An Investigation of Enzymatic Phosphorolysis of α(1→4)-Linked Oligo-d-glucosaminides by Thermostable α-Glucan Phosphorylase Catalysis

Jun Ichi Kadokawa,Kento Yamashita

doi:10.21767/2472-1123.100010

Jun Ichi Kadokawa, Kento Yamashita

Open Access

https://doi.org/10.21767/2472-1123.100010

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Abstract

This paper reports an investigation of enzymatic phosphorolysis of α(1→4)- linked oligo-d-glucosaminide substrates by thermo stable α-glucan phosphorylase (from Aquifex aeolicus VF5) catalysis. α-Glucan phosphorylase catalyzes both phosphorolysis and glucosylation/polymerization at the nonreducing end of α(1→4)-linked glucose substrates depending on conditions. The authors also found that α(1→4)-linked d-glucosaminide polymer (chitosan stereoisomer) was obtained by the thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization of α-d-glucosamine 1-phosphate from a maltooligosaccharide primer. In the present study, we intend to reveal whether the enzyme catalyzes phosphorolysis of substrates containing such d-glucosaminide chains. The α(1→4)-linked oligo-d-glucosaminide substrates elongated from maltotriose were first prepared by the thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization of α-d-glucosamine 1-phosphate from the maltotriose primer and subsequent purification by preparative HPLC. The phosphorolysis of the resulting substrates are conducted in the presence of thermostable α-glucan phosphorylase in phosphate buffer. The analytical results of the products fully supported the occurrence of phosphorolysis at the nonreducing end of both the chains of d-glucosamine-α(1→4)-d-glucosamine and d-glucosamine-α(1→4)-d-glucose sequences.

Highlights

Enzymatic polymerization has been identified as a powerful approach to synthesize polysaccharides composed of stereo- and regio-controlled glycosidic linkages [1,2,3,4,5,6]. α-Glucan phosphorylase is one of the enzymes that have practically been used as a catalyst for enzymatic polymerization to produce well-defined polysaccharides. α-Glucan phosphorylase catalyzes in vivo phosphorolysis of α(1→4)-glucans such as amylose and glycogen at the nonreducing end in the presence of inorganic phosphate (Pi) to produce α-D-glucose 1-phosphate (Glc-1-P) [7,8,9]
Potato α-glucan phosphorylase catalyzes enzymatic glucosaminylations using α-D-glucosamine 1-phosphate (GlcN1-P) and its derivative, N-formyl-α-D-glucosamine 1-phosphate (GlcNF-1-P), as non-native glycosyl donors in acetate buffer to produce oligosaccharides having one GlcN(F) unit at the nonreducing end [20,21], whereas successive glucosaminylations using GlcN-1-P occur by thermostable α-glucan phosphorylase catalysis in acetate buffer, giving rise to α(1→4)-linked oligo-GlcN chains at the nonreducing end of glycosyl acceptors, e.g., Glc3, the smallest glycosyl acceptor for this enzyme
With increasing the donor/acceptor feed ratio, the degree of polymerization (DP) values of the α(1→4)-GlcN chains retained at most five. This result indicated that further chain-elongation was inhibited by Pi produced from GlcN-1-P, because which is a native substrate for phosphorolysis by α-glucan phosphorylase catalysis

Summary

Introduction

Enzymatic polymerization has been identified as a powerful approach to synthesize polysaccharides composed of stereo- and regio-controlled glycosidic linkages [1,2,3,4,5,6]. α-Glucan phosphorylase is one of the enzymes that have practically been used as a catalyst for enzymatic polymerization to produce well-defined polysaccharides. α-Glucan phosphorylase catalyzes in vivo phosphorolysis of α(1→4)-glucans such as amylose and glycogen at the nonreducing end in the presence of inorganic phosphate (Pi) to produce α-D-glucose 1-phosphate (Glc-1-P) [7,8,9]. This result indicated that further chain-elongation was inhibited by Pi produced from GlcN-1-P, because which is a native substrate for phosphorolysis by α-glucan phosphorylase catalysis.

Results

Conclusion