Enzyme-Mediated Reactions of Oligosaccharides and

Chapter 12

Enzyme-Mediated Reactions of Oligosaccharides and Polysaccharides Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 19, 2016 | http://pubs.acs.org Publication Date: February 15, 2001 | doi: 10.1021/bk-2001-0786.ch012

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

Two types of enzymatic reactions are given here as examples of synthetic problems encountered in industry. In the first case, commercially available β-D-galactosidase from Escherichia coli was used as a catalyst to transfer galactose from lactose to oligosaccharides. A preference for galactosyl transfer to the 3 or 4position of the sugar moiety of the oligosaccharide was observed for the products. As expected, only the β-anomer was produced. A wide variety of sugars, including disaccharides, trisaccharides, cellotetraose as well as maltodextrins have been shown to act as acceptors, yielding oligosaccharides. In the second example, α -galactomannan that has been previously treated to contain cationic groups (cationic guar gum) was subjected to treatment with a series of commercial enzymes such as lipases, protease and cellulases. Some enzyme preparations showed significant changes in the viscosities of 1% cationic guar solution. For example, lipases from Aspergillus niger and Aspergillus saitoi and Protease XIII from Rhizopus niveus produced substantial viscosity reduction (0-20% of original viscosity). These examples demonstrate the utility of low -cost enzymes in manipulating polymer structures.

Polysaccharides are used widely in industry for a variety of applications. These polysaccharides are obtained from natural sources, and many of them are approved for food use, e.g., starch, locust bean gum, guar, gum arabic, pectin, agar, alginate, carrageenan, xanthan, dextran, gellan, and pullulan. These are all commercially important materials with desirable properties (1-4). In some applications, however, the properties of natural polysaccharides may need improvements. In these cases, 184

© 2001 American Chemical Society Gross and Scholz; Biopolymers from Polysaccharides and Agroproteins ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 19, 2016 | http://pubs.acs.org Publication Date: February 15, 2001 | doi: 10.1021/bk-2001-0786.ch012

185 modifications of the polysaccharide structures may be necessary. Such modifications have been most often done for starch and cellulose (5). As a result, a wide range of starch and cellulosic derivatives are available commercially. In the past several years a lot of papers have appeared on the use of enzymes and microbes for organic synthesis. Many enzymatic, chemo-enzymatic, and microbial reactions have been developed (6-7). Several of these reactions are applicable toward polysaccharides (8) and in fact some of them were designed specifically for polysaccharides. Through these means, new or improved products or processes may be obtained. In reviewing the literature, there are at least three ways whereby new structures may be generated: 1. Synthesis of polysaccharides, starting with sugars or sugar derivatives. Whereas these processes are likely to result in materials at higher costs than the natural polysaccharides, the resulting materials may have unusual structures or enhanced properties. As an example of this approach, Kobayashi et al. (9) have reported the synthesis of low-molecular-weight cellulose using cellobiosyl fluoride and cellulase. A n alternative method to obtain pure cellulose is to use the bacterium Acetobactor xylinium which yields high-molecular-weight cellulose with favorable tensile and flexural properties (10).

Cellobiosyl

fluoride

Cellulose

Microbial Cellulose

n»ioo

Figure 1. Synthesis of cellulose oligomers and polymers using isolated enzymes and microbes. 2. Degradation of polysaccharides to produce oligosaccharides and lowmolecular-weight materials. As an example, carboxymethylcellulose ( C M C ) can be degraded by cellulase to give a low-molecular-weight polymer (11).

Carboxylmethyl Cellulose CMC

LMW CMC

Figure 2. Degradation of CMC to produce low molecular weight CMC Gross and Scholz; Biopolymers from Polysaccharides and Agroproteins ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

186


3. Modification of the polysaccharide structure by adding selected functional groups that impart desirable properties. One possible example is to acylate a polysaccharide using a protease and vinyl alkanoate (12).

CH (CH2)8COOCH=CH 3

2

CH CHO 3

Figure 3. Acylation of amylose. In the industrial perspective, these various reactions are tools in a "tool-box" whereby an industrial chemist can alter the structure of a compound or a material and thereby optimize the activities or properties of a product. Of course, the commercial viability of such a product depends on a large number of factors: potential market size, performance criteria, price constraints, enzyme and processing cost, availability of competitive products, and proprietary position. In many cases, product development requires the synthesis (or screening) or a large number of structures and some effort in structure/activity or structure/property correlations. In this work two types of reactions will be described as examples of problems encountered in industry. The first is an example of a synthetic reaction (galactosylation) to produce new galactosyl-oligosaccharides. The second example uses degradation by which the polysaccharide properties are modified; cationic guar gum is the example here.

Galactosylation of Oligosaccharides and Polysaccharides Enzymatic synthesis of oligosaccharides and polysaccharides is of growing industrial interest (13). Glycosidases constitute one group of enzymes which may be used for this purpose (7, 14). Whereas glycosidases usually hydrolyze glycosidic linkages in water, under suitable reaction conditions in vitro they can form glycosidic linkages. Hydroxyl-containing molecules other than water, such as alcohols, can serve as acceptors (15-18). The transglycosylation activity of galactosidase has also been used for the synthesis of disaccharides and trisaccharides. For many studies, lactose has been employed as the galactosyl donor since it is inexpensive. Lactose is involved in the synthesis of both industrial materials and a variety of biologically active molecules. For example, Stevenson et al. (19) reported the large-scale production of alkyl galactosides which can be used as substrates for the lipase-catalyzed synthesis of surfactants and emulsifiers. In other reports, lactose and galactosidases were successfully employed for the galactosidation of several drugs, such as genins (20) and chloramphenicol (21).

Gross and Scholz; Biopolymers from Polysaccharides and Agroproteins ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

187


Recently, we screened a variety of carbohydrate enzymes for their ability to transfer galactose (from lactose) onto different oligosaccharides and polysaccharides. During the search, we found the β-galactosidase from E. coli to be particularly facile in transferring galactose onto maltose and cellobiose in high yields. We have applied this reaction to the synthesis of a number of galactosyl-oligosaccharides (e.g., Figure 4). The regioselectivity as well as the enantioselectivity of the transgalactosylation are also examined.

β-Galactosyl-cellobiose

Oligomer

Figure 4. β-Galactosidase

catalyzed synthesis of oligosaccharides.

For the synthesis of D-galactosylcellobiose, the concentrations of lactose and maltose were chosen to be 0.8 M and 0.3 M , respectively. The solvent used was aqueous phosphate buffer at p H 7.0. The progress of the reaction was monitored by T L C (isopropanol/HaO/NRtOH, 7:4:1) and reverse phase H P L C . Figure 5 shows the time course studies of a β-galactosidase-catalyzed reaction. The overall yield of the trisaccharides rises quickly as lactose is consumed but reaches a plateau as the consumption of lactose levels off. Clearly, the effective rate of product hydrolysis increases as the lactose concentration in the reaction mixture decreases. The highest trisaccharide yields (5060%) are obtained i f the reaction is stopped when 50-60% of lactose is consumed. Although the reaction is facile, isolation of the desired product from the reaction


188


mixture can be difficult because the incubation solution contains the starting material, reaction product, and hydrolyzed materials. In the case of the synthesis depicted in Figure 5, the crude trisaccharide was obtained by elution with the same solution that was used for the T L C system from the silica column. The fractions containing the trisaccharide were separated from D-galactose, glucose (from lactose hydrolysis), and cellobiose. These fractions were pooled and concentrated to give a syrup, which showed a single spot on a T L C plate. The trisaccharide was identified to be D galactosyl -(l-4)-p-cellobiose.

Figure 5. Time-course of β-galactosidase-catalyzed transgalactosylation of 0.8 M lactose with 0.3 M cellobiose. The reaction mixture contained 8 mmol lactose, 3 mmol cellobiose in 10 ml of 0.1 M , pH 7.0 phosphate buffer at 37°C. The reaction mixture was withdrawn at certain time intervals and analyzed by reverse phase HPLC Several other enzymes have been screened for the transgalactosylation reaction. The enzymes β-galactosidase from Aspergillus niger and Aspergillus oryzae only hydrolyzed lactose under the same conditions, and no trisaccharides were observed. Neither hydrolysis nor transgalactosylation have been observed when oc-galactosidase from Aspergilluas niger and β-amylase were used. A preference for galactosyl transfer to the 3 or 4-position of the sugar moiety of the oligosaccharide was observed for the products. According to N M R analysis, it is clear that there was no Ι,ό-β-glycosidic linkage in the trisaccharides. In all cases only the β-anomer was produced, as indicated by the absence of α-anomer signal in the


189 anomeric region of N M R spectrum of the product. As an example, the N M R data for lactose/maltose reaction are shown in Figure 6. The C and *H chemical shifts for the starting materials and for the product (D-galactosylmaltose) are given. The observed C - l proton signal appears at 4.25 ppm, indicating the β-confîguration. (The a-anomer has the proton C - l signal higher than 5.1 ppm.) The regioselectivity of the reaction can be seen by the C N M R analysis. The C - l carbon of a 1,6-linkage has normally a signal at 93-95 ppm. The newly-formed glycosidic linkage has a C - l carbon signal appearing at 103 ppm, indicating that the linkage occurs between 1-3 or 1-4. B y comparing with the C N M R spectra of a number of known oligosaccharides, we could ascertain the linkage to be at the 1,4-position. 1 3

1 3


1 3

OH/OH

H-NMR: Ci-H:4.30(P) cr-H:5.15(a,95%)

OH

U\^-0

y^\^0

Ho\^r^°HO^^

Enzyme-Mediated Reactions of Oligosaccharides and

Recommend Documents