Biocatalysis in Polymer Science

Chapter 16

Synthesis and Modification of Carbohydrates through Biotechnology Peter R. Andreana, Wenhua Xie, and Peng George Wang* Downloaded by UNIV OF LEEDS on September 15, 2016 | http://pubs.acs.org Publication Date: December 10, 2002 | doi: 10.1021/bk-2003-0840.ch016

Department of Chemistry, Wayne State University, Detroit, MI 48202

Chemoenzymatic approaches to the modification of polysaccharides plays an important role in discovering novel polymeric structures with varying properties while establishing a non-toxic rapport with the environment. This review will explore the utilization of thermophilic glycosidases, and thermophilic lipases in the ring-opening polymerization/ modification of hydroxy-containing compounds, as well as introduce LiCl as a non-toxic polymer catalyst.

US demand for natural polymers is projected to approach $2.8 billion in 2003 (1). The fastest growth will take place in fermentation products; rapid short term growth will come from the use of these polymers as food ingredients, while in the longer term, strong growth will be fueled by increasing demand for feedstocks in the production of degradable plastics. In addition, protein-based polymers should experience robust growth as baby boomers age and new applications emerge for collagen. While wheat gluten will remain the largest single product by volume, cellulose derivatives (including microcrystalline cellulose) are expected to remain the largest product class in dollar terms. Cellulose ethers constitute a fairly mature market, however, so novel food applications and the advent of new products (e.g., cationic cellulose ethers) should buoy growth somewhat. In addition to fermentation products and protein-based polymers, marine polymers should also fare well. Despite a

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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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189 shake-out in the alginate industry, these products will be among the fastest growing natural polymers. Moreover, significant increases in chitin and chitosan production capacities will allow domestic producers to meet rising US demand. Natural phenolics and polyphenols will exert a moderating effect on the natural polymers market as a whole, given that this segment is dominated by mature products such as tannins and vanillin. Nevertheless, improvements in polyterpene chemistry should serve to spur demand. New technologies have changed the area of natural polymers over the last two decades, and this trend should continue. In order for those products, with the largest market potential (e.g., lactic acid and wheat polymers) to succeed, further advances must be made in order to lower production costs to the point where these biodegradable polymers can compete with petrochemical-derived polymers on the basis of price. The best opportunities for natural polymers will continue to be in food and beverages, led by wheat gluten and xanthan gum. The trend toward healthconsciousness will continue to be a boon to the food and beverages sector, as exudate, vegetable and fermentation gums gain appeal by virtue of their abilities to replace fat, add texture and provide dietary fiber. The fastest growing nonfood markets are expected to be packaging and textiles. The packaging market will be the first beneficiary of the rapid gains made by polylactic acid (PLA) and other carbohydrate-based polymers. Textiles will make more significant gains over the longer term, as biodegradable polymers such at polylactic acid and polytrimethylene terephthalate (PTT) are converted to fibers for use in apparel manufacture. The temptation to improve upon nature has always been great and has rarely been resisted. When scientists link the special properties of these substances (physical properties such as tensile strength and flexibility) to the sizes and functionalities of their molecules the next logical step involved chemical modifications of naturally occurring polymers.

Modification of Carbohydrates Carbohydrates can be modified in order to widen their range of potential application. In this way it is possible to produce a wide range of carbohydrate ingredients with specific functional properties. Naturally, the structure function relationship plays an important role in this. The application of enzymes to industrial productions has developed rapidly since 1960, particularly in the food and beverage industry. Today biotechnology and the use of enzymes has integrated with traditional chemical industry for the production of drugs and vitamins. The substrate and product specificity of enzymes is used for the production of compounds, which only with great difficulty, can be synthesized by conventional organic chemistry. Therefore, the synthesis of biomass-based carbohydrates with specific functional properties for use in drugs, personal care and food products is receiving increasing attention. The functional properties are controlled by the degree of polymerization and the type, position and degree



190 of substitution. These parameters are essential to industrial production and application of carbohydrates. Thermophilic microorganisms produce enzymes with unique characteristics such as high temperature and chemical and pH stability. The enzymes can be applied as biocatalysts in already existing industrial processes to replace the presently used, often polluting, chemical reagents. Thermophilic enzymes are of further interest for production of food additives, such as oligosaccharides. The synthetic potential of sugar-modifying enzymes such as glyeosidases has been investigated for the production of biologically active oligosaccharides. Novel micro-organisms have been isolated and characterized and suitable enzymes identified by screening procedures. Structure and function of the enzymes have been studied with respect to substrate specificity, stability, and product yield. Process improvement as well as the influence of the substrate and of the medium composition have been investigated to obtain novel products and improved yields in synthetic processes.

Thermophilic Glycosidase Library Enzymes evolving from thermophilic and hyperthermophilic organisms have attracted considerable attention due to their potential as biocatalysts with unprecedented properties for industrial applications. The glycosidase CLONEZYME™ library, which currently contains 10 unique thermostable glyeosidases, has been developed by Diversa (San Diego, CA) through cloning and automated high through-put screening systems (2). Each enzyme displays a variety of activities rangingfromgalactosidase, glucosidase to fucosidase. As shown in Scheme 1, a CLONEZYME™ library was screened for the synthesis of iV-acetyllactosamine and lactosamine, whose core structure exists in many glycoproteins and glycolipids (3,4). All thermophilic enzymes from the library were screened as potential catalysts for the glycosylation reaction. Table I lists the percent hydrolysis and yields for the varying thermophiles.

Modification of Hydroxyethylcellulose by Transgalactosylation with β-Galactosidases In recent years, enzymatic transglycosylation catalyzed by glyeosidases has been the focus of considerable interest (5-9). These enzymatic syntheses demonstrate transglycosylation ability toward a wide array of acceptors. By utilizing the thermophilic CLONEZYME™ glycosidase library as well as glyeosidases from three mesophilic sources (A .oryzae, B. circulons, and E. coli), it was demonstrated that hydroxyethylcellulose (HEC) could be transgalactosylated using lactose as the donor Scheme 2 (10). HEC has been


191

0H


HO f HO^±r°v

O2N 0~Γ)>

OH H O - ^ O

O

CLONEZYM ™ E

H

H

HO f

/OH U

1 HO . O H V*—Q

+

H

NHAc

2

3

^OH

O H ΗΟ~*-*Τ*Λ^ΟΗ

OH

.OH

HO-^T^ β

V

CLONEZYME™ 0 1 1

G 1

y

c o s i d a

s e Library

»? /

0

H

ΗθΛ^*^ο~"Τ^--" OH

Η

6

0

^

0

•OH

χ

^

Scheme 1. CLONEZYME™ glycosidase library in the synthesis ofNacetyllactosamine and lactosamine.

Table I. Enzymatic Synthesis with Recombinant Thermophilic Glycosidase CLONEZYME™ Library and Conventional MesophHic β-Galactosidase Yield(%) Enzyme Hydrolysis(%f Gly001-02 92 Gly001-06 46 9 Gly001-07 45 8 Gly001-08 86 Gly001-09 18 61 Hydrolysis was determined according to the free galactose content measured enzymatically by the Hans-Otto Beutler method using galactose dehydrogenase.

a


192


HEC acceptor

HO

Gal-HEC

Scheme 2. Transgalactosylation between lactose and HEC.

widely used as rheology modifiers, thickening agents, protective colloids, and a variety of other applications (11). Since HEC is a statistical polymer, the orientations of the hydroxyl groups are randomly distributed along the polymer backbone. Treatment with the enzymes would result in random transgalactosylation. C NMR spectroscopy is a conventional means to determine the loci of newly formed glycosidic bonds (12) , as well as the degree of substitution on HEC. From initial studies directed at the elucidation of the parent HEC and its galactosylated forms, it was concluded that they were indistinguishable. Thus in order to quantify substituted galactose on the HEC polymer, a highly sensitive enzymatic assay (13) was employed. The galactose oxidase-catalyzed reaction was highly specific for and reportedly more efficient with polymers containing terminal Dgalactose as illustrated in Scheme 3. 13

Hο O r 0 H HO. I \ r\ ^2^0R OH

Galactose Oxidase -

H O

Biocatalysis in Polymer Science

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