This paper reports an investigation of enzymatic phosphorolysis of α(1→4)- linked oligo-d-glucosaminide substrates by thermostable 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.
Jun-ichi Kadokawa, Kento Yamashita, Riko Shimohigoshi and Kazuya Yamamoto
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