Biphasic Enzymatic Reduction of Bile Acids
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Solution Overview
Problem
Current methods for industrial production of ursodeoxycholic acid (UDCA) face challenges such as low yields, stereoselectivity issues, and enzymatic inactivation due to gel formation and substrate concentration limitations, making it difficult to achieve quantitative conversions at economically viable scales.
Innovation Solution
A regioselective enzymatic reduction process using NAD(P)H-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) in a biphasic system with a glucose dehydrogenase (GDH) regeneration system and specific organic solvents, optimizing enzyme activities, substrate concentrations, temperature, and pH to prevent gel formation and achieve high conversions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If substrate concentration is increased to achieve economically viable production, then productivity improves, but gel formation occurs causing enzymatic inactivation
Solution Approach 1:
The reaction system is divided into two separate phases: an aqueous phase containing the enzyme and a organic phase containing the substrate. This segmentation prevents gel formation by keeping the substrate in the organic phase while the enzyme operates in the aqueous phase, allowing high substrate concentrations without enzymatic inactivation.
Solution Approach 2:
A biphasic system acts as an intermediary between the enzyme and substrate, allowing them to interact without direct contact that would cause gel formation. The interface between phases enables substrate transfer to the enzyme without requiring high concentrations in the aqueous phase.
2Productivity
If chemical reduction methods are used, then production scale can be increased, but stereoselectivity decreases leading to mixture formation
Solution Approach 1:
The enzyme catalyzes the reduction reaction with inherent stereoselectivity, making the process self-regulating for producing the desired stereoisomer. The biological catalyst naturally discriminates between stereoisomers, providing high manufacturing precision without requiring complex additional separation steps.
3Productivity
If substrate concentration is increased above 50 mM, then productivity improves, but complete gelification occurs impeding reaction progression
Solution Approach 1:
The system changes the physical state parameters by introducing a biphasic configuration. The substrate concentration in the organic phase can be increased above 50 mM without causing gelification, as the organic solvent prevents the formation of gel structures while still allowing enzyme access to the substrate at the phase interface.
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 process allows for quantitative conversions of 7-keto-lithocholic acid, 7-keto-deoxycholic acid methyl ester, and 7,12-diketo-lithocholic acid methyl ester to UDCA, overcoming previous limitations by maintaining enzymatic activity and preventing gelation, enabling industrial-scale production.
Implementation Method 1
regioselective enzymatic reduction of bile acids, their salts or derivatives... using NAD(P)H-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH)
Implementation Method 2
The HSDH activities demonstrate an almost absolute regioselectivity and stereoselectivity, and can therefore be used to catalyse reactions without resulting in preventive protection of the hydroxyl groups
Implementation Method 3
oxidation of glucose to give glucono-δ-lactone, which spontaneously hydrolyses to give gluconic acid, catalysed by glucose dehydrogenase (GDH)
Implementation Method 4
glucono-δ-lactone, which spontaneously hydrolyses to give gluconic acid
Implementation Method 5
the reaction mixture will be subject to complete gelification... The formation of the gel seems to be caused both by a natural tendency of the bile acids, and in particular of UDCA, to form supramolecular structures
Implementation Method 6
the presence in the reaction environment of the glucose/gluconic acid co-substrates/co-products, which, due to their pronounced hydrophilicity, tend to remove water molecules from the other solutes present
Implementation Method 7
regioselective enzymatic reduction of bile acids... in a biphasic system... making possible to dissolve the starting substrates in elevated quantities, avoid the formation of gel
Data Source
AI summary
New process for the stereo- and regioselective enzymatic reduction of bile acids, their salts or derivatives, such as 7-keto-lithocholic acid, 7- ketodesoxycholic acid methyl ester, or 7,12 diketolithocholic acid methyl ester, in a buffer/organic solvent biphasic system by the action of NAD (P) H-dependent 7β-hydroxysteroid dehydrogenase (7β- HSDH) in the presence of a glucose dehydrogenase (GDH) and glucose as co- substrate for the regeneration of the NAD ( P) H cofactor.