Here we report for the first time on phosphorylation of cotton cellulose using baker’s yeast hexokinase and phosphoryl donor adenosine-50-triphosphate. An enzymatic assay was adopted for determination of the degree of  hosphorylation of cellulose. This functional modification
of cellulose resulted in improved colorability and flame resistance

Azhgozhinova, Galiya S.

Khutoryanskiy, Vitaliy V.

Mun, Grigoriy A.

Nurkeeva, Zauresh S.

Park, Kinam

Shaikhutdinov, Erengaip M.

Macromolecular Rapid Communications

Crossref

Contents: Macromol. Rapid Commun. 16/2002

Complex formation of poly(methacrylic acid) hydrogels with linear poly(ethylene glycol) has been studied at different pH values and in the presence of an electric field. The fastest contraction of the gel samples was observed under simultaneous action of electric field and complex formation

The University of Manchester - Institutional Repository

Collapse of poly (methacrylic acid) hydrogels in response to simultaneous stimulation by an electric field and complex formation

Chain-length-dependent termination rate coefficients of the bulk free-radical polymerization of styrene at 80 °C are determined by combining online polymerization rate measurements (DSC) with living RAFT polymerizations. Full kt versus chain-length plots were obtained indicating a high kt value for short chains (2 × 109 L · mol-1 · s-1) and a weak chain-length dependence between 10 and 100 monomer units, quantified by an exponent of -0.14 in the corresponding power law (kt i,i) = kt 0 · P-b

Vana, P.

Davis, T. P.

Barner-Kowollik, C.

Queensland University of Technology ePrints Archive

Easy access to chain-length-dependent termination rate coefficients using RAFT polymerization

A theoretical model on the basis of the free-volume concept is presented explaining the temperature dependence of photoinduced birefringence in polystyrene films that contain Disperse Red-1. Birefringence increases with temperature up to 180 K as the free volume for isomerization increases, and then decreases as thermally activated processes dominate. The fast process of birefringence decay has a time constant that increases with temperature at low temperatures, due to the change kin photoisomerization

Dall'Agnol, F.

Silva, JR

Zilio, S. C.

Oliveira, O. N.

Giacometti, J. A.

Acervo Digital da Unesp

Temperature dependence of photoinduced birefringence in polystyrene doped with Disperse Red-1

Tzanov, Tzanko

Stamenova, Maria

Paulo, Artur Cavaco

Universidade do Minho: RepositoriUM

Phosphorylation of Cotton Cellulose with Baker’sYeast HexokinaseTzanko Tzanov, Maria Stamenova, Artur Cavaco-Paulo*Departamento de Engenharia Teˆxtil, Universidade do Minho, Campus de Azure´m, 4800-058 Guimara˜es, PortugalFax: þ351 253 510293; E-mail: artur@det.uminho.ptKeywords: enzymes; hexokinases; functionalization of polymersIntroductionThe chemical modification of cotton cellulose, by introduc-ing new functional groups or compounds, could be a way toimprove dye fixation, water and soil-repellence, crease-resistance, handle, flame retardance and others. Hexokinaseenzymes (EC 2.7.1.1.) catalyze phosphoryl transfer fromadenosine-50-triphosphate (ATP)[1–4] to the 6-hydroxylgroup of a number of furanose and pyranose compounds.[5]The ability of hexokinase to initiate the transfer of phos-phate groups suggested the application of this enzyme to thefunctional modification of the C6-hydroxyl groups incellulose. Chemical phosphorylation usually is a rathercomplicated process, requiring several protection and de-protection steps.[6,7] Enzymatic phosphorylation canmake the synthesis more efficient, eliminating many ofthese steps. The introduction of phosphate groups intocellulose will provide a polymer highly reactive towardvarious chemical compounds.Experimental PartBleached, twill weave, 120 g m2, 100% cotton fabric wasused. The enzymatic phosphorylation of the samples wascarried out at 30 8C in an Ahiba Spectradye-Datacolor dyeingapparatus with closed vessels, at 40 r.p.m. for 6 h, with40 U mL1 hexokinase (EC 2.7.1.1., Type IV: from baker’syeast, Sigma; one unit will phosphorylate 1 mmol of D-glucoseper min at pH 7.6 at 25 8C) dissolved in 50 mmol potassiumphosphate buffer (pH 7.6) and 50 mmol of a solution of ATP indeionized water, disodium salt (Sigma), using a liquor to fabricratio of 20:1. Fabrics were first treated with the ATP solutionfor 15 min to ensure the impregnation of the textile material,and then the enzyme was added. After completing the processthe enzymatically treated fabrics were washed under reflux toremove any residual protein.Sigma procedure No. 345-UV for quantitative, ultraviolet,kinetic determination of glucose-6-phosphate dehydrogenase(G-6-PDH, EC 1.1.1.49) in blood at 340 nm, was adopted forthe detection of glucose-6-phosphate (G-6-P) formed in thehexokinase phosphorylation reaction. G-6-PDH is an enzymethat catalyzes the first step in the pentose phosphate shunt,oxidizing glucose-6-phosphate to 6-phosphogluconate (6-PG)and reducing nicotinamide adenine dinucleotide phosphate(NADP) to NADPH, according to Equation (1):G-6-Pþ NADPþ !G-6-PDH 6-PGþ NADPHþ Hþ ð1ÞNADP is reduced by G-6-PDH in the presence of G-6-P. Therate of formation of NADPH (measured spectrophoto-metrically following the increase in absorbance at 340 nm)is proportional to the G-6-PDH activity and thus to theCommunication: Here we report for the first time on phos-phorylation of cotton cellulose using baker’s yeast hexokinaseand phosphoryl donor adenosine-50-triphosphate. An enzy-matic assay was adopted for determination of the degreeof phosphorylation of cellulose. This functional modificationof cellulose resulted in improved colorability and flameresistance.Phosphorylated glucopyranose unit of cellulose.962 Macromol. Rapid Commun. 2002, 23, 962–964Macromol. Rapid Commun. 2002, 23, No. 16  WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2002 1022-1336/2002/1611–962$17.50þ.50/0concentration of G-6-P. The assay mixture contains 1 mLNADP (1.5 mmol L1) and 0.01 mL G-6-PDH (2 U mL1;one unit will oxidize 1.0 mmol of D-glucose 6-phosphate to6-phospho-D-gluconate per min in the presence of NADP atpH 7.4 at 25 8C). The solution was stored at room temperature(18–26 8C) for 5–10 min.Then 2.0 mL G-6-P (1.05 mmol L1)were added and mixed gently. The absorbance of the sample(A) at 340 nm vs water was recorded asAi. The final absorbance(Af) was recorded 5 min later. The G-6-PDH activity wasdefined as: DA min1¼ (Ai–Af)/5. The standard curve ofG-6-PDH activity as a function of G-6-P concentration was set(Y¼ 0.292X, R2¼ 0.981) so that the concentration of asample containing unknown amounts of G-6-P could becalculated. Then 1 g sample of phosphorylated cotton fabricwas completely hydrolyzed to the constituting glucose andpresumably glucose-6-phosphate units with 0.91 g L1 totalcrude cellulase Ecostone L (Ro¨hm Enzyme, Finland), at 37 8C,pH 5, for 24 h. The solution was filtered through an Ultrafree-4centrifugal filter unit for concentration and purification ofbiological samples (Millipore Corp.), in order to separateprotein from sugars. The sample of G-6-P in the Sigma assaywas replaced with equivalent amounts of enzymaticallyhydrolyzed, hexokinase-treated cellulose.Results and DiscussionThe spectrophotometrically detected activity of G-6-PDHtoward the hydrolyzed cotton fabric treated with hexo-kinase is a clear evidence for the occurrence of enzymaticphosphorylation of cellulose. This enzymatic approachprovides an alternative to the 31P NMR or XPS techniques,since the degree of phosphorylation expected on the fabricsis rather low. The enzymatic procedure is reliable due to thehigh specificity of the enzyme toward the substrate underinvestigation. The concentration of G-6-P (0.01986 g L1)in the hydrolyzed cellulose was calculated according to acalibration curve. This value corresponds to a phosphoryla-tion of 0.03% of the glucopyranose units. Phosphorylationoccurs on the primary alcohol groups of cellulose, whichare best available for reaction. The secondary 3-OH groupforms extra hydrogen bonds with the ring oxygen atom, andthe chain is additionally coiled, protecting the 2-OH groups.The expected reaction mechanism is shown in Scheme 1.Conventional dyeing procedures[8] with basic, direct andreactive dyes resulted in an improvement of 10% in terms ofdye fixation on hexokinase-treated cotton. The enzymati-cally phosphorylated fabrics showed nearly threefoldretarded fire propagation[9] (1.1 cm2  s1), as compared tountreated cotton samples (3 cm2  s1), when exposed toflames.ConclusionsThis research reports on a new biosynthetic process for thephosphorylation of cotton cellulose with the enzyme hexo-kinase in the presence of phosphoryl donor ATP. Theenzymatic procedure applied, originally developed for thedetermination of glucose-6-phosphate dehydrogenase, wassufficiently sensitive to detect the occurrence of phospho-rylation, and represents an entirely innovative approach.Phosphorylation of 0.03% of the glucopyranose units in thecellulose provided a textile material with improved color-ability and flame-resistance, which also might be used for alot of chemical synthesis.Scheme 1. Mechanism of hexokinase-catalyzed cellulose phosphorylation.Phosphorylation of Cotton Cellulose with Baker’s Yeast Hexokinase 963Acknowledgement: We thank the HPMF-CT-2000-00868project for providing a grant to Dr. Tzanov.Received: September 18, 2002Revised: October 22, 2002Accepted: October 23, 2002[1] H. K. Chenault, E. S. Simon, G. M. Whitesides, Biotechnol.Genet. Eng. Rev. 1988, 6, 221.[2] G. M. Whitesides, C. H. Wong, Aldrichim. Acta 1983, 16, 27.[3] G. M. Whitesides, C. H. Wong, A. Pollak, ACS Symp. Ser.1982, 185, 205.[4] C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C. H. Wong,O. Abril, G. M. Whitesides, in: Methods Enzymol., Vol. 136,K. Mosbach, Ed., Academic Press Inc., New York 1987,p. 263.[5] R. E. Viola, F. M. Raushel, A. L. Rendina, W. W. Cleland,Biochemistry 1982, 21, 1295.[6] J. V. Edwards, D. R. Yager, I. K. Cohen, R. F. Diegelmann,S. Montante, N. Bertoniere, A. F. Bopp,Wound Repair Regen.2001, 1, 50.[7] S. S. Kim, W. Y. Jeong, B. C. Shin, S. Y. Oh, J. M. Rhee,J. Biomed. Mater. Res. 1998, 40, 401.[8] C. H. Giles, ‘‘A Laboratory Course in Dyeing’’, The Societyof Dyers and Colourists, Bragford, Yorkshire, England 1974,p. 65.[9] Norme franc¸aise NF G 07-184, Textiles-Comportement aufeu, Me´thode de classement en fonction de la surface bruˆle´e,1985.964 T. Tzanov, M. Stamenova, A. Cavaco-Paulo