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Phosphorylase is a type of enzyme-producing sugar phosphates through the reversible phosphorolysis reactions of glycosides, which makes it an important starting enzyme in multi-enzyme systems for rare sugar biomanufacturing. To investigate its application in D-tagatose biosynthesis from maltodextrin using in vitro multi-enzyme cascade biosystem, the α-glucan phosphorylase (αGP; EC 2.4.1.1) from the thermophile D. turgidum DSM 6724 was prepared and characterized. It exhibited the specific activity of 30.28 U/mg at its optimal temperature of 70 °C. Thermostability results revealed that DituαGP could maintain more than 25% of initial activity for 4 h, even at 90 °C. The highest activity was observed at pH 5.5, and most divalent metal ions deactivated the enzyme. DituαGP exhibited great application potential in the multi-enzyme system that about 3.919 g/L of D-tagatose was produced from 150 g/L of maltodextrin within 36 h. DituαGP has played an important role in this biosystem and will also be applied in the synthesis of other rare sugars from maltodextrin., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Tagatose is a rare hexoketose with potential health benefits. Here, an enzyme, GatZ subunit ofd-tagatose-1,6-bisphosphate aldolase, was characterized. GatZ is involved in a multi-enzyme cascade reaction system that can produce tagatose from maltodextrin. It showed maximum activity at 70 °C and a pH 8.0, and required supplementation with 5 mM Mg 2+ to achieve the highest catalytic activity. The K m and V max values of GatZ using fructose 6-phosphate as substrate were 5.66 mM and 0.0329 mmol/L min, respectively. An in vitro multi-enzyme system containing GatZ was constructed, and 1.75 g/L tagatose was produced from 5 g/L maltodextrin after 10 h. This biosystem could potentially enrich the application of C4 epimerases in rare sugar bioproduction., (Copyright © 2020 Elsevier Inc. All rights reserved.)