A natural supply chain: Enzymatic synthesis of ... - Poster Chains 2017.pdf · (UDCA) is the...
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CDCA CA A natural supply chain: Enzymatic synthesis of ursodeoxycholic acid (UDCA) Protein Specific activity Purification Yield (mg) (U/mg) (fold) (%) Cell extract 1050 233.2 - 100.0 HiTrap chelating 232.3 430.4 1.8 40.8 V max (U/mg) k cat (s -1 ) K m (mM) K i (mM) V max /K m CA 278.2 ± 9.6 519.3 ± 17.9 0.960 ± 0.110 / 289.8 ± 43.2 CDCA 488.7 ± 19.1 912.2 ± 35.7 0.218 ± 0.024 11 ± 1.6 2241.7 ± 334.4 NAD + 528.4 ± 5.1 986.3 ± 9.5 0.560 ± 0.017 / 943.6 ± 37.8 NADP + NAD + Vmax (U/mg) Km (mM) Vmax/Km Preference Vmax (U/mg) Km (mM) Vmax/Km Preference WT 0.49 0.012 40.833 408 0.11 1.1 0.100 0.002 ADLN variant 0.01 2.600 0.004 0.019 0.47 2.34 0.201 52.2 In the European project ONE-FLOW (www.one-flow.org) the objective is to develop sustainable catalytic cascade processes in flow-reactors for pharmaceuticals. Here ursodeoxycholic acid (UDCA) is the subject of study. As bile acid it solubilizes cholesterol gallstones and improves liver function in cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. This transformation requires the use of toxic and dangerous reagents, resulting in an overall yield of about 30%. For that reason, studies have been performed on the development of microbial transformations or chemo- enzymatic procedures for the synthesis of UDCA employing CA or chenodeoxycholic acid (CDCA) [1,2]. In particular, we focused on the enzymatic epimerization of CDCA, that represent a fundamental step for a green production of UDCA. HO OH OH O HO OH OH O OH Sm7α-HSDS Cs7β-HSDS A novel NAD + dependent 7α-HSDH from Stenotrophomonas maltophilia was recombinantly expressed, purified and biochemically characterized. This enzyme shows good properties in terms of activity (430 U/mg protein on CDCA as substrate) and stability to cosolvents. Purification of recombinant His-tagged Sm7α-HSDH from E. coli BL21(DE3) cells (7 g corresponding to 1.0 L of fermentation broth). Kinetic parameters of purified recombinant Sm7α-HSDH The NADP + dependent Clostridium sardiniense 7β-HSDH was engineered in order to change the cofactor specificity: VARIANT DESIGN MUTAGENESIS SCREENING ADLN VARIANT Changes cofactor specificity Time (min) Conversion yield (%) Conclusions: • A new compatible set of NAD + dependent enzymes (both recombinant) was developed; • Cs7β-HSDH was succefully engineered for the change of cosubstrate specificity (from NADP + to NAD + ); • Preliminary bioconversions have demonstrated the feasibility of an enzymatic process for UDCA production. Preliminary Bioconversion: Fabio Tonin 1 and Isabel W.C.E. Arends* 1 1 Department of Biotechnology, TU Delft, Van der Maasweg 9, 2629 HZ Delft, The Netherlands. E-mail: [email protected] 2.0 μmol of CDCA (■) in presence of 2.0 μmol of NAD + , were converted in the desired product (UDCA (▲) - 60.0% yield) in 40 min by 1 U of each enzyme. 38% of keto intermediate (7-oxo-LCA (●)) was also obtained. These preliminary data show that the equilibrium of reaction and/or the product inhibition are the main obstacles for the use of this set of enzymes as biocatalyst. a Activity was assayed on 1.25 mM CDCA and 2.5 mM NAD + as substrate in 50 mM KPi buffer, pH 8.0. a Values were calculated considering the homo-tetrameric structure of the enzyme, with a total molecular weight of 112 kDa. 0.0 20.0 40.0 60.0 80.0 100.0 0 50 100 150 200 250 Cholic acid Chenodeoxycholic acid No cofactor regeneration is required[3] HO OH OH O HO O OH O HO OH OH O NADH NADH NAD + NAD + CDCA UDCA 7-oxo-LCA 7α-HSDS 7β-HSDS References: 1. Eggert, T., D. Bakonyi, and W. Hummel, Enzymatic routes for the synthesis of UDCA. Journal of biotechnology, 2014. 191: p. 11-21. 2. Tonin, F., L.G. Otten, and I.W. Arends, Mini-review: bottlenecks in the synthesis of ursodeoxycholic acid (UDCA). In preparation, 2018. 3. Hollmann, F., I.W. Arends, and K. Buehler, Biocatalytic redox reactions for organic synthesis: nonconventional regeneration methods. ChemCatChem, 2010. 2(7): p. 762-782. NADP + NAD + Combination of: • Site saturation mutagenesis • Site directed mutagenesis
Transcript of A natural supply chain: Enzymatic synthesis of ... - Poster Chains 2017.pdf · (UDCA) is the...
CDCA CA
A natural supply chain: Enzymatic synthesis of ursodeoxycholic acid (UDCA)
Protein Specific activity Purification Yield (mg) (U/mg) (fold) (%)
Cell extract 1050 233.2 - 100.0 HiTrap chelating 232.3 430.4 1.8 40.8
Vmax (U/mg) kcat (s-1) Km (mM) Ki (mM) Vmax/Km CA 278.2 ± 9.6 519.3 ± 17.9 0.960 ± 0.110 / 289.8 ± 43.2
CDCA 488.7 ± 19.1 912.2 ± 35.7 0.218 ± 0.024 11 ± 1.6 2241.7 ± 334.4 NAD+ 528.4 ± 5.1 986.3 ± 9.5 0.560 ± 0.017 / 943.6 ± 37.8
NADP+ NAD+
Vmax (U/mg)
Km (mM) Vmax/Km Preference Vmax
(U/mg) Km
(mM) Vmax/Km Preference
WT 0.49 0.012 40.833 408 0.11 1.1 0.100 0.002 ADLN variant 0.01 2.600 0.004 0.019 0.47 2.34 0.201 52.2
In the European project ONE-FLOW (www.one-flow.org) the objective is to develop sustainable catalytic cascade processes in flow-reactors for pharmaceuticals. Here ursodeoxycholic acid (UDCA) is the subject of study. As bile acid it solubilizes cholesterol gallstones and improves liver function in cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. This transformation requires the use of toxic and dangerous reagents, resulting in an overall yield of about 30%. For that reason, studies have been performed on the development of microbial transformations or chemo-enzymatic procedures for the synthesis of UDCA employing CA or chenodeoxycholic acid (CDCA) [1,2]. In particular, we focused on the enzymatic epimerization of CDCA, that represent a fundamental step for a green production of UDCA.
HO OH
OH
O
HO OH
OH
OOH
Sm7α-HSDS Cs7β-HSDS A novel NAD+ dependent 7α-HSDH from Stenotrophomonas maltophilia was recombinantly expressed, purified and biochemically characterized. This enzyme shows good properties in terms of activity (430 U/mgprotein on CDCA as substrate) and stability to cosolvents.
Purification of recombinant His-tagged Sm7α-HSDH from E. coli BL21(DE3) cells (7 g corresponding to 1.0 L of fermentation broth).
Kinetic parameters of purified recombinant Sm7α-HSDH
The NADP+ dependent Clostridium sardiniense 7β-HSDH was engineered in order to change the cofactor specificity:
VARIANT DESIGN
MUTAGENESIS
SCREENING ADLN VARIANT
Changes cofactor specificity
Time (min)
Conv
ersio
n yi
eld
(%) Conclusions:
• A new compatible set of NAD+ dependent enzymes (both recombinant) was developed;
• Cs7β-HSDH was succefully engineered for the change of cosubstrate specificity (from NADP+ to NAD+);
• Preliminary bioconversions have demonstrated the feasibility of an enzymatic process for UDCA production.
Preliminary Bioconversion:
Fabio Tonin1 and Isabel W.C.E. Arends*1
1Department of Biotechnology, TU Delft, Van der Maasweg 9, 2629 HZ Delft, The Netherlands. E-mail: [email protected]
2.0 μmol of CDCA (■) in presence of 2.0 μmol of NAD+, were converted in the desired product (UDCA (▲) - 60.0% yield) in 40 min by 1 U of each enzyme. 38% of keto intermediate (7-oxo-LCA (●)) was also obtained. These preliminary data show that the equilibrium of reaction and/or the product inhibition are the main obstacles for the use of this set of enzymes as biocatalyst.
aActivity was assayed on 1.25 mM CDCA and 2.5 mM NAD+ as substrate in 50 mM KPi buffer, pH 8.0.
aValues were calculated considering the homo-tetrameric structure of the enzyme, with a total molecular weight of 112 kDa.
0.0
20.0
40.0
60.0
80.0
100.0
0 50 100 150 200 250
Cholic acid Chenodeoxycholic acid
No cofactor regeneration is required[3]
HO OH
OH
O
HO O
OH
O
HO OH
OH
O
NADH NADH NAD+ NAD+ CDCA UDCA
7-oxo-LCA
7α-HSDS 7β-HSDS
References: 1. Eggert, T., D. Bakonyi, and W. Hummel, Enzymatic routes for the synthesis of UDCA. Journal of biotechnology, 2014. 191: p. 11-21. 2. Tonin, F., L.G. Otten, and I.W. Arends, Mini-review: bottlenecks in the synthesis of ursodeoxycholic acid (UDCA). In preparation, 2018. 3. Hollmann, F., I.W. Arends, and K. Buehler, Biocatalytic redox reactions for organic synthesis: nonconventional regeneration methods. ChemCatChem, 2010. 2(7): p. 762-782.
NADP+ NAD+
Combination of: • Site saturation mutagenesis • Site directed mutagenesis
