Amylose phosphorylases

For the hydrosilylation reaction various rhodium, platinum, and cobalt catalysts were employed. For the further chain extension the OH-functionalities were deprotected by KCN in methanol. The final step involved the enzymatic polymerization from the maltoheptaose-modified polystyrene using a-D-glucose-l-phosphalc dipotassium salt dihydrate in a citrate buffer (pH = 6.2) and potato phosphorylase (Scheme 59). The characterization of the block copolymers was problematic in the case of high amylose contents, due to the insolubility of the copolymers in THF. 

In the field of polymer science, the most extensively used transferase is phosphorylase (systematic name (1 4)-a-D-glucan phosphate a-D-glucosyltransferase EC 2.4.1.1). Although this enzyme is responsible for the depolymerization of linear a-( 1 4) glycosidic chains in vivo it can also be used to synthesize linear a-( 1 4) glycosidic chains (amylose) in vitro. 

In vivo linear a-l,4-glucans are synthesized from ADP-glucose by the enzyme glycogen synthase [94-97]. The enzyme, as well as the monomer, are quite sensitive and therefore most researchers (at least in the field of polymer science) prefer to use phosphorylase for the synthesis of amylose. 

The glycogen phosphorolysis of phosphorylase can be reverted, which makes it possible to enzymatically polymerize amylose as well as hybrid structures with amylose as outlined in the following section. 

Enzymatic Polymerization of Amylose with Glycogen Phosphorylase... 

The fact that glycogen phosphorylase can be used to polymerize amylose was first demonstrated by Schaffner and Specht [110] in 1938 using yeast phosphorylase. Shortly after, the same behavior was also observed for other phosphorylases from yeast by Kiessling [111, 112], muscles by Cori et al. [113], pea seeds [114] and potatoes by Hanes [115], and preparations from liver by Ostern and Holmes [116], Cori et al. [117] and Ostern et al. [118]. These results opened up the field of enzymatic polymerizations of amylose using glucose-1-phosphate as monomer, and can be considered the first experiments ever to synthesize biological macromolecules in vitro. 

Recently Kuriki and coworkers succeeded in producing glucose-1-phosphate in situ during the enzymatic polymerization of amylose. By using sucrose phosphorylase or cellobiose phosphorylase, the monomer was produced during the polymerization from inorganic phosphate and sucrose or cellobiose, respectively [119-121]. 

One of the remarkable properties of phosphorylase is that it is unable to synthesize amylose unless a primer is added (poly- or oligomaltosaccharide) ... 

The strict primer dependence of the glycogen phosphorylases makes them ideal candidates for the synthesis of hybrid structures of amylose with non-natural materials... 

Arsenate similarly replaces phosphate in various phosphorolysis reactions, so that sucrose phosphorylase catalyzes the hydrolysis of sucrose in its presence (23), potato phosphorylase can hydrolyze amylose and amylopectin (24), nucleoside phosphorylase can hydrolyze inosine... 

Fig. 33. Experimentally observed molecular-weight dependence of (S2)z for amylose chains grafted onto glycogen (filled circles) and partially debranched amylopectin (open circles). Grafting was achieved by potato phosphorylase (15 rays for glycogen and 12000 per amylopectin molecule), and by muscle phosphorylase (400 rays per glycogen molecule)142,143)...

Formation of glycogen chains by glycogen muscle phosphorylase and starch amylose by potato phosphorylase. 

Earlier studies on the properties of phosphorylases isolated from various sources have indicated that their subunits are similar in size with about 100,000 daltons.15-17 The reaction proceeds in a rapid equilibrium random Bi-Bi mechanism as has been shown by kinetic studies with rabbit skeletal muscle phosphorylases a18-20 and b,21,22 rabbit liver enzyme,23 potato tuber enzyme,24 and the enzyme from E. coli.25) In contrast, the substrate specificities for various glucans differ considerably depending on the enzyme sources. The rabbit muscle enzyme has high affinity for branched glucans such as glycogen and amylopectin but low affinity for amylose and maltodextrin.26,27 The potato tuber enzyme can act on amylose, amylopectin, and maltodextrin but only poorly on glycogen,28,29 while the E. coli enzyme shows high affinity for maltodextrin.10 ... 

One possible way to solve this problem is to combine another enzyme, like SP, which produces a-GlP for GP. Waldmann and colleagues reported the combined use of SP and GP for the production of amylose from sucrose (1986). In this system, SP catalyzes the phosphorolysis of sucrose to produce a-GlP and fructose, and the a-GlP is next used as a substrate of GP to produce amylose. An interesting feature of this SP-GP system is that Pi produced in the second GP reaction is used as a substrate for the first SP reaction. The cooperative action by the two phosphorylases proceeds continuously with a constant Pi concentration, without any inhibition caused by an accumulation of Pi. Based on this SP-GP system, we have now established the process to manufacture essentially linear amylose with controlled molecular size, by using thermostable variants of SP and GP (Yanase et al., 2007 Ohdan et al., 2007). 

Ohdan, K., Fujii, K., Yanase, M.,Takaha,T., and Kuriki,T. 2007. Phosphorylase coupling as a tool to convert cellobiose into amylose. 1 Biotechnol., 127,496-502. 

The substrates, either 1 mg/ml amylose (c.l. 405) or amylopectin (c.l. 21), were incubated with 5 units/ml maize-branching enzyme (measured as by stimulation of phosphorylase a Guan and Preiss. 1993). The decrease in absorbance by the iodine/glucan complex was measured at 660 nm. 

The rate of synthesis, or degradation, of amylose depends on the degree of polymerization of the polysaccharide the action is faster on the species of lower molecular weight. It is thought that inactive enzyme—substrate complexes can form between phosphorylase and internal sections of D-glucosidic chains, and the result of this is an effective diminution in the concentration of enzjune free to attack chain ends. As the inactive complexes are more likely to form with the longer molecules of amylose, the rate of reaction decreases with increasing molecular size of the polysaccharide. 

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