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Regulating substrate preference of phosphatase by reshaping the "cap domain" for multi-enzymatic biosynthesis of high-purity monosaccharides. | LitMetric

Regulating substrate preference of phosphatase by reshaping the "cap domain" for multi-enzymatic biosynthesis of high-purity monosaccharides.

Biotechnol Bioeng

Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.

Published: December 2022

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.

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Source
http://dx.doi.org/10.1002/bit.28244DOI Listing

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