LAUSR

Phosphatases are a class of enzymes catalyzing the cleavage of monophosphate ester bonds from the phosphorylated substrates. They have important applications in construction of in vitro multi‐enzymatic system for monosaccharides. However, the enzymes generally show substrate ambiguity, which has become a bottleneck for efficient biosynthesis of target products with high purity. In this study, semirational design was performed on phosphatase from Thermosipho atlanticus (Ta‐PST). The hotspot amino acid residues forming a “cap domain” were identified and selected for saturation mutagenesis. The mutant F179T and F179M showed improved substrate preference toward fructose‐6‐phosphate and mannose‐6‐phosphate, respectively. Coupling with other enzymes involved in the multi‐enzymatic system under optimized conditions, the application of F179T led to fructose yield of 80% from 10 g/L maltodextrin and the ratio between the target product and by‐product glucose was increased from 2:1 to 19:1. On the other hand, the application of F179M led to mannose yield of 59% with ratio of mannose to the by‐products glucose and fructose increased from 1:1:1 to 14:2:1. Moreover, the molecular understanding of the beneficial substitution was gained by structural analysis and molecular dynamic simulations, giving important guidance to regulate the enzyme's substrate preference.