by

Lane 1 Lane 4Lane 3Lane 2

1

234

Figure 8 Demonstration of endoxylanolytic nature of crudexylanase preparation of strain X1 Lane 1 indicates standard D-xylose Lanes 2 and 3 represent the methanol extract of control andtreated xylan while Lane 4 represents crude xylanase respectivelyNumbers (1ndash4) represent compounds present in crude xylanase-treated xylan

11 to 136 after 3 h treatment at 50∘C and 60∘C respectively[7 11] While in the present study the treatment for 3 h at60∘C using crude xylanases from Providencia sp strain X1 ledto 285 reduction in kappa number

4 Conclusions

The pulp and paper industry requires cellulase-free andthermoalkalophilic xylanase that can be produced at low costIn the present study the isolated bacterial strain X1 producesxylanases using wheat bran as a substrate The xylanasesproduced by present isolate are cellulase-free and are alkaliand thermotolerant They are also tolerant to higher ligninconcentrations as well as its degradation products and tometal ions and solvents The significant reduction in kappa


500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)

0 1 2 3Treatment time (h)

16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)

Reducing sugar120581 number

Figure 9 Effect of crude xylanase preparation treatment on level ofthe reducing sugars released and kappa number of kraft pulp

number of pulp by crude xylanase preparation suggestedProvidencia sp strain X1 to be a potential source of xylanasefor pulp biobleaching

Acknowledgments

The authors A Raj S Kumar and M Kumar gratefullyacknowledge the support and encouragement provided bythe Director of CSIR-Indian Institute of Toxicology ResearchLucknow India The authors acknowledge CSIR TASKFORCE project INDEPTH (BSC 0111) The present paper isIITR paper no 538

References

[1] S S Dhiman J Sharma and B Battan ldquoIndustrial applicationsand future prospects of microbial xylanases a reviewrdquo BioRe-sources vol 3 no 4 pp 1377ndash1402 2008

[2] A Sanghi N Garg J Sharma K Kuhar R C Kuhad and V KGupta ldquoOptimization of xylanase production using inexpensiveagro-residues by alkalophilic Bacillus subtilisASH in solid-statefermentationrdquoWorld Journal ofMicrobiology and Biotechnologyvol 24 no 5 pp 633ndash640 2008

[3] B K Bajaj and N P Singh ldquoProduction of xylanase from analkalitolerant Streptomyces sp 7b under solid-state fermenta-tion its purification and characterizationrdquo Applied Biochem-istry and Biotechnology vol 162 no 6 pp 1804ndash1818 2010

[4] K S Kumar A Manimaran K Permaul and S Singh ldquoPro-duction of 120573-xylanase by a Thermomyces lanuginosus MC 134mutant on corn cobs and its application in biobleaching ofbagasse pulprdquo Journal of Bioscience and Bioengineering vol 107no 5 pp 494ndash498 2009

[5] Q K Beg M Kapoor L Mahajan and G S HoondalldquoMicrobial xylanases and their industrial applications a reviewrdquoApplied Microbiology and Biotechnology vol 56 no 3-4 pp326ndash338 2001

[6] S Subramaniyan and P Prema ldquoBiotechnology of microbialxylanases enzymology molecular biology and applicationrdquoCritical Reviews in Biotechnology vol 22 no 1 pp 33ndash64 2002

[7] B Battan J Sharma S S Dhiman and R C Kuhad ldquoEnhancedproduction of cellulase-free thermostable xylanase by Bacilluspumilus ASH and its potential application in paper industryrdquoEnzyme and Microbial Technology vol 41 no 6-7 pp 733ndash7392007

[8] C A Poorna and P Prema ldquoProduction of cellulase-freeendoxylanase from novel alkalophilic thermotolerent Bacilluspumilus by solid-state fermentation and its application inwastepaper recyclingrdquo Bioresource Technology vol 98 no 3 pp485ndash490 2007

[9] J Kiddinamoorthy A J Anceno G D Haki and S K RakshitldquoProduction purification and characterization of Bacillus spGRE7 xylanase and its application in eucalyptus kraft pulpbiobleachingrdquoWorld Journal ofMicrobiology and Biotechnologyvol 24 no 5 pp 605ndash612 2008

[10] A Dhillon J K Gupta B M Jauhari and S Khanna ldquoAcellulase-poor thermostable alkalitolerant xylanase producedby Bacillus circulans AB 16 grown on rice straw and itsapplication in biobleaching of eucalyptus pulprdquo BioresourceTechnology vol 73 no 3 pp 273ndash277 2000

[11] I Sindhu S Chhibber N Capalash and P Sharma ldquoProductionof cellulase-free xylanase from Bacillus megaterium by solidstate fermentation for biobleaching of pulprdquo Current Microbi-ology vol 53 no 2 pp 167ndash172 2006

[12] B Berg B von Hofsten and G Pettersson ldquoGrowth andcellulase formation by Cellvibrio fulvusrdquo Journal of AppliedBacteriology vol 35 no 2 pp 201ndash214 1972

[13] R M Teather and P J Wood ldquoUse of congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982

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

[15] G I Barrow and R K A Feltham Cowan and Steelrsquos Manualfor the Identification of Medical Bacteria Cambridge UniversityPress New York NY USA 3rd edition 1993

[16] M-J Tseng M-N Yap K Ratanakhanokchai K L Kyu andS-T Chen ldquoPurification and characterization of two cellulasefree xylanases from an alkaliphilic Bacillus firmusrdquo Enzyme andMicrobial Technology vol 30 no 5 pp 590ndash595 2002

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

[18] 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

[19] B K Kubata T Suzuki H Horitsu K Kawai and KTakamizawa ldquoPurification and characterization of Aeromonascaviae ME-1 xylanase V which produces exclusively xylobiosefrom xylanrdquo Applied and Environmental Microbiology vol 60no 2 pp 531ndash535 1994

[20] ldquoTappi method T 236 cm-8rdquo in Kappa Number of Pulp TappiPress Atlanta Ga USA 1985

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


[22] A Sharma S Adhikari and T Satyanarayana ldquoAlkali-thermo-stable and cellulase-free xylanase production by an extremethermophile Geobacillus thermoleovoransrdquo World Journal ofMicrobiology and Biotechnology vol 23 no 4 pp 483ndash4902007

[23] M Kapoor L M Nair and R C Kuhad ldquoCost-effective xylan-ase production from free and immobilized Bacillus pumilusstrain MK001 and its application in saccharification of Prosopisjuliflorardquo Biochemical Engineering Journal vol 38 no 1 pp 88ndash97 2008

[24] K R Babu and T Satyanarayana ldquoProduction of bacterialenzymes by solid state fermentationrdquo Journal of Scientific andIndustrial Research vol 55 no 1 pp 464ndash467 1996

[25] A K Badhan B S Chadha K G Sonia H S Saini and M KBhat ldquoFunctionally diverse multiple xylanases of thermophilicfungus Myceliophthora sp IMI 387099rdquo Enzyme and MicrobialTechnology vol 35 no 5 pp 460ndash466 2004

[26] K K Wong L U Tan and J N Saddler ldquoMultiplicity of beta-14-xylanase in microorganisms functions and applicationsrdquoMicrobiological Reviews vol 52 no 3 pp 305ndash317 1988

[27] R Bernier Jr H Driquez and M Desrochers ldquoMolecularcloning of a Bacillus subtilis xylanase gene in Escherichia colirdquoGene vol 26 no 1 pp 59ndash65 1983

[28] P Sa-PereiraM Costa-Ferreira andM R Aires-Barros ldquoEnzy-matic properties of a neutral endo-13(4)-120573-xylanase Xyl II fromBacillus subtilisrdquo Journal of Biotechnology vol 94 no 3 pp 265ndash275 2002

[29] J X Heck S H Flores P F Hertz and M A Z Ayub ldquoOpti-mization of cellulase-free xylanase activity produced by Bacilluscoagulans BL69 in solid-state cultivationrdquo Process Biochemistryvol 40 no 1 pp 107ndash112 2005

[30] A Sanghi N Garg V K Gupta A Mittal and R C KuhadldquoOne-step purification and characterization of cellulase-freexylanase produced by alkalophilic Bacillus subtilis ashrdquo Brazil-ian Journal of Microbiology vol 41 no 2 pp 467ndash476 2010

[31] R D Kamble and A R Jadhav ldquoIsolation purification andcharacterization of xylanase produced by a new species ofBacillus in solid state fermentationrdquo International Journal ofMicrobiology vol 2012 Article ID 683193 8 pages 2012

[32] S Nakamura K Wakabayashi R Nakai R Aono and KHorikoshi ldquoPurification and some properties of an alkalinexylanase from alkaliphilicBacillus sp strain 41M-1rdquoApplied andEnvironmental Microbiology vol 59 no 7 pp 2311ndash2316 1993

[33] A Gessesse and B A Gashe ldquoProduction of alkaline xylanaseby an alkaliphilic Bacillus sp isolated from an alkaline sodalakerdquo Journal of Applied Microbiology vol 83 no 4 pp 402ndash406 1997

[34] N Annamalai R Thavasi S Jayalakshmi and T Balasubrama-nian ldquoThermostable and alkaline tolerant xylanase productionby Bacillus subtilis isolated form marine environmentrdquo IndianJournal of Biotechnology vol 8 no 3 pp 291ndash297 2009

[35] A Khasin I Alchanati and Y Shoham ldquoPurification and char-acterization of a thermostable xylanase fromBacillus stearother-mophilus T-6rdquoApplied and Environmental Microbiology vol 59no 6 pp 1725ndash1730 1993

[36] C Tachaapaikoon K L Kyu andK Ratanakhanokchai ldquoPurifi-cation of xylanase from alkaliphilic Bacillus sp K-8 by usingcorn husk columnrdquo Process Biochemistry vol 41 no 12 pp2441ndash2445 2006

[37] G Mamo R Hatti-Kaul and B Mattiasson ldquoA thermostablealkaline active endo-120573-1-4-xylanase from Bacillus halodurans

S7 purification and characterizationrdquo Enzyme and MicrobialTechnology vol 39 no 7 pp 1492ndash1498 2006

[38] R Khandeparkar and N B Bhosle ldquoApplication of thermoal-kalophilic xylanase from Arthrobacter sp MTCC 5214 inbiobleaching of kraft pulprdquo Bioresource Technology vol 98 no4 pp 897ndash903 2007

[39] BM Faulet SNiamke J TGonnety and L P Kouame ldquoPurifi-cation and characterization of two thermostable cellulase-freexylanases fromworkers of the termiteMacrotermes subhyalinus(Isoptera Termitidae)rdquo International Journal of Tropical InsectScience vol 26 no 2 pp 108ndash120 2006

[40] V J H Sewalt W G Glasser and K A Beauchemin ldquoLigninimpact on fiber degradation 3 reversal of inhibition of enzy-matic hydrolysis by chemical modification of lignin and byadditivesrdquo Journal of Agricultural and Food Chemistry vol 45no 5 pp 1823ndash1828 1997

[41] D Morrison J S van Dyk and B I Pletschke ldquoThe effectof alcohols lignin and phenolic compounds on the enzymeactivity of Clostridium cellulovorans XynArdquo BioResources vol6 no 3 pp 3132ndash3141 2011

[42] F Kaya J A Heitmann and T W Joyce ldquoInfluence of ligninand its degradation products on enzymatic hydrolysis of xylanrdquoJournal of Biotechnology vol 80 no 3 pp 241ndash247 2000

[43] A Berlin M Balakshin N Gilkes et al ldquoInhibition of cellulasexylanase and 120573-glucosidase activities by softwood lignin prepa-rationsrdquo Journal of Biotechnology vol 125 no 2 pp 198ndash2092006

[44] E Ximenes Y Kim N Mosier B Dien and M LadischldquoDeactivation of cellulases by phenolsrdquo Enzyme and MicrobialTechnology vol 48 no 1 pp 54ndash60 2011

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


of xylanaseThewheat bran has been found to be the best sub-strate for higher xylanase production by Bacillus spp [2 7 8]

Although a highly purified enzyme is a prerequisite forcharacterization and structural studies however from appli-cation perspective using purified enzyme is not economicalfor applications like biobleaching [9] Also it has beenalready demonstrated that hydrolysis product profile of crudeand purified enzyme preparations are similar [10] Thususing agroresidues as substrate for microbial productionof xylanase with a combination of all characters that iscellulase-free functions at high pH and temperature couldbe one of the ways towards substantially reducing the enzymecost as verified by recent publications [7 11] For this reasonthere is still a need of continued search for more efficientxylanolytic bacterial strains that can produce enzymes suit-able for pulp biobleaching In the present study we screeneda new Providencia sp strain X1 from saw-dust decomposingsite It produces multiple xylanases when grown on wheatbran mediumThe crude enzyme preparation was alkali- andthermotolerant in nature

2 Materials and Methods

21 Chemicals Chemicals like birchwood xylan 35-dinitro-salicylic acid (DNSA) carboxymethyl cellulose (CMC)Congo red and D-xylose were purchased from Sigma (StLouis MO USA) Other reagents used in this work were ofanalytical grade

22 Isolation of Xylanolytic Bacteria Serial dilutions of 10(wv) soil sample collected from saw-dust decomposing sitelocated around a saw mill in Lucknow Uttar Pradesh Indiawere used for screening of the bacteria Agar platesweremadeby using modified Bergrsquos mineral salts (BMSs) medium thatcontained (gL) NaNO

330 K

2HPO405 MgSO

4sdot7H2O 02

MnSO4sdotH2O 002 FeSO

4sdotH2O 002 and CaCl

2sdot2H2O 002

agar powder 160 and yeast extract 50 [12] adjusted to pH70 by using 20 Na

2CO3 and 10 birchwood xylan (wv)

as a source of carbon After incubation at 35∘C for 48 h sixbacterial colonies of distinct morphology (named as X1-X6)were isolated and their purity was verified by microscopicobservation after Gram-staining

23 Screening of Xylanase Producing Isolates All isolatedxylanolytic bacteria were further tested for their xylanaseactivity by growing them on agar plate and in broth mediumAll six isolates were grown on BMS agar plate which con-tained birchwood xylan (10 wv) After incubation for 48 hat 35∘C xylan hydrolysis zone was measured by Congo redstainingmethod [13]These bacterial isolates were rescreenedby growing in BMS broth that contained birchwood xylan(10 wv) at 35∘C for 48 h at 120 rpm The xylanase activityin culture supernatant was measured by using the DNSAmethod [14]

24 Characterization of Bacterium The selected bacteriumwas characterized by conventional and molecularapproaches Morphological and biochemical tests were

performed according to standard method [15] Physiologicaltests namely thermotolerance and halotolerance assayswere carried out on Petri dishes For thermotolerance assay100 120583L culture broths were added to 90mL distilled waterand the dilution tubes were heated at 80 90 and 100∘C inwater bath for 30min Samples (50 120583L) were spread ontonutrient agar plates and incubated at 35∘C for 48 h Similarlyhalotolerance was assayed by adding 80 NaCl (wv) to themedia Molecular characterisation of bacterium was doneby 16S rRNA gene sequencing Total DNA was isolated frombacterium using kit (UltraClean Microbial DNA isolationKit MO BIO USA) The 16S rRNA gene of DNA samplewas PCR amplified using 16S rRNA universal primers27F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and 1492R(51015840-TACGGTTACCTTGTTACGACTT-31015840) at annealingtemperature of 56∘C (35 cycles) The PCR product waspurified by gel extraction (Gel extraction Kit Qiagen) andwas sequenced in an ABI 3130 genetic analyzer using BigDye Terminator version 31 cycle sequencing kit The taskof sequencing was outsourced to Ms Eurofins Genomica professional company in Bangalore India The 16S rRNAgene sequences were compared with available sequencesusing NCBI-BLAST A phylogenetic tree was constructedusing Bio Informatics Bacterial Identification (BIBI) software(httpumr5558-sud-str1univ-lyon1frlebibilebibicgi)

25 Xylanase Production by Bacterium Xylanase was pro-duced by selected bacterium in liquid culture condition usingBMS broth containing wheat bran and birchwood xylan asproduction substrate Erlenmeyer conical flasks (250mL)containing 100mL of BMS broth (pH 80) supplemented with10 (wv) of wheat bran or xylan were autoclaved for 20minat 121∘C The pH of the medium was adjusted using 20Na2CO3 Flasks were inoculated with 10mL of overnight

grown pre-culture with an inoculum size of 1 times 106 CFUmLonnutrient agar plate and incubated at 35∘C 120 rpm for 120 h(Innova New Brunswick USA) The culture broths werewithdrawn every 24 h and measured for change of bacterialgrowth and enzyme activity

26 Measurement of Bacterial Growth Culture broth (2mL)waswithdrawn tomeasure bacterial growth by taking absorb-ance at 620 nm in a glass cuvette with 10 cm path length (UV-visible 2300 spectrophotometer Techcomp Korea)

27 Estimation of Enzyme Activity The culture broth wascentrifuged at 10000timesg for 30min and xylanase activityin culture supernatant was determined by measuring thereleased reducing sugars formed by enzymatic hydrolysis ofbirchwood xylan Briefly 05mL of supernatant and 15mL of10 (wv) birchwood xylan prepared in 100mM phosphatebuffer pH 80 was incubated at 50∘C for 15minThe reactionwas terminated by adding 20mL of 35-dinitrosalicylic acid(DNS) reagent The color development due to formation ofreducing sugars wasmonitored at 540 nm [14] and quantifiedby comparisonwith the standard graph of D-xylose One unit(IU) of xylanase activity was defined as the amount of enzymethat liberated 1 120583Mof reducing sugars equivalent to D-xylose









4 MnSO

4 MgCl

2 ZnSO

4

CuSO4 and CaCl









X1 25 14 18 174 plusmn 04

X2 13 13 10 96 plusmn 02

X3 25 25 10 64 plusmn 01

X4 24 13 18 94 plusmn 02

X5 14 14 10 46 plusmn 01






(OD620


























Strain X1 JN384121

















0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5

Xyla

nase

activ

ity (I

Um

L)


Gro

wth

(A620

nm)

(a)

Xyla

nase

activ

ity (I

Um

L)

0

10

20

30

40

50

0

05

1

15

2

25


Gro

wth

(A620

nm)

(b)


30

200

150100

8060

40

(kDa) M 21


6 (35kDa)7 (33kDa)




44kDa

33kDa30

200150100

8060

40

(kDa) M 21





0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty


30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty






FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44








0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 MnSO

4 MgCl

2 ZnSO

4

CuSO4 and CaCl









X1 25 14 18 174 plusmn 04

X2 13 13 10 96 plusmn 02

X3 25 25 10 64 plusmn 01

X4 24 13 18 94 plusmn 02

X5 14 14 10 46 plusmn 01






(OD620


























Strain X1 JN384121

















0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5

Xyla

nase

activ

ity (I

Um

L)


Gro

wth

(A620

nm)

(a)

Xyla

nase

activ

ity (I

Um

L)

0

10

20

30

40

50

0

05

1

15

2

25


Gro

wth

(A620

nm)

(b)


30

200

150100

8060

40

(kDa) M 21


6 (35kDa)7 (33kDa)




44kDa

33kDa30

200150100

8060

40

(kDa) M 21





0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty


30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty






FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44








0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




X1 25 14 18 174 plusmn 04

X2 13 13 10 96 plusmn 02

X3 25 25 10 64 plusmn 01

X4 24 13 18 94 plusmn 02

X5 14 14 10 46 plusmn 01






(OD620


























Strain X1 JN384121

















0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5

Xyla

nase

activ

ity (I

Um

L)


Gro

wth

(A620

nm)

(a)

Xyla

nase

activ

ity (I

Um

L)

0

10

20

30

40

50

0

05

1

15

2

25


Gro

wth

(A620

nm)

(b)


30

200

150100

8060

40

(kDa) M 21


6 (35kDa)7 (33kDa)




44kDa

33kDa30

200150100

8060

40

(kDa) M 21





0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty


30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty






FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44








0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Strain X1 JN384121

















0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5

Xyla

nase

activ

ity (I

Um

L)


Gro

wth

(A620

nm)

(a)

Xyla

nase

activ

ity (I

Um

L)

0

10

20

30

40

50

0

05

1

15

2

25


Gro

wth

(A620

nm)

(b)


30

200

150100

8060

40

(kDa) M 21


6 (35kDa)7 (33kDa)




44kDa

33kDa30

200150100

8060

40

(kDa) M 21





0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty


30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty






FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44








0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40

50

0

05

1

15

2

25

0 1 2 3 4 5

Xyla

nase

activ

ity (I

Um

L)


Gro

wth

(A620

nm)

(a)

Xyla

nase

activ

ity (I

Um

L)

0

10

20

30

40

50

0

05

1

15

2

25


Gro

wth

(A620

nm)

(b)


30

200

150100

8060

40

(kDa) M 21


6 (35kDa)7 (33kDa)




44kDa

33kDa30

200150100

8060

40

(kDa) M 21





0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty


30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty






FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44








0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100

4 5 6 7 8 9 10 11pH

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty


30 40 50 60 70 80 90 100Temperature (∘C)

0

20

40

60

80

100

Rela

tive x

ylan

ase a

ctiv

ity (

)

ActivityStabilty






FeSO4 95 plusmn 45

ZnSO4 95 plusmn 32

MgCl2 93 plusmn 41

CuSO4 78 plusmn 30

MnSO4 35 plusmn 44








0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100Re

lativ

e xyl

anas

e act

ivity

()

0000 0125 0250 0500 0750 1000Lignin (mgmL)

(a)

0

50

100

150

200

Rela

tive x

ylan

ase a

ctiv

ity (

)


0mgmL50mgmL

25mgmL100mgmL

(b)






1

234



4 Conclusions



500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


500

400

300

200

100

0

Redu

cing

suga

r (120583

gm

L)


16

14

12

10

8

6

4

2

0

120581nu

mbe

r (po

ints)




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 characterization of a new providencia...

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