Research Article Investigation of Bacterial Cellulose...

Research ArticleInvestigation of Bacterial Cellulose BiosynthesisMechanism in Gluconoacetobacter hansenii

Bhavna V Mohite and Satish V Patil

School of Life Sciences North Maharashtra University PO Box 80 Jalgaon 425001 India

Correspondence should be addressed to Satish V Patil satishpatil7gmailcom

Received 28 January 2014 Accepted 25 February 2014 Published 16 March 2014

Academic Editors J Ruiz-Herrera and T P West

Copyright copy 2014 B V Mohite and S V Patil This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study explores the mechanism of cellulose biosynthesis in Gluconoacetobacter hansenii The cellulose synthase enzymewas purified as membrane fraction and solubilized by treatment with 01 digitonin The enzyme was separated by native-gelelectrophoresis and 120573-D-glucan analysis was carried out using in vitro gel assay The cellulose synthase has glycoprotein natureand composed two polypeptide subunits of 93 KDa and 85KDa The confirmation of 120573-14-glucan (cellulose) was performed inwhole and hydrolyzed monomeric sugar form Tinopal and Congo red were used for cellulose detection on the gel Thus the invitro cellulose synthesis assay with cell free enzyme fraction was attempted to improve the understanding of cellulose biosynthesis

1 Introduction

Cellulose is one of the most abundant macromolecule onearth In spite of the importance of cellulose its mechanismof biosynthesis is poorly understood Acetobacter xylinumbecome amodel system to study the synthesis of cellulose [1]The firstmajor advance in the area of (1-4)-120573-glucan synthesiscame from a prokaryotic organism Acetobacter xylinum[2] The cellulose synthase from this organism uses uridinediphosphate-glucose (UDP-glucose) directly as a substratefor polymerization in vivo and in vitro [2]

In the past decade a number of new developments inthe biological and cytological aspects of cellulose biosyn-thesis from Acetobacter have led to better understandingof this process Cellulose biosynthesis is an exciting areaof study with lots of challenges and opportunities [3] Theenzymatic pathway for cellulose synthesis in A xylinum hasbeen extensively investigated and four essential enzymaticsteps have been identified [4] Cellulose synthase is theonly enzyme known to be unique to the cellulose syntheticpathway Biosynthesis of cellulose essentially proceeds bypolymerization of glucose residues using an activated sub-strate (UDP-glucose) In Acetobacter xylinum the enzymecellulose synthase is present on the cytoplasmic membraneand the cellulose product is obtained extracellularly [5]

Successful purification of cellulose synthase and beingacquainted with its properties would augment our under-standing of molecular mechanism of cellulose synthesis [6]Partially purified membrane protein was used for assayingcellulose synthase activity UDP-glucose is the direct sub-strate fromwhich glycosyl residues transfer to the nonreduc-ing end of the growing (1rarr 4)-120573-glucan chainThe formationof cellulose that is the polymerization of glucose appears tobe catalyzed by the gene product of the bcsA gene productof the bcs (bacterial cellulose synthase operon) [7] There is acoupling of cellulose synthesis and translocation in which thenascent polysaccharide is extended by one glucose moleculeat a time [8]

In the present study in vitro 120573-14-glucan synthesis wasdirected towards understanding the mechanism of cellu-lose biosynthesis Later conformation of formed cellulosewas detected with its intact form and after hydrolysis intomonomer sugar In addition we illustrated the electropho-retic separation of cellulose synthase and determination of itsmass and glycoprotein nature

2 Materials and Methods

21Microorganism Gluconoacetobacter hanseniiNCIM2529was grown in Luria Bertani (LB) broth (Himedia Mumbai

Hindawi Publishing CorporationISRN MicrobiologyVolume 2014 Article ID 836083 7 pageshttpdxdoiorg1011552014836083

2 ISRNMicrobiology

India) on shaker at 28∘C overnight The cell pellet wasseparated by centrifugation at 18000 g for 20min at 4∘C

22 Preparation of Membrane Fractions Solubilization ofEnzyme The cell pellet was suspended in 50mM Tris-HCLpH 75 containing 5mMEDTA followed by a brief sonicationand recentrifugation at 10000timesg for 5min at 4∘C to removecell wall Membrane pellet was collected and used as sourceof enzyme

Enzyme was solubilized by resuspending the membranesin 005M Tris-HCL pH 75 containing 22mMMgCl

2 1 mM

EDTA and 01 (wv) digitonin The suspension was placedin a sonic bath for 5min at 1ndash4∘C and then stirred on ice for30min followed by centrifugation at 100000timesg for 1 h Thesupernatant represented the solubilized enzyme [6]

23 Native Polyacrylamide Gel Electrophoresis The digitoninsupernatant was applied directly as enzyme sample afterapplication of samples (5ndash10 120583L each containing 10ndash50 120583gprotein) electrophoresis was carried out using the discontin-uous buffer system without the addition of SDS A BangaloreGenei vertical minislab gel apparatus was used the small sizeof the gels (8 cm times 7 cm) reduces the volume of solutionrequired in the activity assay and enhances diffusion ofsubstances (eg substrate) between gel and solution Stackinggel (45) and separating gel (10) acrylamide were usedNative-gel electrophoresis was performed at 4∘C at 10 to15mAmp per gel The 50 v current was applied initially andthen increased to 100 v after the electrophoresis run reachedup to the separating gel The position of bands for molecularweight was determined by comparisonwithmolecular weightmarkers (high range molecular weight markers 29 to 250KDcat 105977 Merck inst) Among the bands obtained a bandof cellulose synthase complex (predicted molecular masssim175 KDa) was selected according to previous report [9] andit was further purified by using gel extraction kit (BangloreGenei)

24 120573-D-Glucan Product (Cellulose) Assay in Native GelFollowing electrophoresis gel lane was cut into sectionscontaining sample lane and rinsed in 100mL of 10mM Tris-HCL (pH 75) for 30min and buffer was changed after 15minEach gel section was then placed in a plastic petri dishand incubated for enzyme activity at room temperature in50mM Tris-HCL (pH 75) 3mM NaN

3 and UDP-glucose

(SiscoResearch Laboratories India) Unreacted substratewasremoved from gel sections by rinsing twice for 20min eachin 100mL of 5mM EDTA 10mM Tris-HCL (pH 75) The 120573-glucan product in the gel was visualized with Congo red afterstaining in 50mL of 01 Congo red for 30min and destain-ing for 20min in 100mL of 10mM Tris-HCL (pH 75) buffer

An alternative method was employed to detect the 120573-glucan product After reaction with the substrate UDP-glucose the 120573-glucan product was visualized by its flu-orescence under UV light staining with Tinopal (441015840-distyrylbiphenyl sodium sulfonate salt Sigma-Aldrich) insolution of 50mL of 001 Tinopal blue in K

2PO4buffer pH

82 for 10min The gel was then washed in water and storedin the same solution

25 Analysis of 120573-Glucan Product (Cellulose) The product(cellulose) formation was confirmed by two major ways(i) detection of cellulose and (ii) detection of cellulosedegradation product (monomer sugar)

(i) Detection of Cellulose The formation of reaction prod-uct (cellulose) in cellulose synthase assay was detected byfollowing ways (a) The thin layer chromatogram of UDP-glucose and reaction product was developed with methanolas solvent system TLC was visualized under short UVfor fluorescence The UV-visible spectrum of UDP-glucoseand reaction product was also carried out by Nanodropspectrophotometer (ND 1000 Nanodrop technology USA)(b) The second one is reaction with Congo red (i) Thereaction product (cellulose) and substrate (UDP-glucose) ascontrol were streaked on silica gel plate and the plate wassprayed with Congo red solution to observe the characteristicbinding of Congo red (ii) The reaction product was mixedwith Congo red solution and centrifuged for 10min at 5000 gThe colour of precipitated cellulose at bottom was observedBinding of Congo red was also confirmed by measuring thespectrum by UV-visible Nanodrop spectrophotometer

(ii) Detection of Cellulose Degradation Product (MonomerSugar) The degradation product of cellulose that ismonomer glucose sugar was detected for the confirmation ofcellulose as reaction product in cellulose synthase assay Thereaction product was precipitated with isopropyl alcohol andused for further assays

The reaction product was used as substrate for cellulaseassay Reaction mixture precipitate was treated with cellulaseenzyme (Himedia Mumbai India) and allows it to reactfor 60min at 50∘C in a water bath for the hydrolysisAfter hydrolysis of reaction product with cellulase enzymereducing sugar was measured by Millers method [10] Thehydrolysed product of cellulose was confirmed as glucoseby thin layer chromatogram (TLC) and Fourier transforminfrared chromatography (FTIR) The hydrolysis product ofcellulose assay was spotted on TLC (Silica gel Hi-250 F) platewith glucose as standard and chromatogram was run withchloroform glacial acetic acid water (30 35 5) as solventsystem Detection was carried with chromogenic iodine(CIR) as spraying reagent The FT-IR transmission spectrumof the cellulase enzyme treated product was studied to provethe purity of the reaction product (14-120573-D-glucan) made upof only glucosemonomer comparedwith glucose as standard

26 SDS PAGE Electrophoresis of Purified Enzyme ComplexThe protein band from native PAGE was further char-acterized by sodium dodecyl sulphate-polyscrylamide gelelectrophoresis (SDS-PAGE) with 10 SDS The sample wasdissolved in sample buffer containing 10 SDS 02M Tris-HCL (pH 68) 10mM-mercaptoethanol and 20 glycerolwith 005 bromophenol blue The incubation was carriedout on ice for 1 h to avoid smearing protein bands due toheating Gel was stained overnight with 0125 Coomassiebrilliant blue R-250 prepared in 40methanol and 10 aceticacid and destained for 2 h with the same lacking Coomassie

ISRNMicrobiology 3

blue The position of bands for molecular weight was deter-mined by comparison with molecular weight markers (highrange molecular weight markers 29 to 250KD cat 105977Merck inst)

27 Glycoprotein Identification Thenative gel electrophoresiswas run and a lane was cut and incubated in the fixingsolution (25 isopropanol 10 acetic acid and 65 water)for 2 h with gentle shaking The fixation was repeated foranother hour with change of fixing solution Then gel wastransferred into equilibrium solution (02 thymol blue (wv)in fixing solution) and incubated for 2 h Then equilibriumsolution was totally decanted and staining solution (80sulphuric acid 20 ethanol) was added and incubated thegel for 3 h Reddish brown colour bands of correspondingglycoprotein lighten up and are documented by photography

3 Results and Discussion

Many different approaches have been studied to investigatesynthesis of cellulose [11 12] Significant efforts have beenmade to achieve in vitro synthesis of cellulosewithmembranepreparations of various degrees of purity from differentorganisms [13ndash15] In the present study we demonstrated themechanism behind cellulose synthesis and polymerization inG hansenii NCIM 2529 The role of cellulose synthase wasdetected and characterized using an in situ assay followingsolubilization and electrophoretic separation in nondenatur-ing polyacrylamide gelsThe enzymewhich is extraordinarilyunstable in extracts at ambient temperaturemaintains at leastsome activity in the gel assay Therefore native gel assay wasused rather than solution assay

31 Determination of Cellulose Synthase Activity When cellu-lose biogenesis was considered as a universal phenomenonmuch interest was obviously focused upon the cellulosesynthase since this may well be the only enzyme uniqueto this process [16] The cellulose synthase is most probablyan integral membrane protein that occurs exclusively in themembrane-associated fraction as determined for a varietyof A xylinum strains and Agrobacterium tumefaciens [17]Successful separation of cellulose synthase and its activitydepends on efficient membrane solubilization and extractionof active proteins [18] Digitonin was reported as the best forsolubilization activity for Acetobacter cellulose synthase [19]compared with Triton Zwittergent and cholate and henceit was used in this study Brief sonication and extraction ofAcetobacter membranes with buffer containing digitonin (1ndash10) result in effective solubilization of the UDP-glucose l4-P-D-glucan 4-P-D-glucosyltransferase (cellulose synthase)with very good recovery of activity [19] After electrophoreticseparation of proteins in the membrane fractions by non-denaturing polyacrylamide gel electrophoresis (PAGE) theproduct of 120573-glucan synthase activity can be demonstratedby the cellulose synthase enzyme assay by providing UDP-glucose as a substrate and cofactorsThe in vitro assay systemincludes synthesis of alkali insoluble120573-14-glucan (cellulose)from UDP-glucose which is added as the sole exogenoussubstrate at rate comparable (40) to that of the whole cell

[20] UDP-glucose has been characterized as glucosyl donorfor cellulose synthesis [21] According to Delmer et al [22] invitro rate of cellulose synthase is half of that rate observed invivo The in vitro synthesis of cellulose was confirmed by (a)detecting the cellulose formed and (b) detecting the hydroly-sis product of cellulose that is monomer sugar glucose

32 Detection of Cellulose Formation

(a) The UDP-Glucose Gives Fluorescence Compared with theReaction Product (Cellulose) (Figure 1(a)) The UDP-glucosewhen reacting with cellulose synthase from the membranefraction results into cellulose which did not fluoresce underUV light this gives a primary indication of conversionof UDP-glucose into cellulose The UDP-glucose and itstransformation into productwere confirmedby observing thedecrease in concentration of UDP-glucose after reaction withmembrane fraction enzyme (Figure 1(b))

(b) Detection with Congo Red Binding of dye with cellulose(i) when the reaction mixture (product) was streaked onTLC plate along with UDP-glucose as substrate control andsprayed with Congo red solution red colour was developedonly with the reaction product when compared to control(Figure 2(a)) This happens due to characteristic bindingaffinity between cellulose and Congo red (ii) The precipi-tated red coloured cellulose (product) was observed due tocharacteristic binding of Congo red to cellulose (Figure 2(b))As the polymerization further proceeded the precipitate ofthe reaction product (polysaccharide cellulose) appeared inthe solution This indicated the self-assembly of cellulose(water insoluble) synthesized by in vitro enzymatic polymer-ization [23] (iii)The precipitated reaction product and UDP-glucose with Congo red were compared spectrophotomet-rically which indicate that Congo red shows binding withthe reaction product that is cellulose not with UDP-glucose(Figure 2(c)) This confirmed formation of cellulose as reac-tion product by catalysis with cellulose synthase enzymeseparated from membrane fraction UDPG and UDPG +enzyme fraction (= BC) reactedwithCongo red and had beenmeasured on Nanodrop spectrophotometer (Figure 2(c))

(c) Detection of Cellulose Synthase Product (Cellulose) in theNative Gel Following separation on nondenaturing gel theactivity of cellulose synthase can be analyzed by incubatingwashed gel with appropriate substrate and effector subse-quent removal of unused substrate by washing followedby the use of suitable detection procedure The gel assay ishighly sensitive than solution assay as the product formedwas detected on the gel itself without loss of product [6]Figures 3(a) and 3(b) demonstrate such an assay for detectionof formed cellulose by binding with Congo red and TinopalCBS respectively The Congo red reacts with cellulose andgives red coloured band on gel Tinopal reacted with celluloseand was detected under UVwith fluorescence due to bindingwith optical brightener TinopalThe insolubility of produced120573-glucan helps to immobilize in the gel matrix thus suggest-ing that this product was 120573-14-glucan

4 ISRNMicrobiology

(a) (b)

Figure 1 (a) Thin layer chromatogram showing UV fluorescence by UDP-glucose (b) UV-Vis spectrum of UDP-glucose and reactionproduct

(a) (b)

UDPG + congo red UDPG with reaction product (BC)+ congo red

Congo red

(c)

Figure 2 (a) Characteristic binding of Congo red with reaction product (b) Precipitate of red colored cellulose (c) Spectrum of UDPG andreaction product with Congo red compared with spectrum of Congo red

ISRNMicrobiology 5

(a) (b)

Figure 3 Detection of cellulose on native gel by (a) Congo red and (b) Tinopal CBS under UV light

(a)

1114

990

1103

308

991

40

500750100012501500175020002500300035004000(1cm)

10

20

30

40

50

60

3490

31

2357

09

2332

02

2069

69

1635

69

1385

90

1272

10

1127

43

1071

49

895

00

730

08

506

33

T(

)

(b)

Figure 4 (a) Thin layer chromatogram of cellulase hydrolyzed reaction product (T) compared with glucose (G) as standard (b) FT-IRspectrum of cellulase treatment reaction product

Detection by Analyzing the Degradation Product (MonomerSugar) of120573-Glucan Product (Cellulose)The formation of reac-tion product as cellulose was also confirmed by detecting thedegradation product of cellulose that is monomeric form ofsugar (glucose)The cellulose was precipitated with isopropylalcohol and hydrolyzed with cellulase enzyme by cellulaseassay The detection of glucose as a result of hydrolysis ofcellulose confirms the purity of cellulose formed [24]

Cellulase Assay The cellulase assay was performed withthe product of in vitro assay as substrate (cellulose) Incellulase hydrolysis assay glucose liberated fromcellulosewasquantified byMillers method with dinitrosalicylic acid whichspecifically reacts with the free sugar The concentration ofreducing sugar after cellulase hydrolysis was much higher(115 120583gmL) compared with unhydrolyzed reaction product[25]The positive cellulase assay confirmed that the substratefor cellulase assay was of polymeric 120573-14-glucan nature

The hydrolysis product of cellulose (ie glucose) wasdetected by thin layer chromatography (TLC) and Fourier

transform infra red spectroscopy (FTIR) The hydrolysis ofcellulose with cellulase results into release of glucose asmonomer sugar

(i) In TLC the Rf valuewas correspondingwith standardglucose that confirmed that the reaction productunder cellulase assay was cellulose (Rf value oftest- 063 matching with Rf value of glucose- 061)(Figure 4(a))

(ii) The cellulase treated reaction product wasmatched with glucose based on FT-IR spectrum(Figure 4(b)) FTIR spectrum of aqueous solution ofcellulase enzyme hydrolysate was well defined andshows intense and characteristic bands in the regionbetween 1200 and 900 cmminus1 The characteristic bandsof glucose have specific maxima at 991 1033 10781107 and 1149 cmminus1 with the peak at 1033 cmminus1 havingthe highest absorption which is a characteristic to theCndashO stretch vibration Glucose has endocyclic CndashOlocated at around 1080 cmminus1 The peak at 1033 cmminus1

6 ISRNMicrobiology

1 2

9385

(kD

a)

(kD

a)

205

97

68

43

29

(a) (b)

Figure 5 (a) SDS-PAGE of cellulose synthase (lane 1) purified fromG hansenii stained by Coomassie brilliant blue compared with highrange molecular weight marker (lane 2) (b) Glycoprotein band ofcellulose synthase on native gel

was considered for the identification of cellulasehydrolytic activity and the rate of glucose release[26 27]

33 SDS PAGE of Purified Cellulose Synthase The bandsdetected by Coomassie brilliant blue staining was comparedwith high range molecular weight marker Two distinctbands were observed corresponding to molecular weight of93KDa and 85KDa (Figure 5(a) lane 1) compared to highrange molecular weight marker showing bands of 205 9768 43 and 29KDa (Figure 5(a) lane 2) This result stronglyimplicated the involvement of two subunit polypeptide withcellulose synthase [28] and this work as catalytic subunitof cellulose synthase [29] Multiple catalytic subunits arerequired for cellulose synthesis in Arabidopsis [30 31]

34 Glycosylated Nature of Cellulose Synthase Enzyme Com-plex The cellulose synthase enzyme on a gel was stainedwith thymol blue for determination of its glycoprotein natureThe glycoprotein nature of the protein is detected by stainingmethod where amino group of amino acid and aldehyde orketo group of the sugar in the protein leads to the reddishbrown colour of the band Glycoprotein band was visualizedin reddish brown colour which confirmed the glycoproteinnature of cellulose synthase enzyme (Figure 5(b))

4 Conclusion

The present study attempted to reveal the mechanism behindbacterial cellulose production and polymerization Extrac-tion and purification of membrane protein were carriedout with its elctrophoretic separation The two subunitpolypeptides of 93KDa and 85KDa with glycoprotein nature

implicated cellulose synthase The in vitro 120573-14-glucan gelassay was carried out to determine role of cellulose synthaseand further 120573-glucan product was analyzed Formation of 120573-glucan product was confirmed inwhole and hydrolyzed sugarmonomer Thus the mechanism of bacterial cellulose forma-tion was elucidated with involvement of cellulose synthasethat would facilitate understanding of cellulose synthesiswhich could be employed for more productivity of cellulosefrom cellulose producing strains

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author Bhavna V Mohite is greatly acknowledged Uni-versity Grants Commission New Delhi for Research fellow-ship in Sciences for Meritorious students (RFSMS)

References

[1] D P Delmer ldquoCellulose biosynthesisrdquo Annual Review of PlantPhysiology vol 38 pp 259ndash290 1987

[2] P Ross H Weinhouse Y Aloni et al ldquoRegulation of cellulosesynthesis in Acetobacter xylinum by cyclic diguanylic acidrdquoNature vol 325 pp 279ndash281 1987

[3] L Lei S Li and Y Gu ldquoCellulose synthase complexes compo-sition and regulationrdquo Frontiers in Plant Science vol 3 article75 pp 1ndash6 2012

[4] M Swissa Y Aloni H Weinhouse and M Benizman ldquoInter-mediary steps in Acetobacter xylinum cellulose synthesis stud-ies with whole cells and cell-free preparations of the wild typeand a celluloseless mutantrdquo Journal of Bacteriology vol 143 no3 pp 1142ndash1150 1980

[5] I M Saxena T Dandekar and R M Brown Jr Mechanismsin cellulose biosynthesis [PhD thesis] University of Texas atAustin Austin Tex USA 2000

[6] M PThelen and D P Delmer ldquoGel-electrophoretic separationdetection and characterization of plant and bacterial UDP-glucose glucosyl transferasesrdquo Plant Physiology vol 81 no 3pp 913ndash918 1986

[7] M Benziman and R Tal ldquoCellulose synthase associated pro-teinsrdquo US Patent no 5382656 1995

[8] J L Morgan J Strumillo and J Zimmer ldquoCrystallographicsnapshot of cellulose synthesis and membrane translocationrdquoNature vol 10 no 493 pp 181ndash186 2013

[9] IM Saxena and RM Brown Jr ldquoIdentification of a second cel-lulose synthase gene (acsAII) inAcetobacter xylinumrdquo Journal ofBacteriology vol 177 no 18 pp 5276ndash5283 1995

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

[11] R D Peterson The Physical Biology of Plant Cell Walls Chap-man amp Hall London UK 1994

[12] A Frey-Wyssling ldquoBiochemistry of the cell wallrdquo in The PlantCell Wall p 114 Gebruder Borntraeger Berlin Germany 1996

[13] G Maclachlan ldquoDoes 120573-glucan synthesis need a primerrdquo inCellulose and Other Natural Polymer Systems R M Brown JrEd pp 227ndash339 Plenum press New York NY USA 1982

ISRNMicrobiology 7

[14] S M Read and D P Delmer ldquoBiochemistry and regulationof cellulose synthesis in higher plantsrdquo in Biosynthseis andBiodegradation of Cellulose C Haigler and P Weimer Eds pp177ndash200 Marcel Dekker New York NY USA 1991

[15] K Okuda L Li K Kudlicka R M Brown Jr and S Kuga ldquo120573-Glucan synthesis in the cotton fiber I Identification of 120573-14-and 120573-13-glucans synthesized in vitrordquo Plant Physiology vol101 no 4 pp 1131ndash1142 1993

[16] M Benziman P Ross and R Mayer ldquoCellulose biosynthesisand function in bacteriardquo Microbiological Reviews vol 55 no1 pp 35ndash38 1991

[17] M Benziman and D Amikam ldquoCyclic diguanylic acid andcellulose synthesis in Agrobacterium tumefaciensrdquo Journal ofBacteriology vol 171 no 12 pp 6649ndash6655 1989

[18] R M Brown Jr ldquoCellulose and callose biosynthesis in higherplantsrdquo Plant Physiology vol 115 no 2 pp 643ndash656 1997

[19] M Benziman D Delmer Y Aloni and R Cohen ldquoSolubiliza-tion of the UDP-glucose 14-120573-glucan 4-120573-d-glucosyltransfer-ase (cellulose synthase) from Acetobacter xylinum A compari-son of regulatory properties with those of themembrane-boundform of the enzymerdquo The Journal of Biological Chemistry vol258 no 7 pp 4419ndash4423 1983


[21] L Glaser ldquoThe synthesis of cellulose in cell-free extracts ofAcetobacter xylinumrdquo The Journal of Biological Chemistry vol232 pp 627ndash636 1958

[22] D P Delmer P Ohana L Gonen and M Benziman ldquoIn vitrosynthesis of cellulose in plants still a long way to gordquo PlantPhysiology vol 103 no 2 pp 307ndash308 1993

[23] S Koizumi Y Tomita T Kondo and T Hashimoto ldquoWhat fac-tors determine hierarchical structure of microbial cellulosemdashinterplay among physics chemistry and biologyrdquo Macromolec-ular Symposia vol 279 no 1 pp 110ndash118 2009

[24] H-Y Su T-M Lee Y-L Huang et al ldquoIncreased celluloseproduction by heterologous expression of cellulose synthasegenes in a filamentous heterocystous cyanobacterium witha modification in photosynthesis performance and growthabilityrdquo Botanical Studies vol 52 no 3 pp 265ndash275 2011

[25] F Ito Y Amano M Shiroishi et al ldquoAccumulation of cello-oligosaccharides during bacterial cellulose production by Ace-tobacter xylinumrdquo Journal of Applied Glycoscience vol 52 no 1pp 27ndash30 2005

[26] C Adina F Florinela T Abdelmoumen and S CarmenldquoApplication of FTIR spectroscopy for a rapid determination ofsome hydrolytic enzymes activity on sea buckthorn substraterdquoRomanian Biotechnological Letters vol 15 no 6 pp 5738ndash57442010

[27] httpterachemuteeIR spectraindexphpoption=com con-tentampview=categoryampid=26ampItemid=31

[28] F C Lin and R M Brown ldquoPurification of cellulose synthasefrom Acetobacter xylinumrdquo in Cellulose and Wood Chemistryand Technology C Schuerch Ed pp 473ndash492 John Wiley ampSons New York NY USA 1989

[29] F C Lin R M Brown Jr R R Drake Jr and B E HaleyldquoIdentification of the uridine 51015840-diphosphoglucose (UDP-Glc)binding subunit of cellulose synthase in Acetobacter xylinumusing the photoaffinity probe 5-azido-UDP-GlcrdquoThe Journal ofBiological Chemistry vol 265 no 9 pp 4782ndash4784 1990

[30] N G Taylor S Laurie and S R Turner ldquoMultiple cellulosesynthase catalytic subunits are required for cellulose synthesisin Arabidopsisrdquo The Plant Cell vol 12 no 12 pp 2529ndash25392000

[31] M Fujita R Himmelspach J Ward et al ldquoThe anisotropy1D604N mutation in the Arabidopsis cellulose synthase1 cat-alytic domain reduces cell wall crystallinity and the velocity ofcellulose synthase complexesrdquo Plant Physiology vol 162 no 1pp 74ndash85 2013

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Enzyme Research



Microbiology

2 ISRNMicrobiology

India) on shaker at 28∘C overnight The cell pellet wasseparated by centrifugation at 18000 g for 20min at 4∘C

22 Preparation of Membrane Fractions Solubilization ofEnzyme The cell pellet was suspended in 50mM Tris-HCLpH 75 containing 5mMEDTA followed by a brief sonicationand recentrifugation at 10000timesg for 5min at 4∘C to removecell wall Membrane pellet was collected and used as sourceof enzyme

Enzyme was solubilized by resuspending the membranesin 005M Tris-HCL pH 75 containing 22mMMgCl

2 1 mM

EDTA and 01 (wv) digitonin The suspension was placedin a sonic bath for 5min at 1ndash4∘C and then stirred on ice for30min followed by centrifugation at 100000timesg for 1 h Thesupernatant represented the solubilized enzyme [6]

23 Native Polyacrylamide Gel Electrophoresis The digitoninsupernatant was applied directly as enzyme sample afterapplication of samples (5ndash10 120583L each containing 10ndash50 120583gprotein) electrophoresis was carried out using the discontin-uous buffer system without the addition of SDS A BangaloreGenei vertical minislab gel apparatus was used the small sizeof the gels (8 cm times 7 cm) reduces the volume of solutionrequired in the activity assay and enhances diffusion ofsubstances (eg substrate) between gel and solution Stackinggel (45) and separating gel (10) acrylamide were usedNative-gel electrophoresis was performed at 4∘C at 10 to15mAmp per gel The 50 v current was applied initially andthen increased to 100 v after the electrophoresis run reachedup to the separating gel The position of bands for molecularweight was determined by comparisonwithmolecular weightmarkers (high range molecular weight markers 29 to 250KDcat 105977 Merck inst) Among the bands obtained a bandof cellulose synthase complex (predicted molecular masssim175 KDa) was selected according to previous report [9] andit was further purified by using gel extraction kit (BangloreGenei)

24 120573-D-Glucan Product (Cellulose) Assay in Native GelFollowing electrophoresis gel lane was cut into sectionscontaining sample lane and rinsed in 100mL of 10mM Tris-HCL (pH 75) for 30min and buffer was changed after 15minEach gel section was then placed in a plastic petri dishand incubated for enzyme activity at room temperature in50mM Tris-HCL (pH 75) 3mM NaN

3 and UDP-glucose

(SiscoResearch Laboratories India) Unreacted substratewasremoved from gel sections by rinsing twice for 20min eachin 100mL of 5mM EDTA 10mM Tris-HCL (pH 75) The 120573-glucan product in the gel was visualized with Congo red afterstaining in 50mL of 01 Congo red for 30min and destain-ing for 20min in 100mL of 10mM Tris-HCL (pH 75) buffer

An alternative method was employed to detect the 120573-glucan product After reaction with the substrate UDP-glucose the 120573-glucan product was visualized by its flu-orescence under UV light staining with Tinopal (441015840-distyrylbiphenyl sodium sulfonate salt Sigma-Aldrich) insolution of 50mL of 001 Tinopal blue in K

2PO4buffer pH

82 for 10min The gel was then washed in water and storedin the same solution

25 Analysis of 120573-Glucan Product (Cellulose) The product(cellulose) formation was confirmed by two major ways(i) detection of cellulose and (ii) detection of cellulosedegradation product (monomer sugar)

(i) Detection of Cellulose The formation of reaction prod-uct (cellulose) in cellulose synthase assay was detected byfollowing ways (a) The thin layer chromatogram of UDP-glucose and reaction product was developed with methanolas solvent system TLC was visualized under short UVfor fluorescence The UV-visible spectrum of UDP-glucoseand reaction product was also carried out by Nanodropspectrophotometer (ND 1000 Nanodrop technology USA)(b) The second one is reaction with Congo red (i) Thereaction product (cellulose) and substrate (UDP-glucose) ascontrol were streaked on silica gel plate and the plate wassprayed with Congo red solution to observe the characteristicbinding of Congo red (ii) The reaction product was mixedwith Congo red solution and centrifuged for 10min at 5000 gThe colour of precipitated cellulose at bottom was observedBinding of Congo red was also confirmed by measuring thespectrum by UV-visible Nanodrop spectrophotometer

(ii) Detection of Cellulose Degradation Product (MonomerSugar) The degradation product of cellulose that ismonomer glucose sugar was detected for the confirmation ofcellulose as reaction product in cellulose synthase assay Thereaction product was precipitated with isopropyl alcohol andused for further assays

The reaction product was used as substrate for cellulaseassay Reaction mixture precipitate was treated with cellulaseenzyme (Himedia Mumbai India) and allows it to reactfor 60min at 50∘C in a water bath for the hydrolysisAfter hydrolysis of reaction product with cellulase enzymereducing sugar was measured by Millers method [10] Thehydrolysed product of cellulose was confirmed as glucoseby thin layer chromatogram (TLC) and Fourier transforminfrared chromatography (FTIR) The hydrolysis product ofcellulose assay was spotted on TLC (Silica gel Hi-250 F) platewith glucose as standard and chromatogram was run withchloroform glacial acetic acid water (30 35 5) as solventsystem Detection was carried with chromogenic iodine(CIR) as spraying reagent The FT-IR transmission spectrumof the cellulase enzyme treated product was studied to provethe purity of the reaction product (14-120573-D-glucan) made upof only glucosemonomer comparedwith glucose as standard

26 SDS PAGE Electrophoresis of Purified Enzyme ComplexThe protein band from native PAGE was further char-acterized by sodium dodecyl sulphate-polyscrylamide gelelectrophoresis (SDS-PAGE) with 10 SDS The sample wasdissolved in sample buffer containing 10 SDS 02M Tris-HCL (pH 68) 10mM-mercaptoethanol and 20 glycerolwith 005 bromophenol blue The incubation was carriedout on ice for 1 h to avoid smearing protein bands due toheating Gel was stained overnight with 0125 Coomassiebrilliant blue R-250 prepared in 40methanol and 10 aceticacid and destained for 2 h with the same lacking Coomassie

ISRNMicrobiology 3











4 ISRNMicrobiology

(a) (b)


(a) (b)


Congo red

(c)


ISRNMicrobiology 5

(a) (b)


(a)

1114

990

1103

308

991

40

500750100012501500175020002500300035004000(1cm)

10

20

30

40

50

60

3490

31

2357

09

2332

02

2069

69

1635

69

1385

90

1272

10

1127

43

1071

49

895

00

730

08

506

33

T(

)

(b)








6 ISRNMicrobiology

1 2

9385

(kD

a)

(kD

a)

205

97

68

43

29

(a) (b)





4 Conclusion





Acknowledgment


References














ISRNMicrobiology 7


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRNMicrobiology 3











4 ISRNMicrobiology

(a) (b)


(a) (b)


Congo red

(c)


ISRNMicrobiology 5

(a) (b)


(a)

1114

990

1103

308

991

40

500750100012501500175020002500300035004000(1cm)

10

20

30

40

50

60

3490

31

2357

09

2332

02

2069

69

1635

69

1385

90

1272

10

1127

43

1071

49

895

00

730

08

506

33

T(

)

(b)








6 ISRNMicrobiology

1 2

9385

(kD

a)

(kD

a)

205

97

68

43

29

(a) (b)





4 Conclusion





Acknowledgment


References














ISRNMicrobiology 7


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 ISRNMicrobiology

(a) (b)


(a) (b)


Congo red

(c)


ISRNMicrobiology 5

(a) (b)


(a)

1114

990

1103

308

991

40

500750100012501500175020002500300035004000(1cm)

10

20

30

40

50

60

3490

31

2357

09

2332

02

2069

69

1635

69

1385

90

1272

10

1127

43

1071

49

895

00

730

08

506

33

T(

)

(b)








6 ISRNMicrobiology

1 2

9385

(kD

a)

(kD

a)

205

97

68

43

29

(a) (b)





4 Conclusion





Acknowledgment


References














ISRNMicrobiology 7


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRNMicrobiology 5

(a) (b)


(a)

1114

990

1103

308

991

40

500750100012501500175020002500300035004000(1cm)

10

20

30

40

50

60

3490

31

2357

09

2332

02

2069

69

1635

69

1385

90

1272

10

1127

43

1071

49

895

00

730

08

506

33

T(

)

(b)








6 ISRNMicrobiology

1 2

9385

(kD

a)

(kD

a)

205

97

68

43

29

(a) (b)





4 Conclusion





Acknowledgment


References














ISRNMicrobiology 7


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 ISRNMicrobiology

1 2

9385

(kD

a)

(kD

a)

205

97

68

43

29

(a) (b)





4 Conclusion





Acknowledgment


References














ISRNMicrobiology 7


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRNMicrobiology 7


























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 Investigation of Bacterial Cellulose...

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