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Phosphatase of the haloacid dehalogenase ( HAD) -like hydrolase superfamily has a broad substrate spectrum and diverse catalytic functions. Identification and characterization of thermostable phosphatases provides great potential for exploring their applications in biochemical production. Herein, the phosphatase from Acidothermus cellulolyticus (AcPase) was investigated. The gene encoding AcPase was codon-optimized and synthesized, and was then transformed into E. coli Rosetta ( DE3 ). The recombinant enzyme was subsequently expressed and purified by His Trap affinity chromatography. Studies of enzymatic properties revealed that AcPase was a Mg2+-dependent HAD-like phosphatase which functioned optimally at 65 X and pH 6. 0. AcPase was thermostable at 45-65 X,with a half-life of 17. 4 h at 45 X. At 50 X,AcPase exhibited a relatively high specific activity for D-glucose-6-phosphate (0. 68 U/mg). An in vitro synthetic enzymatic biosystem containing four enzymes including AcPase was therefore constructed to transform cellulose into glucose in one pot,achieving a yield of 97. 6%. To sum up, based on the characterization of a phosphatase which prefers D-glucose-6-phosphatase, this study suggests a new strategy for the complete conversion of cellulose. [ABSTRACT FROM AUTHOR]

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis , was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli , and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D -tagatose 6-phosphate, and was relatively specific for this substrate than for D -glucose 1-phosphate, D -glucose 6-phosphate, and D -fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D -tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D -tagatose in the future. • We examined the enzymatic properties of a putative phosphatase from M. marburgensis. • This enzyme was thermostable and relatively specific for D -tagatose 6-phosphate. • This enzyme was applied in an in vitro synthetic biosystem to produce D -tagatose. [ABSTRACT FROM AUTHOR]