Thermostable α-Glucan Phosphorylase-Catalyzed Enzymatic Copolymerization to Produce Partially 2-Deoxygenated Amyloses

Jun-ichi Kadokawa,Kazuya Yamamoto,Shota Nakamura

doi:10.3390/pr8091070

Abstract

α-Glucan phosphorylase catalyzes the enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce α(1→4)-glucan—i.e., amylose. In this study, by exploiting the weak specificity for the substrate recognition of a thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5), we investigated the enzymatic copolymerization of 2-deoxy-α-d-glucose 1-phosphate (dGlc-1-P), which was produced in situ from d-glucal, with Glc-1-P to obtain non-natural heteropolysaccharides composed of α(1→4)-linked dGlc/Glc units—i.e., partially 2-deoxygenated amylose. The reactions were carried out at different monomer feed ratios using a maltotriose primer at 40 °C for 24 h. The products were precipitated from the reaction medium, isolated by centrifugation, and subjected to 1H NMR spectroscopic and powder X-ray diffraction measurements to evaluate their chemical and crystalline structures, respectively. Owing to its amorphous nature, the partially 2-deoxygenated amylose with adapted unit ratios formed a film when subjected to a casting method.

Highlights

Polysaccharides are widely distributed and play important roles in nature as structural materials, suppliers of energy, and key materials for specific biological and vital functions [1].Natural polysaccharides provide a great variety of chemical structures owing to the many types of monosaccharide units and different stereo- and regio-arrangements of the glycosidic linkages in the polymeric chains, which contributes to their many different in vivo functions [2]
Since it is well recognized that the enzymatic method is a useful approach to generate well-defined oligo- and polysaccharides [6,7,8,9,10], we investigated α-glucan phosphorylase-catalyzed enzymatic reactions using several monosaccharide 1-phosphates as substrates to obtain non-natural oligoand polysaccharides [11,12,13,14,15]. α-Glucan phosphorylase catalyzes the enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce α(1→4)-glucan—i.e., amylose—upon the liberation of inorganic phosphate (Pi) [16,17,18,19,20,21,22,23]
We reported that the enzymatically synthesized 2-deoxyamylose spontaneously formed an antiparallel double-helical crystalline structure, which was completely different from the native parallel double helix built from amylose [33]

Summary

Introduction

Natural polysaccharides provide a great variety of chemical structures owing to the many types of monosaccharide units and different stereo- and regio-arrangements of the glycosidic linkages in the polymeric chains, which contributes to their many different in vivo functions [2]. Α-Glucan phosphorylase catalyzes the enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce α(1→4)-glucan—i.e., amylose—upon the liberation of inorganic phosphate (Pi) [16,17,18,19,20,21,22,23].

Methods

Discussion

Conclusion