The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform

Olga Bortolini - One of the best experts on this subject based on the ideXlab platform.

Giancarlo Fantin - One of the best experts on this subject based on the ideXlab platform.

Marco Fogagnolo - One of the best experts on this subject based on the ideXlab platform.

Dirk Weuster-botz - One of the best experts on this subject based on the ideXlab platform.

  • Dynamic mechanistic modeling of the multienzymatic one‐pot reduction of Dehydrocholic Acid to 12‐keto ursodeoxycholic Acid with competing substrates and cofactors
    Biotechnology progress, 2015
    Co-Authors: Boqiao Sun, Florian Hartl, Kathrin Castiglione, Dirk Weuster-botz
    Abstract:

    Ursodeoxycholic Acid (UDCA) is a bile Acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of Dehydrocholic Acid to 12-keto-ursodeoxycholic Acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile Acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile Acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions.

  • Novel whole-cell biocatalysts with recombinant hydroxysteroid dehydrogenases for the asymmetric reduction of Dehydrocholic Acid
    Applied Microbiology and Biotechnology, 2012
    Co-Authors: Michael Braun, Boqiao Sun, Bernd Anselment, Dirk Weuster-botz
    Abstract:

    Ursodeoxycholic Acid is an important pharmaceutical so far chemically synthesized from cholic Acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of Dehydrocholic Acid to 3,12-diketo-ursodeoxycholic Acid was observed with the selected recombinant E. coli strain. In the optimal, slightly Acidic pH range Dehydrocholic Acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial Dehydrocholic Acid concentration, and initial formate concentration. Complete conversion of 72 mM Dehydrocholic Acid was thus made possible at pH 6.4 in a whole-cell batch process within a process time of 1 h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50 mM Dehydrocholic Acid directly to 12-keto-ursodeoxycholic Acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic Acid.

  • One-step synthesis of 12-ketoursodeoxycholic Acid from Dehydrocholic Acid using a multienzymatic system.
    Applied microbiology and biotechnology, 2012
    Co-Authors: Luo Liu, Michael Braun, Dirk Weuster-botz, Gabi Gebhardt, Ralf Gross, Rolf D. Schmid
    Abstract:

    12-ketoursodeoxycholic Acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic Acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of Dehydrocholic Acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic Acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.

Boqiao Sun - One of the best experts on this subject based on the ideXlab platform.

  • dynamic mechanistic modeling of the multienzymatic one pot reduction of Dehydrocholic Acid to 12 keto ursodeoxycholic Acid with competing substrates and cofactors
    Biotechnology Progress, 2015
    Co-Authors: Boqiao Sun, Florian Hartl, Kathrin Castiglione, Dirk Weusterbotz
    Abstract:

    Ursodeoxycholic Acid (UDCA) is a bile Acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of Dehydrocholic Acid to 12-keto-ursodeoxycholic Acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile Acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile Acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions.

  • Dynamic mechanistic modeling of the multienzymatic one‐pot reduction of Dehydrocholic Acid to 12‐keto ursodeoxycholic Acid with competing substrates and cofactors
    Biotechnology progress, 2015
    Co-Authors: Boqiao Sun, Florian Hartl, Kathrin Castiglione, Dirk Weuster-botz
    Abstract:

    Ursodeoxycholic Acid (UDCA) is a bile Acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of Dehydrocholic Acid to 12-keto-ursodeoxycholic Acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile Acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile Acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions.

  • Novel whole-cell biocatalysts with recombinant hydroxysteroid dehydrogenases for the asymmetric reduction of Dehydrocholic Acid
    Applied Microbiology and Biotechnology, 2012
    Co-Authors: Michael Braun, Boqiao Sun, Bernd Anselment, Dirk Weuster-botz
    Abstract:

    Ursodeoxycholic Acid is an important pharmaceutical so far chemically synthesized from cholic Acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of Dehydrocholic Acid to 3,12-diketo-ursodeoxycholic Acid was observed with the selected recombinant E. coli strain. In the optimal, slightly Acidic pH range Dehydrocholic Acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial Dehydrocholic Acid concentration, and initial formate concentration. Complete conversion of 72 mM Dehydrocholic Acid was thus made possible at pH 6.4 in a whole-cell batch process within a process time of 1 h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50 mM Dehydrocholic Acid directly to 12-keto-ursodeoxycholic Acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic Acid.