Biocatalysis in Polymer Science - American Chemical Society

Chapter 20

Lipase-Catalyzed Grafting Reactions on Polysaccharides

Downloaded by TUFTS UNIV on September 23, 2016 | http://pubs.acs.org Publication Date: December 10, 2002 | doi: 10.1021/bk-2003-0840.ch020

Qu-Ming Gu Hercules Incorporated Research Center, 500 Hercules Road, Wilmington, DE 19808-1599

Hydrophobic modification of polysaccharides is usually done by chemical means. In this work we have used enzymes, especially lipases, to carry out this hydrophobic modification. One example is the modification of hydroxyethylcellulose (HEC) using lipase-catalyzed transesterification reaction with vinyl stearate. The reaction conditions employed are much milder than those needed for the chemical synthesis. Similarly, this reaction has been applied to cationic guar (galactomannan substituted with 2-hydroxypropyl-3-trimethylammonium chloride), which is being used as an industrial thickening agent. Lipase-catalyzed reaction with vinyl stearate leads to stearoyl-cationic guar, which exhibits a moderate increase in solution viscosity when compared to the unmodified cationic guar. A similar reaction has been used to graft the acrylic functionality on HEC, using vinyl acrylate and lipase. The formation of ester bonds on HEC and on cationic guar has been confirmed by IR and solid state C-NMR analysis. 13

Polysaccharides are sometimes modified with hydrophobic groups to provide surface active and interfacial properties". These modifications are usually achieved via chemical reactions". Thus, hydrophobic alkyl ether derivatives, which are widely used for industrial applications, are often 1

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© 2003 American Chemical Society

Gross and Cheng; Biocatalysis in Polymer Science ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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synthesized under rather harsh caustic conditions . Hydrophobic alkyl ester derivatives can be made by the use of alkyl carboxylic anhydrides and alkyl acyl chloride . In contrast to the traditional chemical approaches, several papers have recently reported the use of enzymes (lipase or protease) to graft alkyl esters on mono- or disaccharides" . Such reactions on polysaccharides have been much less investigated. Gross and Dordick, et al reported the successful grafting of the caproyl group onto amylose using vinyl caprate and subtilisin Carlsberg. In this work, we have extended this reaction and shown that we can graft alkyl esters and acrylic groups onto cationic guar and hydroxyethyl cellulose (HEC). Guar gum is a carbohydrate polymer containing galactose and mannose as the structural building blocks. It is useful as a thickening agent for water in various industrial applications . Etherification of guar with cationic reagent 2hydroxypropyl-3-trimethylammonium chloride gives a cationic guar, which has extended the usefulness of the guar gum . Hydrophobic modification of the cationic guar may enhance the rheology of the cationic guar aqueous solution and also provide interfacial properties. As part of our investigation of enzymecatalyzed modifications of polysaccharides, we attempted the hydrophobic esterification of cationic guar with vinyl alkylate using a lipases or a protease as the catalyst. The resulting material was stearoyl cationic guar that showed both cationic and hydrophobic properties and exhibited a moderate increase in solution viscosity comparing to the unmodified cationic guar. Similarly, HEC was modified hydrophobically through the same enzymatic reaction using vinyl stéarate. The reaction conditions employed were much milder than those used for the chemical synthesis. Commercial hydrophobically modified HEC is often used as a thickening agent in paint, construction, and personal care . 6

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Experimental

Materials Vinyl stéarate, Ν,Ν-dimethylacetamide (DMAc), t-butyl methyl ether, and methanesulfonic acid were obtained from Aldrich. Lipases from Pseudomonas fluorescens (lipase AK) and from Pseudomonas cepacia (Lipase PS) were obtained from Amano Enzyme USA. Lipase Β from Candida antarctica (Novozym 435) came from Novozymes. The cationic guar and hydroxyethyl cellulose (HEC) used for the study came from Hercules Incorporated.


245 Hydrophobic Modification of Cationic Guar in DMAc A cationic guar (N-Hance 3000, Hercules, 5g) having a degree of substitution of 0.3 was suspended in 100 ml of DMAc. 0.8 g of vinyl stéarate was added, followed by 0.4 g of lipase AK. After thorough mixing, a few drop of methanesulfonic acid was added. The resulting solution became very viscous. The mixture was then incubated at 50°C for 24 hours and then treated with isopropyl alcohol (IPA). The resulting precipitate was washed extensively with IPA and dried in air. The yield was 5.1 g. C-NMR (solid state, 75.5Hz): 614.0 (-CH ), 23 (-CH ), 30 (-OOCCH -); IR: 1740 cm-1 (ester) and 1670 cm-1 (water or acid). 13


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Hydrophobic Modification of Cationic Guar in t-Butyl Methyl Ether A cationic guar sample (N-Hance 3000, Hercules, 10 g) is suspended in 100 ml of t-butyl methyl ether. 2 g of vinyl stéarate was added, followed by 0.5 g of lipase PS. The mixture was stirred at 50°C for 72 hours. The product was recovered by filtration and washed with IPA and hexane. After air-drying, the yield was 10.5 g. The product was a water-soluble material. C-NMR (solid state, 75.5Hz): Ô14.0 (-CH ), 24 (-CH -); IR: 1735 cm-1 (ester). 13

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Hydrophobic Modification of Hydroxyethylcellulose (HEC) in DMAc A hydroxyethylcellulose (Natrosol, Pharm-250MR, Hercules, 4 g) was suspended in 20 ml of DMAc, followed by the addition of 1 g of vinyl stéarate and 0.5 g of lipase PS. After thorough mixing in 5-10 minutes, the resulting mixture turned into slurry that was subsequently incubated at 50°C for 48 hours. The yellowish slurry was treated with acetone/IPA (1:1) to give precipitates. After washing with IPA and air-drying, 3.8 g of the hydrophobically modified HEC was obtained as a white solid. IR: 1750 cm-1.

Brookfield Viscosity Measurement As an example, viscosity of the aqueous solutions of the stearoyl-cationic guar was measured at 1% concentration at pH 6.5 and room temperature. A L V type Brookfield viscometer was used for the measurement with the spindle speed


246 set at 30 rpm. The viscosity of the hydrophobically modified cationic guar varies with the degree of esterification (Table I).

Table I. Brookfield Viscosity of Stearoy 1-Cationic Guar


Cationic Guar Solubility in water Unmodified Yes Stéarate ester Yes Stéarate ester No Stéarate ester Yes

pH of the solution 6.0 6.0 5.0, 6.0 6.0

Viscosity (cpsat ΙΨο) 2,000 5,600 0 4,200

DE* 0 0.05-0.2 >0.5 0.05-0.1

*Degree of esterification, estimated by IR spectral analysis by comparing the carbonyl signals intensity of a known mixture of methyl palmitate/cationic guar with that of the enzymatically modified products.

Results and Discussion The reaction of cationic guar with vinyl stéarate in the presence of a lipase gave a hydrophobically modified polysaccharide. As shown in Figure 1, the product contains an ester functionality that absorbs at 1740 cm' in the IR spectrum. The starting material vinyl stéarate has a distinct IR absorption at 1775 cm' that was not observed in the spectrum of the product, indicating all the unreacted vinyl stéarate had been washed away during the work-up. Introduction of the ester bonds onto the guar gum has also been confirmed by solid state CNMR analysis, which shows a clear signal at 14.0 ppm, corresponding to -CH , and signals at 23 and 30 ppm corresponding to the CH of the long chain fatty ester. A control experiment was performed without the use of die enzyme. The product did not show any detectable IR absorption in the region of 1735-1750 cm-1 or higher, suggesting ester formation was low or negligible in the absence of the enzyme. The degree of substitution (DS) mainly depends on the amount of vinyl stéarate used in the starting reaction mixture and reaction time. The more vinyl stéarate used, the higher the degree of substitution of the product was. The enzyme use level also has an effect on the DS of the final product. When too much hydrophobe (>0.5 DS) is grafted onto the cationic guar, the product usually becomes water insoluble. It was found that the enzymatic reaction performed very well when the starting material was dissolved or swollen in DMAc. When using an organic solvent such as t-butyl methyl ether in which cationic guar was merely suspended, the reaction took a longer time and the degree of esterification was 1

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247 also relatively low. However, as the lipases are relatively stable in a non-polar organic solvent environment, a slow but steady increase in the DS of the product could be achieved after long time incubation. Interestingly, when the same enzymatic reaction was applied to guar gum instead of its derivative cationic guar, the ester formation was very low. One interpretation of this result is that transesterification of the cationic guar with vinyl stéarate occurs mostly at the 2hydroxy functionality of the cationic moiety on the polysaccharide (Figure 2). Further study will be carried out to investigate the regioselectivity of this lipasecatalyzed modification of cationic guar. We screened several commercially available lipases and proteases. The results indicated that the lipases from Pseudomonus sp., such as lipase AK and lipase PS from Amano Co., were active in DMAc and t-butyl methyl ether in catalyzing the transesterification of cationic guar and HEC with vinyl stéarate. The lipase ΒfromCandida antarctica (Novozym 435, Novozymes) was less active. Other lipases and proteases including immobilized Alcalase from Novozymes and subtilisin from Sigma were inactive under the reaction conditions. Suitable suspending media or solvents were hydrocarbons and polar aprotic solvents. The solvent mostly used in this work was DMAc since it solubilized cationic guar in the presence of a small amount of organic acid. It also dissolved HEC easily. The temperature of the reaction was optimally between 40-55°C. The hydrophobically modified stearoyl-polysaccharides usually have a higher viscosity than the starting material if the products are water-soluble. As shown in Table 1, the viscosity of the cationic guar increased 2-3 times after stearoyl modification. This viscosity increase also confirms covalent attachment of the C18 hydrophobe onto cationic guar. In order to study the generality of this method, the same lipase-catalyzed reaction has been applied to other polysaccharides. As depicted in Figure 3, cellulose ether hydroxylethylcellulose (HEC) that contains primary alcohol farther away from the polysaccharide backbone, has been modified covalently through transesterification using vinyl stéarate as an acyl donor. Both Lipase PS and Novozym 435 have showed activity when DMAc was used as a solvent. In all other solvents such as t-butyl methyl ether, t-butanol and chloroform the formation of the ester material was also observed but with much lower yield according to IR analysis. At a low substrate concentration (

Biocatalysis in Polymer Science - American Chemical Society

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