Polyester Coating of Cellulose Fiber Surfaces Catalyzed by a

Biomacromolecules 2004, 5, 106-112

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Polyester Coating of Cellulose Fiber Surfaces Catalyzed by a Cellulose-Binding Module-Candida Antarctica Lipase B Fusion Protein Malin T. Gustavsson,† Per Valdemar Persson,‡ Tommy Iversen,‡ Karl Hult,† and Mats Martinelle*,† Royal Institute of Technology, Department of Biotechnology, AlbaNova University Centre, SE-106 91 Stockholm, Sweden, and STFI, Swedish Pulp and Paper Research Institute, PO Box 5604, SE-114 86 Stockholm, Sweden Received July 17, 2003; Revised Manuscript Received October 15, 2003

A new approach to introduce polymers to cellulosic materials was developed by using the ability of a cellulosebinding module-Candida antarctica lipase B conjugate to catalyze ring-opening polymerization of -caprolactone in close proximity to cellulose fiber surfaces. The -caprolactone was introduced to the cellulose surfaces either by simple addition of liquid monomer or through gas phase. The effects of water activity and temperature on the lipase-catalyzed polymerization process were investigated. Analysis showed that the water content in the system primarily regulated the obtained polymer molecular weight, whereas the temperature influenced the reaction rate. The hydrophobicity of the obtained surfaces did not arise from covalent attachment of the poly(-caprolactone) to the surface hydroxyl groups but rather from surfacedeposited polymers which could be readily extracted. The degree of lipase-catalyzed hydrolysis through introduction of water to the polymer-coated cellulose fiber surfaces was also investigated and shown to be significant. Introduction Thermoplastic composites reinforced with cellulose fibers are increasingly gaining importance and the production and use of these materials have been described.1 Because the nature of cellulose fibers makes them inherently incompatible with hydrophobic polymers, several physical and chemical methods have been developed to improve the fiber-matrix interface in order to obtain a good composite material, as recently reviewed by Bledzki and Gassan.2 A different approach to introduce hydrophobic polymers into cellulose materials could be to perform the polymerization reaction in close proximity to the cellulose surfaces. By using such techniques, the problems involved in mixing pre-made long hydrophobic polymers with the hydrophilic cellulosic material might be overcome. The use of biotechnological tools for such an approach would be advantageous because many enzymes are highly specific and work under environmentally sound conditions. Lipases constitute a class of enzymes comprising an attractive choice of biocatalyst because they are generally very stable, and a variety of polyesters with different characteristics can be generated using several reaction types, as recently reviewed by Uyama and Kobayashi.3 The lipase-catalyzed ring-opening polymerization of lactones is a well-characterized process first described by two separate research groups: Uyama and Kobayashi4 and Knani * To whom correspondence should be addressed. Phone: +46 8 553 783 84. Fax: +46 8 553 784 68. E-mail: [email protected]. † Royal Institute of Technology. ‡ Swedish Pulp and Paper Research Institute.

et al.5 in 1993. It offers the advantage that polymers with high molecular weights can be obtained without the production of leaving group water molecules that can limit the conversion of monomer. The first step of the polymerization reaction is the initiation, where the catalytic serine reacts with the carbonyl carbon of the lactone monomer to form an acyl-enzyme intermediate, which subsequently is attacked by water to release the hydroxycarboxylic acid. In the propagation step, the terminal hydroxyl of a formed acid of any degree of polymerization attacks the acyl-enzyme intermediate to prolong the polymer chain. Water has multiple roles in the reactions in regulating lipase activity while also functioning as the polymerization initiator. A certain initial amount of water is needed for the initiation of the ring-opening polymerization reaction, whereas excessive amounts favors the undesired hydrolysis of ester bonds. Several research groups have studied the kinetics of the polymerization reaction, and parameters that influence the reaction rate and/or the obtained molecular weight of the produced polymer have been investigated in detail.5-13 So far, most ring-opening polymerizations have been performed either neat or in a suitable organic solvent and in mixed suspensions with the immobilized enzyme. Nonaqueous biocatalysis can also be performed in vapor-phase systems, in which substrates come into contact with the immobilized enzyme from gases. Several enzymes have been employed, including alcohol oxidases, alcohol dehydrogenases, and lipases.14-17 In the present study, the ability of cellulose-bound Candida antarctica lipase B to catalyze the ring-opening polymerization of -caprolactone on cellulose surfaces was investi-

10.1021/bm034244y CCC: $27.50 © 2004 American Chemical Society Published on Web 11/14/2003

Enzymatic Polyester Coating of Cellulose Fiber Surfaces

gated. The lipase was efficiently immobilized onto the fiber surfaces by fusing its N-terminus to the cellulose-binding module (CBM) of Neocallimastix patriciarum cellulase A (Cel6A), thereby creating the enzyme conjugate CBMCALB, as described previously.18,19 The produced polymer was characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and gel permeation chromatography (GPC). Experimental Section Preparation of CBM-CALB. The fusion protein CBMCALB (variant 418) was produced using Pichia pastoris as a host in high-cell density bioreactor cultures.20 The enzyme was purified essentially as previously described,19 except that 50 mM NH4HCO3, pH 7.8, was used as mobile phase in the gel filtration step to accommodate subsequent protein lyophilization. Cellulose-Binding Isotherms. Whatman No. 1 filter paper disks (1.5 cm in diameter, average mass 15 mg) were used as a high purity cellulose surface for the study. Dilutions of 15 µM enzyme stock solutions were made in 10 mM potassium phosphate buffer, pH 7.0. Filters were incubated together with 0.5 mL of enzyme solution by shaking at 160 rpm and 20 °C for 60 min. All samples were filtered through Millex PVDF Durapore filters, 0.45 µM (Millipore, Bedford, U.S.A.), and the amount of bound and free fusion protein was determined by comparing the optical density measured at 280 nm of the filtrate with the value obtained before incubation with the cellulose material. Polymerization onto Paper Surfaces. Using the isotherm for CBM-CALB binding to Whatman No. 1 filter papers, the conditions were chosen so that the binding of the enzyme to a cellulose surface reached saturation (0.5 mL of a solution containing 10 µM enzyme per paper). Controls were prepared with solutions without the enzyme but otherwise treated the same way. The papers were subsequently washed in water to remove excess enzyme and placed in chambers over saturated solutions of KCl (aw ) 0.84) or LiCl (aw ) 0.11). The systems were allowed to reach equilibrium for 48 h before initiation of the reaction. The seven-membered lactone -caprolactone (-CL) (Aldrich Chemical Co.) was distilled under reduced pressure in a nitrogen atmosphere prior to use. To start the reactions, 20 mg (0.18 mmol) of -CL was added to each filter paper surface, and the reaction chambers were incubated at 20 or 60 °C. Filter paper samples were withdrawn at different time points, and the produced polymer was extracted using acetonitrile as the solvent. The possibility to perform CBM-CALB catalyzed ringopening polymerization of -CL subjected to the enzyme from a gaseous phase was also investigated. Whatman No. 1 filter papers with bound CBM-CALB were placed in closed chambers over saturated KCl or LiCl solutions together with containers of -CL and were allowed to equilibrate at room temperature for 48 h before incubation at 80 °C. Samples were withdrawn at different time points, and the papers were weighed in order to estimate the amount of produced polymer, which was subsequently extracted using acetonitrile as solvent.

Biomacromolecules, Vol. 5, No. 1, 2004 107

Instrumental Methods. The progress of the polymerization was monitored by MALDI-TOF MS and GPC. Extraction samples were withdrawn for MALDI-TOF MS analysis and added to an equal volume of matrix composed of gentisic acid dissolved in a 1:1 mixture of methanol and water. A 0.5 µL aliquot was applied to the sample probe, the solvent was evaporated in vacuo, and the probe was inserted into the mass spectrometer (Hewlett-Packard G20205 A LD- TOF system). GPC was used to determine the monomer conversion and the average molecular weight of the polymer product. Extracted samples were diluted with tetrahydrofuran and filtered through a 0.45 µm PTFE membrane before injection. The GPC system was equipped with a Waters HPLC pump 510 connected to a Waters 410 differential refractometer and a Rheodyne 7125 injector (20 µL loop). Three columns from Waters (Ultrastyragel 50 Å, 100 Å and 500 Å) connected in series were used for the separation. Tetrahydrofuran was used as eluent at a flow rate of 1 mL/min. The GPC system was calibrated using polystyrene standards from Machery Nagel, 370-266,000 D. The conversion of monomer could be estimated by comparing the obtained results with the analysis of calibration samples containing -CL. The Whatman No. 1 filter papers were analyzed before and after extraction by Fourier transform infrared (FT-IR) spectroscopy on a PerkinElmer FT-IR 1725X. Potassium bromide tablets were prepared containing finely cut paper samples (5% w/w) and the total transmittance of 16 scans was measured in single beam mode. Micrographs of modified cellulose surfaces and the corresponding control samples were taken using a Zeiss Axioplan 2 optical microscope in reflected light using dark field technique. Water Resistance Analysis of Polymer-Coated Paper Surfaces. The hydrophobicity of poly(-CL)-coated filter papers was evaluated by contact angle measurements using a DAT 1100 system (FIBRO System, Sweden). Water droplets (3 µL) were added to the modified paper surfaces and monitored over time intervals of 60 s. The analysis was performed in triplicates and two droplets were added at different positions on all samples. In addition, 10 µL water droplets were manually placed on the surfaces using a syringe, and the time required for the paper sample to completely absorb the water was measured. Results and Discussion Binding of CBM-CALB to Whatman No. 1 Filter Papers. To perform the polymerization reaction in close proximity to cellulose-fiber surfaces, the cellulose-binding module-Candida antarctica lipase B conjugate CBM-CALB was utilized.18,19 Whatman No. 1 filter paper disks were used as the high purity cellulose surface for the study. The N-terminal CBM efficiently bound the fusion enzyme to the filter papers, whereas wild-type CALB did not show any binding affinity to this material (Figure 1). The conditions for the ring-opening polymerization reactions were chosen so that the binding of CBM-CALB to the cellulosic material reached saturation, which resulted in a load of approximately 0.2 µmol (7.5 mg) fusion enzyme per gram of filter paper.

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Figure 1. Equilibrium binding isotherms of wild-type CALB (]) and CBM-CALB ([) were established using Whatman No. 1 filter paper disks as cellulose substrate.

Gustavsson et al.

Figure 3. Molecular weight distribution of extracted poly(-CL) relative polystyrene calibration. The samples were withdrawn after 48 h reactions at 60 °C. The curves reveal the impact of water activity on the polymer product pattern; mainly oligomers and polymers of low molecular weight were produced at high aw, whereas a low aw afforded polymers of Mw ) 41 000. Table 1. Molecular Weight and Molecular Weight Distribution of Produced Poly(-CL), aw ) 0.11, According to GPC Analysis

Mwa (D)

Mwa/Mnb

time (h)

20 °C

60 °C

20 °C

60 °C

8 24 48

9500 18 000 20 000

33 000 37 000 41 000

1.9 1.9 2.1

3.6 5.7 3.1

a M 2 b w ) ΣniMi /ΣniMi. Mn ) ΣniMi/Σni, where ni is the number of molecules of molecular weight Mi.

Figure 2. Monomer conversion monitored by GPC. Paper samples were obtained after incubation at aw ) 0.11, 20 °C ([); aw ) 0.84, 20 °C (]); aw ) 0.11, 60 °C (9); aw ) 0.84, 60 °C (0). The reactions at 20 °C were performed and analyzed in duplicates.

CBM-CALB Catalyzed Ring-Opening Polymerization on Paper Surfaces. First, the reaction where -caprolactone (-CL) was added to the cellulose-bound CBM-CALB via a syringe was investigated. GPC analysis showed that the reaction temperature had a stronger impact on the monomer conversion rate than the water activity (aw) (Figure 2). After 8 h at 60 °C, all of the monomer had been converted, whereas the corresponding consumption at 20 °C was less than 50%. Low reaction water content resulted in a slightly higher conversion rate at 20 °C according to GPC analysis. One explanation could be that hydrolysis was favored over ester bond formation at high reaction water contents. The product molecular weight proved to be highly dependent on the water activity. Figure 3 shows GPC curves of the polymerization performed at 60 °C after a 48 h reaction. The catalysis performed at low water activity (aw ) 0.11) produced predominantly polymeric product, whereas mainly oligomers were detected when the reaction was run in high wateractivity atmospheres (aw ) 0.84). This can be explained by the fact that at a high water activity there are more water molecules present in the system to initiate polymer formation, which would result in a higher relative amount of short

polymers and oligomers. Another possible explanation is that hydrolysis will be favored over ester bond formation when water is abundant in the reaction system. GPC analysis also showed that a reaction temperature of 60 °C resulted in polymers with higher molecular weights compared with reactions carried out at 20 °C (Table 1). One explanation for the observation could be that the mobility of the polymer chains increases with temperature and that they therefore become more accessible for the enzyme. The GPC analysis was complemented with MALDI-TOF MS analysis to provide fundamental information about the mass of the repeating unit and the present end group, as well as supporting information about possible trends in the polymerization reactions. The results from MALDI-TOF MS analysis showed that, at any time point and temperature, products with higher degrees of polymerization (DP) were obtained on surfaces of papers that were incubated in chambers equilibrated to aw ) 0.11 as compared with experiments where aw ) 0.84, as seen in Figure 4. No significant difference in DP of samples incubated at different temperature and equal water activity was observed. This does not contradict the results from GPC, which indicated a difference in average molecular weight between samples incubated at equal aw but at different temperatures (Table 1), because MALDI-TOF MS tends to discriminate against high-molecular weight fractions in broad distributions.21,22 Moreover, a high relative amount of oligomer chains with a DP < 6 was detected in the MALDI-TOF MS analysis of samples incubated for 48 h at 20 °C, aw ) 0.11 (Figure 4A). This was not observed in the corresponding reaction at 60 °C (Figure 4B), which suggests an increase in reaction rate with elevated temperature. Mei et al. investigated the water-



Figure 4. MALDI-TOF MS analysis of extracted polymers obtained after reactions at various temperatures, water activities, and reaction times ([ 4 h; 0 24 h; 2 48 h). The surface area of each peak in a spectrum was normalized against the area of the first detected oligomer peak (DP ) 4).

temperature relationship for lipase-catalyzed ring-opening polymerizations of lactones. They reported the observation that the temperature primarily influenced the reaction rate and that the polymer molecular weight was regulated by the water activity.10 These observations are in agreement with the results obtained in the present study. In all experiments performed in the present study, solely water-initiated linear polymers were detected. However, other reports describe the formation of macrocycles when reactions are carried out as bulk polymerizations.21,23 The absence of cyclic structures might be explained by the fact that the polymerization was carried out in close proximity to cellulose fiber surfaces without agitation. Whatman No. 1 filter papers were also coated with CBMCALB produced polymers by allowing -CL to come into contact with the surface-bound enzyme from the gas phase. Figure 5 describes the poly(-CL) produced on CBM-CALB containing paper surfaces after incubation at 80 °C. The distributions according to MALDI-TOF MS are similar to those obtained with the conventional addition of monomer. Paper samples withdrawn at different time points were weighed before extraction in order to estimate the amount of produced poly(-CL) on the surface (Figure 6). At low water activity, the polymerization reaction was fast, and after 24 h, no significant increase in the amount of polymer was observed. The reaction conditions resulted in a production of approximately 20 mg of polymer on the filter surfaces. At this point, the cellulose-bound biocatalyst was likely to be covered with polymer, thereby obstructing additional monomer molecules to reach the active site of the enzyme.

At high water activity, only a low amount of polymer was produced on a paper surface, which could be caused by the fact that hydrolysis is favored over ester bond formation when water is abundant. Another possible explanation is the loss of enzyme activity. Studies describing lipase-catalyzed transesterifications of gaseous substrates report a significant loss in enzyme activity when reactions are performed at elevated temperatures and in high water-activity atmospheres.17,24 The observations were explained by the fact that enzymes with more free water molecules are less thermally stable.25 No polymer product was obtained from control samples lacking bound enzyme, neither in the case of simple addition of monomer followed by incubation at 20 or 60 °C nor from the controls subjected to -CL through gas phase. Whatman No. 1 filter papers containing added wild-type CALB were coated with polymers in a similar fashion as the described CBM-CALB samples. Therefore, the CBM is not directly needed for the polymerization reaction, but results in an efficient targeting of the lipase to cellulosic surfaces. Analysis of Hydrophobicity. Whatman No. 1 filter papers coated with polymers were subjected to drops of water, and the time required for complete adsorption was determined. Measurements of water drop contact angles were performed on the papers which showed water resistance according to the initial drop tests. The results are summarized in Figure 7 and Table 2. A significant hydrophobicity of paper surfaces was observed when the CBM-CALB catalyzed polymerization was performed at low water-activity atmospheres in combination with high reaction temperatures. The resulting

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Figure 7. Water drop tests of poly(-CL)-coated Whatman No. 1 filter papers. The samples were obtained after 48-h reactions performed at different water-temperature conditions. Table 2. Water Resistance Analysis of Poly(-CL)-Coated Paper Surfaces contact angle

awa 0.84 0.84 0.84 0.11 0.11 0.11

tempa

(°C)

20 60 80b 20 60 80b

adsorption time

t)0

t ) 60 s

0-5 s 20-25 s 90-100 s 5-10 min 20-25 min 40-50 min

n.d. 60 ° n.d. n.d. 80 ° 75 °

n.d. 0° n.d. n.d. 80 ° 75 °

a Samples obtained after reactions at different conditions. b -CL introduced to a paper surface through gas phase.

Figure 5. MALDI-TOF MS analysis of extracted polymers from reactions where -CL was introduced to the enzyme through gas phase. Each system was incubated at 80 °C for 4 h ([), 24 h (0), or 48 h (2) at different water activities. The surface area of each peak in a spectrum was normalized against the area of the first detected oligomer peak (DP ) 4).

Figure 6. Amount of produced polymer obtained from reactions where -CL was introduced to the system through gas phase, ([) aw ) 0.11 (0) aw ) 0.84.

contact angle values were initially relatively high, 75-80°, and remained constant throughout the measurements. Furthermore, the droplets in the most successful case (gas-phase addition of monomer, aw ) 0.11, 80 °C) stayed unaffected almost 1 h before complete absorption. When the polymerization reaction was performed at high water-activity atmospheres, only minor improvements in hydrophobicity were noted as compared with control samples.

Hydrolysis of Produced Polymers. The CBM-CALB fusion enzyme remained bound to the cellulose fibers after the completed reaction. The polymers are thus potentially susceptible to hydrolysis upon addition of water. To investigate the extent of hydrolysis, the poly(-CL)-coated paper samples were wetted with water and incubated at room temperature for 2.5 h before extraction and MALDI-TOF MS analysis. Comparisons were made with extracted poly(-CL) from sample duplicates that were not subjected to water. Figure 8 shows the analysis of extracted polymers obtained from coated filter papers prepared in aw ) 0.11 at 20 or 60 °C. A decrease in DP of extracted polymers after wetting with water can be observed, showing that CBMCALB remained bound and active at the cellulose surface. Similar changes in molecular weight distribution were observed for all reaction conditions of this study (data not shown). The results further indicate that the low amount of polymer produced in the reaction performed at 80 °C, aw ) 0.84 (Figure 6) could not be explained by a complete loss in enzyme activity. Light Microscopy and FT-IR Analysis of Paper Surfaces. Light microscopy analysis of the surface of a poly(-CL)-modified Whatman No. 1 filter paper (48 h, 60 °C, aw ) 0.11, Figure 9A) was performed, and the result was compared with the analysis of a control sample (Figure 9B). It can be concluded that the modified cellulose surface was coated with polymer to the extent that no individual fibers could be observed. Fourier transform infrared (FT-IR) spectroscopy was used in order to detect polymers present on a filter paper surface.



Figure 9. Micrographs of modified (A) and unmodified (B) Whatman No. 1 filter papers showing the extent of polymer deposition on the cellulose surfaces.

enzyme complexes and the hydroxyl groups present at the cellulose fiber surface. Figure 8. MALDI-TOF MS analysis of extracted polymers from poly(-CL)-coated papers subjected to water for 2.5 h (9) compared with control samples ([). The surface area of each peak in a spectrum was normalized against the area of the first detected oligomer peak (DP ) 4).

Analysis of CBM-CALB modified filter papers before extraction of polymers resulted in a signal at approximately 1730 cm-1, which corresponds to the presence of carbonyl groups (data not shown). No such signal could be observed in the spectrum achieved for a paper after extraction, showing that the polymers were not covalently attached to the cellulose surface hydroxyl groups but rather present as a polymer layer which could be readily extracted. Conclusions This study demonstrates a novel enzymatic approach to coat cellulose fiber surfaces with hydrophobic polyesters. Although the reaction was performed on solid cellulose surfaces where the substrates and products only were allowed to enter and exit the enzyme active site through simple diffusion without any mixing in the system, the ring-opening polymerization of lactones was successfully catalyzed by CBM-CALB. The resulting polymers were in no case found covalently attached to cellulose fibers. The reactions were performed using high CBM-CALB concentrations, resulting in a saturation of available CBM binding sites on the cellulose fiber surface. Further studies are necessary to investigate if such a high level of CBM-CALB coating prevented chemical reactions between activated monomer-

Acknowledgment. We are grateful for VINNOVA and IRECO Holding AB for financial support. Anders Uhlin is thanked for technical assistance with GPC analysis. Prof. Tuula T. Teeri and Dr. Harry Brumer are thanked for fruitful discussions and help with experimental design. References and Notes (1) Mohanty, A. K.; Misra, M.; Drzal, L. T. Compos. Interfaces 2001, 8. (2) Bledzki, A. K.; Gassan, J. Prog. Polym. Sci. 1999, 24, 221. (3) Uyama, H.; Kobayashi, S. J. Mol. Catal. B-Enzym. 2002, 19, 117. (4) Uyama, H.; Kobayashi, S. Chem. Lett. 1993, 1149. (5) Knani, D.; Gutman, A. L.; Kohn, D. H. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 1221. (6) Matsumoto, M.; Odachi, D.; Kondo, K. Biochem. Eng. J. 1999, 4, 73. (7) Kumar, A.; Gross, R. A. Biomacromolecules 2000, 1, 133. (8) Dong, H.; Cao, S. G.; Li, Z. Q.; Han, S. P.; You, D. L.; Shen, J. C. J. Polym. Sci. Part A: Polym. Chem. 1999, 37, 1265. (9) Macdonald, R. T.; Pulapura, S. K.; Svirkin, Y. Y.; Gross, R. A.; Kaplan, D. L.; Akkara, J.; Swift, G.; Wolk, S. Macromolecules 1995, 28, 73. (10) Mei, Y.; Kumar, A.; Gross, R. A. Macromolecules 2002, 35, 5444. (11) Alston, M. J.; Freedman, R. B. Biotechnol. Bioeng. 2002, 77, 641. (12) Bisht, K. S.; Henderson, L. A.; Gross, R. A.; Kaplan, D. L.; Swift, G. Macromolecules 1997, 30, 2705. (13) Deng, F.; Gross, R. A. Int. J. Biol. Macromol. 1999, 25, 153. (14) Barzana, E.; Klibanov, A. M.; Karel, M. Appl. Biochem. Biotech. 1987, 15, 25. (15) Barzana, E.; Karel, M.; Klibanov, A. M. Biotechnol. Bioeng. 1989, 34, 1178. (16) Lamare, S.; Legoy, M. D. Trends Biotechnol. 1993, 11, 413. (17) Parvaresh, F.; Robert, H.; Thomas, D.; Legoy, M. D. Biotechnol. Bioeng. 1992, 39, 467. (18) Gustavsson, M.; Lehtio, J.; Denman, S.; Teeri, T. T.; Hult, K.; Martinelle, M. Protein Eng. 2001, 14, 711. (19) Rotticci-Mulder, J. C.; Gustavsson, M.; Holmquist, M.; Hult, K.; Martinelle, M. Protein Expres. Purif. 2001, 21, 386.

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(20) Jahic, M.; Gustavsson, M.; Jansen, A. K.; Martinelle, M.; Enfors, S. O. J. Biotechnol. 2003, 102, 45. (21) Wahlberg, J.; Persson, P. V.; Olsson, T.; Hedenstro¨m, E.; Iversen, T. Biomacromolecules 2003, 4, 1068. (22) Nielen, M. W. F. Mass. Spectrom. ReV. 1999, 18, 309. (23) Co´rdova, A.; Iversen, T.; Hult, K.; Martinelle, M. Polymer 1998, 39, 6519.

Gustavsson et al. (24) Cameron, P. A.; Davison, B. H.; Frymier, P. D.; Barton, J. W. Biotechnol. Bioeng. 2002, 78, 251. (25) Volkin, D. B.; Staubli, A.; Langer, R.; Klibanov, A. M. Biotechnol. Bioeng. 1991, 37, 843.

BM034244Y

Polyester Coating of Cellulose Fiber Surfaces Catalyzed by a

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