Mutated Sucrose Phosphorylase Kojibiose Selectivity
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Solution Overview
Problem
Current methods for producing kojibiose are inefficient and costly due to low yields and the generation of product mixtures, with existing enzymes having low selectivity for kojibiose production and requiring multi-step chemical processes or the use of hazardous reagents.
Innovation Solution
Genetically modified sucrose phosphorylases with specific mutations, such as L341I_Q345S or L341I_Y344A_Q345N, are used to convert sucrose into kojibiose with high selectivity and purity through a transglycosylation reaction, followed by a yeast treatment for purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type sucrose phosphorylase is used for transglycosylation with glucose, then kojibiose is produced, but maltose side-products are formed reducing overall yield
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the sucrose phosphorylase sequence (positions 134, 135, 193, 234, 341, 342, 343, 344, 345) to alter the enzyme's catalytic properties. These mutations change the enzyme's substrate binding characteristics and transition state stabilization, thereby improving selectivity for kojibiose over maltose while maintaining high productivity.
2Manufacturing precision
If chemical synthesis methods are used to produce kojibiose, then pure product can be obtained, but multiple hazardous reagents and multi-step processes are required
Solution Approach 1:
The patent replaces complex chemical synthesis mechanisms with a simplified enzymatic catalysis system. The genetically modified sucrose phosphorylase performs the transglycosylation reaction in a single step under mild conditions, eliminating the need for multiple chemical protection/deprotection steps, hazardous reagents, and complex purification procedures required in traditional chemical synthesis.
3Productivity
If other enzymes like α-glucosidase or glucansucrase are used, then kojibiose can be produced, but yields are low and product mixtures are generated
Solution Approach 1:
The patent optimizes the enzymatic parameters by engineering sucrose phosphorylase with specific amino acid mutations that enhance its catalytic efficiency for kojibiose production. The mutated enzyme exhibits improved substrate binding affinity and transition state stabilization specifically for the kojibiose-forming reaction, achieving both high yield and high selectivity unlike other enzymes.
4Manufacturing precision
If isolation from partial acetolysis of dextran is used, then kojibiose can be obtained, but a multi-step process with several chemical reagents is involved
Solution Approach 1:
The patent replaces the complex chemical isolation process from dextran acetolysis with a straightforward enzymatic synthesis approach. The genetically modified sucrose phosphorylase directly converts sucrose and glucose into kojibiose in a single reaction step, eliminating multiple chemical treatment steps including acetolysis, hydrolysis, and extensive purification procedures.
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
The modified sucrose phosphorylases achieve a high activity ratio of kojibiose to maltose formation, allowing for the production of highly pure kojibiose with increased yield and reduced side-products, making the process more efficient and cost-effective.
Implementation Method 1
genetically modified sucrose phosphorylases with specific mutations, such as L341I_Q345S or L341I_Y344A_Q345N, are used to convert sucrose into kojibiose with high selectivity and purity through a transglycosylation reaction
Implementation Method 2
followed by a yeast treatment for purification
Data Source
AI summary
The present invention relates to the production of the disaccharide kojibiose which is known to be a powerful prebiotic. The invention indeed discloses the generation of genetically modified sucrose phosphorylases which convert—via a transglycosylation reaction—sucrose into kojibiose in a very efficient manner. Hence, the present invention relates to a cost-effective production method of kojibiose which is useful within industry.