Synthesis of highly ordered cellulose II in vitro using cellodextrin phosphorylase
Graphical abstract
Introduction
Cellulose is the most abundant biological macromolecule on earth and is a linear homo-polysaccharide, in which d-glucose units are linked via a β-(1→4)-glycosidic bond. This polysaccharide occurs widely in various organisms, but most cellulose exists as a component of plant cell walls, together with other polymers.1 Humans have long utilized cellulose either in the form of composites with other polymers such as wood or in the form of fibers such as paper and clothes. In recent years, attempts have been made to modify the structure of cellulose itself to make tailor-made cellulosic materials for various applications.
In plant cell walls, cellulose is synthesized from UDP-glucose by cellulose synthase (EC 2.4.1.12), which is localized in the plasma membrane as a highly ordered multi-enzyme complex called the terminal complex.2 Cellulose is elongated to a degree of polymerization (DP) of more than several thousand, and multiple cellulose chains are immediately assembled into microfibrils. Consequently, natural cellulose molecules are oriented in parallel to one another with the same polarity; this form is called cellulose I.3, 4, 5 It has been shown that cellulose can exist in a variety of alternative crystalline allomorphs, which differ in their unit cell dimensions, chain packing schemes, and hydrogen-bonding relationships.6, 7, 8, 9, 10, 11, 12 One of the major crystalline allomorphs is cellulose II, in which all the cellulose chains are in an anti-parallel arrangement.6, 7, 13, 14 Cellulose II is easily obtained by mercerization of native cellulosic material. It is also produced by regeneration from solution and utilized on an industrial scale.
Synthesis of cellulose in vitro is a different approach to prepare cellulose, and it is expected to afford pure cellulose uncontaminated with other polymers such as hemicellulose and lignin. In addition, an in vitro synthetic approach will allow for greater control of the structure of cellulose than is the case with natural microfibrils. Chemical synthesis of cellulose; however, faces the difficulty of controlling glycosidic bond formation because the monomer units often carry several hydroxyl groups with similar reactivity. To solve this problem, enzymatic catalysis, where the reaction proceeds under mild conditions with high catalytic activity and high selectivity, has been employed.15, 16, 17, 18 Kobayashi et al.19, 20 have reported the synthesis of cellulose in vitro by utilizing a transglycosylation reaction of β-cellobiosyl fluoride catalyzed by cellulase. They obtained crystalline celluloses having DP ∼22, with the structure of cellulose II.
Sugar phosphorylases are another choice to synthesize glycosyl linkages. These enzymes catalyze the reversible phosphorolysis of their particular substrates to form monosaccharide 1-phosphates, and their reverse reaction has often been utilized in syntheses of glycosides.21, 22, 23 Cellodextrin phosphorylase (CDP; EC 2.4.1.49), for example, phosphorolyzes cello oligosaccharides with the degree of polymerization greater than or equal to three, but does not react with cellobiose. CDP has been found only in Clostridia24, 25, 26, 27 and utilized for the preparation of cello oligosaccharide derivatives.28, 29, 30, 31, 32 Samain et al.,33 reported the preparation of crystalline cellodextrins from cellobiose and α-glucose 1-phosphate (αG1P), and the products showed electron diffraction patterns resembling those of cellulose II.
In the present study, we prepared cello oligosaccharides having an average DP of nine using glucose as a primer, even though it had been believed not to act as an acceptor of CDP, and succeeded synthesizing large crystals of cellulose II with high crystallinity.
Section snippets
Synthesis of cellulose by CDP
Purified CDP was incubated with a high concentration of αG1P and glucosyl acceptors (glucose or cellobiose). As shown in Figure 1, a significant amount of precipitate was observed in the reaction mixture when glucose was used as the acceptor, whereas the amount of precipitate was much smaller in the reaction mixtures using cellobiose as an acceptor or without glucosyl acceptor.
Figure 2 shows the HPLC profiles of the supernatants of the reaction mixtures using cellobiose and glucose as the
Discussion
In the present study, we synthesized cello oligosaccharide having an average DP of nine with CDP by using glucose, a very poor substrate for the enzyme, as an acceptor. For the synthetic reaction, the affinities of cellobiose and cello oligosaccharides as glucosyl acceptors are all very high compared to those of glucose, so a large difference of affinities seems to be the key to the effective production of crystalline cellulose.
A possible mechanism of the production of insoluble cello
Enzyme preparation and assay of recombinant CDP
Purified CDP from C. thermocellum YM4 was prepared by using an Escherichia coli strain containing a plasmid including cdp gene, as described previously.27 The enzyme activity was determined by quantifying the amount of inorganic phosphate produced from 10 mM αG1P and 10 mM cellobiose in 50 mM MOPS buffer, pH 7.5, at 37 °C. One unit of the enzyme activity was defined as the amount of enzyme that produced 1 μmol of inorganic phosphate per minute under the conditions employed. The amount of inorganic
Acknowledgments
The synchrotron radiation experiments were performed at BL38B1 in Spring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; 2007B1349). We thank Professor T. Iwata for his preliminary SAXS experiments at Spring-8. This study was supported by a grant from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists to M.H. (Grant No. 19·4869).
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