Alpha-glucose-1-phosphatase Enzymatic Transphosphorylation
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Solution Overview
Problem
Current methods for biocatalytic synthesis of sugar phosphates face limitations due to restricted substrate scope, high costs of phospho-activated donor substrates, and enzyme inhibition, leading to inefficient production and limited use in sugar phosphate synthesis.
Innovation Solution
The use of an enzyme with alpha-glucose-1-phosphatase activity for enzymatic transphosphorylation reactions between various phosphate donors and sugar substrates, allowing for flexible production of sugar phosphates with improved yields and reduced secondary hydrolysis of products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If phosphotransferase reactions are used for sugar phosphate synthesis, then a large diversity of sugar phosphate products can be accessed, but high costs of phospho-activated donor substrate and enzyme inhibition by dephosphorylation product necessitate continuous regeneration of the phosphoryl donor
Solution Approach 1:
The patent introduces phosphatase as an intermediary enzyme that catalyzes transphosphorylation reactions using inexpensive inorganic phosphate donors. This mediator enables direct phosphorylation of sugar substrates without requiring complex phospho-activated donors like ATP, thereby simplifying the overall process while maintaining versatility in sugar phosphate production
Solution Approach 2:
The patent changes the chemical parameters of the phosphoryl donor from complex organic phosphates (ATP, GTP) to simple inorganic phosphate. This parameter change reduces cost and eliminates the need for continuous cofactor regeneration systems, simplifying the process while maintaining product diversity through enzyme specificity
2Quantity of substance
If phosphatase-catalyzed transphosphorylation is used, then inexpensive phosphoryl donor substrates can be used, but high preponderance of donor substrate hydrolysis and fast secondary hydrolysis of sugar phosphate product occur
Solution Approach 1:
The patent employs dynamic control of reaction conditions including pH optimization and temperature control to favor transphosphorylation over hydrolysis. By adjusting these parameters, the system dynamically shifts the reaction equilibrium toward product formation while minimizing unwanted hydrolysis of both donor substrate and product
Solution Approach 2:
The patent implements continuous removal of product from the reaction mixture through various methods (extraction, precipitation, or coupled enzymatic reactions). This continuous action prevents secondary hydrolysis by maintaining low product concentration, thereby reducing product loss while sustaining high donor substrate efficiency
3Manufacturing precision
If glycoside phosphorylase reactions are used, then phosphorylation occurs exclusively at the sugar's anomeric position, but the narrow substrate scope restricts production to just a few glycosyl phosphates
Solution Approach 1:
The patent utilizes phosphatase enzymes with broad substrate specificity that can accept diverse sugar substrates and produce phosphorylated products at various positions. This universal enzyme catalyzes transphosphorylation reactions for multiple sugar types (hexoses, pentoses, amino sugars) while maintaining position specificity through enzyme active site geometry, thereby achieving both precision and versatility
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient and flexible production of sugar phosphates with high yields, overcoming the limitations of existing methods by utilizing a broad range of phosphate donors and sugar substrates, and minimizing product loss through secondary hydrolysis.
Implementation Method 1
enzymatic transphosphorylation comprising the following steps: (i) providing one or more sugar substrates, (ii) providing one or more phosphate donor substrates, (iii) providing an alpha-glucose-1-phosphatase or an enzyme having alpha-glucose-1-phosphatase activity, (iv) incubating the one or more sugar substrates provided in step (i) and the one or more phosphate donor substrates provided in step (ii) with the alpha-glucose-1-phosphatase or, respectively, the enzyme having alpha-glucose-1-phosphatase activity provided in step (iii) under conditions which allow transphosphorylation
Implementation Method 2
The use of an enzyme with alpha-glucose-1-phosphatase activity for enzymatic transphosphorylation reactions between various phosphate donors and sugar substrates
Data Source
AI summary
The present invention relates to methods of producing sugar phosphates by enzymatic transphosphorylation, wherein the phosphoryl transfer from phosphate donor substrates to sugar substrates is catalyzed by an enzyme having alpha-glucose-1-phosphatase activity. Furthermore, the present invention relates to a biocatalyst having sucrose phosphorylase as well as alpha-glucose-1-phosphatase activity and its use. The production of an enzyme having alpha-glucose-1-phosphatase activity and its use in the transphosphorylation of sugar substrates is also presented herein.