Chapter 17
Enzyme-Catalyzed Reactions of Polysaccharides H. N. Cheng and Qu-Ming Gu Hercules Incorporated Research Center, 500 Hercules Road, Wilmington, DE 19808-1599
Enzyme-catalyzed reactions have been used to modify polysaccharides in the industrial context in order to improve the end-use properties. Typical reactions include molecular weight reduction, addition of charged or polar functional groups, hydrophobic modification, derivatization to form reactive functionalities, and synthesis of reactive oligomers. Many of these reactions are reviewed in this paper, using primarily hydrolases and oxidoreductases.
Introduction Polysaccharides are important articles of commerce. They are used variously as thickeners, gelling agents, stabilizers, interfacial agents, flocculants and encapsulants, in applications such as foods, coatings, construction, paper, pharmaceuticals, and personal care (1). For some applications, polysaccharides need chemical modifications in order to improve their end-use properties. Indeed, many chemical reactions have been devised to modify the polysaccharides (2). A substantial understanding has been obtained of the structure-property correlations for these modified polysaccharides. Whereas chemical means have been successful in modifying polysaccharides, enzymatic reactions may be useful to complement and to supplement these
© 2003 American Chemical Society
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204 means. Since polysaccharides are natural materials, many enzymes are available in nature that can carry out biotransformations (3). In addition, enzymes are often specific (regio-, enantio-, or chemospecific); thus many enzyme-catalyzed reactions can provide products with well-defined or stereospecific Structures (4). Another benefit is the mild conditions under which most enzymatic reactions can be done, often leading to products with less color or odor, and decreased occurrence of by-products. In this paper, several useful enzyme-catalyzed reactions of polysaccharides are reviewed. For convenience, these reactions are grouped intofivecategories: 1) molecular weight reduction, 2) addition of charge, 3) addition of polar group, 4) hydrophobic modification, and 5) formation of reactive oligomers. In order to limit the scope of this paper, only derivatives of cellulose and guar are covered, with an emphasis on the work done at Hercules Incorporated.
Results and Discussion Molecular Weight Reduction In many applications, it has been found necessary to lower the molecular weights of polysaccharides. In commercial processes, low molecular weights are often achieved via two chemical means, namely, hydrolysis with mineral acids and oxidation with hydrogen peroxide (5). However, these methods sometimes produce colored products or undesirable by-products, e.g., oxidized species and/orfragmentswith very low molecular weights. Interestingly, the use of enzymes can often obviate these difficulties. An example of a low-molecular-weight polysaccharide is guar. It is known from the literature that in the presence of a mannanase, guar can be degraded in molecular weight (6). Low-molecular-weight guar has been reported to be useful as a dietaryfiberand as a medically efficacious substance (7). In a combinatorial experiment (8), the degradation of guar was reported in the presence of four enzymes (lipase, hemicellulase, pectinase, and protease). As expected, hemicellulase showed a large decrease in molecular weight. The combination of hemicellulase and protease gave the lowest molecular weights achieved. This result is perhaps not surprising in view of the fact that guar contains up to 7% proteins. Thus, the protein in guar is likely to contribute to the observed molecular weight of guar. The use of enzymes to degrade cellulosic derivatives is well known (9). A recent example was reported by Sau (10). Through the use of the enzyme
205 cellulase, a number of cellulosic derivatives were degraded to lower molecular weights. The resulting materials are reported to be biostable; i.e., their resistance towards biological degradation has been increased.
Addition of a Polar Substituent The nature of a substituent on cellulose has a large effect on the properties of the cellulosic derivative. Thus, hydroxyethylcellulose (HEC) is soluble in water at all temperatures, but methylcellulose starts to gel at about 45°C, and hydroxypropylcellulose precipitatesfromwater at 40-45°C. An example of the effect of polar groups on solubility is shown for the following acetylation reaction. ο
Lipase Vinyl acetate 1
OH
^
HEC
^s AcetyM4EC
HO
J n
Figure 1. Lipase-catalyzed synthesis of acetylated HEC The reaction is very facile and readily takes place in the presence of a lipase. HEC is water-soluble, but when acetylated, the resulting polymer becomes much less soluble in water. A good example of the manipulation of substituents is the addition or removal of galactosefrompolysaccharide side chains. Galactose side chains can be removedfromgalactomannans through a-galactosidase (11). Conversely, galactose can be added through the use of lactose and β-galactosidase. This can be achieved either at the chain ends of an oligosaccharide (12), or at the end of ethylene oxide units on HEC (13). Another way to add the galactose to a polysaccharide is to use glycidyl galactose (vide infra).
Addition of Charge For some industrial applications, a charged polysaccharide can be highly desirable. For example, an anionic polymer may bind specifically to cationic
206 materials, whereas a cationic polymer may bind to anionic substrates. The coaddition of both a polycation and a polyanion may produce a polyelectrolyte complex (either stoichiometric or non-stoichiometric) which can be used as gelling agents, encapsulants, andflocculants(14). An example of the addition of a cationic charge to a polysaccharide is shown for the following reactions with carboxymethylceliulose (CMC), where X = (CH^andn^l As reported earlier (15), the reaction with CMC (where R=H) could proceed at 5-20% yield for subtihsin Carlsberg, papain, and proteasefromAspergillus saitoi in Ν,Ν-dimethylformamide (DMF) solvent. The reaction with CMC ester (R=methyl or ethyl) was, however, more facile, and higher yields were achieved. An example of the addition of an anionic group is shown for the following acylation reaction. Lipase AK (Pseudomonas φ., from Amano) was found to have excellent activity for this reaction, giving maleated guar. A related reaction can be carried out using succinic anhydride. Earlier, guar had been shown to react with succinic anhydride to give succinated guar in the presence of a lipase (8). The reaction is also possible with HEC, to give the corresponding succinated HEC.
Hydrophobic Modification Hydrophobic modification is a common method to synthesize surfactants or rheology modifiers. For rheology modifiers, the hydrophobes associate with one another in aqueous solutions at low concentrations, and thereby increase the viscosity of the solution at low shearfrequencies(16). A number of hydrophobically modified water-soluble polymers are commercially available, e.g., HMHEC (17), HEUR (18), and HASE (19). An enzyme can also be used as a catalyst to put a hydrophobic unit onto a cellulose derivative. For example, a stearic ester can be grafted onto HEC through the use of vinyl stéarate (20). Since vinyl stéarate is expensive, a more commercially viable route is to use methyl stéarate. The latter reaction also works, except that the reaction rate is slower.
Formation of Reactive Oligomers Enzymatic reactions can be used under mild reaction conditions to generate reactive functionalitiesfromsaccharides and polysaccharides. An example is the guar aldehyde where only an enzymatic process (through galactose oxidase) can produce the reactive aldehyde functionality in the C6 position of galactose
3
2
OH
R = H,CH3.CH CH
OCHjCOOR
Figure 2. Aminolysis of CMC and CMC esters
O^HN-X-NHa
^1
IN) Ο
Figure 3. Synthesis of maleated guar
IN)
00
Ο
209
HEC
StearoyWEC
Figure 5. Hydrophobic modification of HEC
(21,22). A more general reaction is the use of vinyl acrylate and a lipase to add an acrylate functionality onto HEC. This HEC-acrylate can then be used for further reactions (20). A reactive compound can also be madefroma monosaccharide. Thus, one can use lactose and β-galactosidase to form glycidyl galactose (8). The glycidyl galactose can then be used to react (via epoxide ring-opening reaction) with an amine or an alcohol. This is one way to add a galactose moiety to a polysaccharide. An interesting structure can be made via the oxidation of N-allylamine to form the corresponding epoxide in organic solvents in the presence of an enzyme from Aspergillus saitoi. Under basic pH conditions, the compound automatically oligomerizes to a trimer product.
Figure 6. Synthesis of glycidyl galactose using β-galactosidase
2
HN OH
Figure 7. Oligomerization of allylamine using an epoxidase
Aspergillus saitoi enzyme
Ρ] H
OH
H
212 Under neutral pH conditions in the presence of water, epoxidation of allylamine leads to the enantiomerically pure 3-amino-l,2-propanediol, which is a useful building block for many analogs of phosphatidic acids.
Comments The polysaccharide modification reactions delineated above may be summarized in Table 1. Both hydrolase and oxidoreductase enzymes have been used in the presence of organic solvents, water, or mixed solvents. In order to achieve optimal yield, each reaction needs to be separately studied, and the reaction parameters systematically varied to produce the best results. The key parameters are the enzyme used, the solvent medium, reactant and enzyme concentrations, and temperature (in that order). Enzyme screening is a major part of this optimization process. Recent developments in directed evolution and gene shuffling will be of great help in producing improved enzymes (23). Table 1. Summary of enzyme-catalyzed reactions cited in this work Polymer HEC
CMC Guar
Reaction Type MW control charge hydrophobe polar group reactive functionality charge MW control reactive functionality
Reactant
Enzyme
Solvent
-
cellulase lipase
water DMAc
lipase lipase lipase
DMAc DMAc DMAc or DMF DMF/water water water
succinic anhydride vinyl stéarate vinyl acetate vinyl acrylate diamine oxygen
lipase or protease mannanase galactose oxidase
EXPERIMENTAL Materials Vinyl stéarate, vinyl acetate, succinic anhydride, N-allylamine, maleic anhydride, Ν,Ν-dimethylacetamide (DMAc) and t-butyl methyl ether were
213 obtainedfromAldrich. LipasesfromPseudomonasfluorescens(Lipase AK) and from Pseudomonas cepacia (Lipase P) were obtained from Amano Enzyme USA. LipasefromCandida antarctica (Novozym® 435) camefromNovozymes A/S. ProteasefromAspergillus saitoi was purchasedfromSigma Co. Neutral guar, cationic guar, hydroxyethylcellulose (HEC), and carboxymethylcellulose (CMC) used for the study are the products of Hercules Incorporated. Brookfield Viscosity Measurements The Brookfield viscosities of aqueous solutions of the modified polysaccharides were measured at 1% concentration at pH 6.5 and room temperature. A LV type Brookfield viscometer was used with the spindle speed set at 30 rpm. Preparation of Succinated HEC A sample of HEC (Natrosol® Pharm-250MR, Hercules Incorporated, 5 g) was suspended in 50 ml of DMAc. To this solution 1 g of succinic anhydride and 0.2 g of Lipase Ρ (Pseudomonas cepacia) were added. The mixture was warmed up to 50°C which turned into a slurry in about 10 minutes. The resulting slurry was incubated at 50°C for 16 hours and then treated with acetone to precipitate the product. After washing with acetone and air-drying, 4.9 g of HEC acetate was obtained as a white solid, IR: 1750 cm-1. The control sample (without enzyme) gave also a band at 1750 cm-1 with about 30% of the intensity. 9
Preparation of Maleated Guar Flake guar (Hercules Incorporated, 1 g) and maleic anhydride (0.5 g) were mixed in 10ml t-butyl methyl ether. Lipase Ρ (Pseudomonas cepacia, 0.2 g) was added and the mixture was stirred for 48 hrs at 50°C. After the mixture was cooled down, isopropanol (20 ml) was added, and the solid residue washed with isopropanol three times and dried under vacuum to give 0.95 g of the maleated guar. IR spectral analysis of the product showed the presence of a strong ester band at 1755 cm", whereas the spectrum of the control sample (without enzyme) gave a much smaller band at 1755 cm' . 1
1
Preparation of Acetylated HEC A sample of HEC (Natrosol® Pharm-250MR, Hercules Incorporated, 5 g) was suspended in 50 ml of DMAc, followed by the addition of 1 g of vinyl acetate and 0.5 g of Lipase Ρ (Pseudomonas cepacia). After thorough mixing in 10 minutes, the resulting mixture gradually turned into a slurry. The mixture was
214 subsequently incubated at 50°C for 24 hours. The slurry was treated with isopropanol (EPA) to precipitate the product. After washing with IPA and airdrying, 4.5 g of HEC acetate was obtained as a white solid. IR: 1745 cm-1. The control sample (without enzyme) gave also a band at 1745 cm-1 but with only 10% of the intensity. The acetylated HEC was visibly less soluble in water, giving a cloudy solution at 1%. Hydrophobic Modification of HEC An HEC sample (Natrosol® Pharm-250MR, Hercules Incorporated, 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 P. After thorough mixing in 5-10 minutes, the resulting mixture turned into a 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". The DS (degree of substitution) was estimated to be 0.1, based on IR and comparing with the carbonyl intensities of known mixtures of methyl palmitate/cationic guar. The Brookfield viscosity of the product was significantly higher than the starting materials, indicating that the hydrophobe has been grafted onto the HEC. (It is known that the Brookfield viscosity of a hydrophobically modified polysaccharide varies with the degree of substitution of the hydrophobe.) 1
Oligomerization of N-Allylamine N-Allylamine (0.4 ml) was dissolved in a mixture of 4 ml of toluene and 30 μΐ water, and an Aspergillus saitoi protease containing epoxidase activity (Sigma, 20 mg) was added. The mixture was stirred at 37°C for 16 hours when the solution became cloudy and the viscosity increased. TLC indicated a major new spot formed. The material was purified byflashchromatography (silica gel, dichloromethane/methanol, 8:2) to give a yellowish liquid product. The yield was 0.2 g. The trimer structure was confirmed as the one shown in Figure 7. H NMR (D 0, 300MHz): 5.80-5.65 ppm (m, 1H, -CH=), 4.95-4.80 ppm (d, 2H, CH2=), 3.85-3.70 ppm (m, 2H, N-CH -C=C), 3.65-3.00 ppm (m, 10H); C (D 0, 75.5MHz) 135.7 ppm (-CH=), 116.9 ppm (CH =), 72.8 ppm (C-OH), 72.5 ppm (C-OH). MS (m/z): 204 (Μ*+1). 1
2
1 3
2
2
2
Acknowledgements. The authors wish to acknowledge the technical assistance of S. Mitai, G. F. Tozer, and A. J. Walton, and helpful discussions with colleagues at Hercules Incorporated. This is Hercules Incorporated Contribution Number 2388.
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