Coupled Biocatalytic Conversion of CDCA to UDCA
Solution Overview
Problem
Current methods for biocatalytic preparation of ursodeoxycholic acid (UDCA) and related compounds face challenges due to substrate inhibition, incomplete enzymatic oxidation, and complex cofactor regeneration processes, leading to inefficiencies and increased costs.
Innovation Solution
A coupled, self-sufficient biocatalytic process using novel combinations of 7α-HSDH and 7β-HSDH enzymes that utilize the same cofactor system, such as NAD+/NADH or NADP+/NADPH, to achieve complete conversion of chenodeoxycholic acid (CDCA) to UDCA, with enzymes like the NADP+-dependent 7α-HSDH from Clostridium difficile and mutants of Escherichia coli 7α-HSDH being used for enhanced activity and reduced substrate inhibition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional 7α-HSDH enzymes are used for biocatalytic preparation of UDCA, then the oxidation reaction can proceed, but substrate inhibition occurs leading to incomplete conversion
Solution Approach 1:
The patent changes the cofactor parameter from NAD+ to NADP+ in the 7α-HSDH enzyme system. This parameter change eliminates substrate inhibition and enables complete conversion of CDCA to UDCA, resolving the contradiction between productivity and reliability.
2Stability of the object's composition
If complex cofactor regeneration processes are used, then cofactor availability is maintained, but process complexity and costs increase
Solution Approach 1:
The patent employs a self-sufficient cofactor regeneration system where the 7β-HSDH enzyme automatically regenerates NADP+ from NADPH during the reduction step. This eliminates the need for external complex regeneration processes while maintaining cofactor availability throughout the coupled reaction.
3Productivity
If multiple different cofactor systems are used in the enzymatic process, then enzyme activity can be optimized, but cofactor regeneration becomes more complex
Solution Approach 1:
The patent achieves universality by selecting a pair of enzymes (7α-HSDH and 7β-HSDH) that both operate with the same NADP+/NADPH cofactor system. This allows a single cofactor regeneration mechanism to serve both oxidation and reduction steps, simplifying the overall system while maintaining high enzyme activity.
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 a more efficient and cost-effective biocatalytic conversion of CDCA to UDCA with improved enzyme activity and reduced substrate inhibition, allowing for complete reaction completion and simplified cofactor regeneration.
Implementation Method 1
a) said 7α-HSDH catalyzes the oxidation of said CDCA compound of general Formula (2) to the corresponding intermediate 7-KLCA compound of general Formula (3)
Implementation Method 2
In one aspect, a gene encoding a novel 7α-specific NADP+-dependent HSDH from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized.
Implementation Method 3
a2) said 7β-HSDH catalyzes the reduction of said 7-KLCA compound of general Formula (3) as formed in reaction step a) to said UDCA compound of general Formula (1) and under regeneration of the cofactor consumed in reaction step a)
Implementation Method 4
wo 2011/064404 describes a novel 7β-HSDH from Collinsella aerofaciens ATCC 25986
Implementation Method 5
a coupled, self-sufficient biocatalytic process using novel combinations of 7α-HSDH and 7β-HSDH enzymes that utilize the same cofactor system, such as NAD+/NADH or NADP+/NADPH, to achieve complete conversion of chenodeoxycholic acid (CDCA) to UDCA
Data Source
AI summary
The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP+-dependent 7α-HSDH of E. coli and their application for the oxidation of bile acids.