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Bioprocess development for the syntheses of selected phosphorylated metabolites
Citation Link: https://doi.org/10.15480/882.1386
Publikationstyp
Doctoral Thesis
Publikationsdatum
2017
Sprache
English
Author
Advisor
Referee
Streit, Wolfgang
Title Granting Institution
Technische Universität Hamburg-Harburg
Place of Title Granting Institution
Hamburg
Examination Date
2016-07-26
Institut
An in vitro synthesis of optically pure phosphorylated metabolites is useful in various natural biological and synthetic processes. The aim of this thesis was to develop bioprocesses for the syntheses of sn-glycerol-3-phosphate (sn-G3P), L-glyceraldehyde-3-phosphate (L-GAP) and
D-glyceraldehyde-3-phosphate (D-GAP).
Asymmetric phosphorylation of glycerol catalyzed by glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) utilizing ATP as a phosphoryl donor and Mg2+ as an essential activator was used for the synthesis of sn-G3P. This enzyme exhibits maximum activity at the optimum Mg2+ to ATP molar ratio of [0.12 to 0.3]. The enzyme shows Michaelis-Menten kinetics with respect to glycerol as well as ATP maintaining constant Mg2+ to ATP molar ratio and two-step kinetics with respect to ATP at a fixed concentration of Mg2+. Detailed kinetics and mechanistic analyses were performed applying 31P and 1H NMR. The two-step kinetics with respect to ATP at the fixed Mg2+ concentration is due to the formation of multiple Mg-ATP complexes. The active site of glycerol kinase exhibits different catalytic property with respect to different Mg-ATP complex species. Validation of reaction kinetics models shows 96.8% and 98.8% 2-D correlation of experimental and numerically simulated data matrices.
Glycerol kinase from Cellulomonas sp. catalyzed phosphorylation of L-glyceraldehyde by ATP was developed for the synthesis of L-GAP. L-GAP decomposes at the reaction of pH 8 and shows a half-life of 6.86 h. The enzyme exhibits maximum activity at the optimum Mg2+ to ATP molar ratio of 0.7. Validation of reaction kinetics model shows 99.9% 2-D correlation of experimental and numerically simulated data matrices. Experimental and numerical evaluations of different reactor types reveal that batch-wise operation is the most convenient process for the synthesis of L-GAP.
A one-pot enzymatic reaction sequence was designed for the synthesis of D-GAP using
fructose-1,6-bisphosphate aldolase from rabbit muscle (RAMA) (EC 4.1.2.13),
sn-glycerol-3-phosphate dehydrogenase (sn-G3PDH) from rabbit muscle (EC 1.1.1.8) and formate dehydrogenase from Candida boidinii (FDH) (EC 1.2.1.2). The reaction sequence circumvents the thermodynamic limitation of aldol cleavage of D-fructose-1,6-bisphosphate
(D-F16BP) that leads to 100% conversion of D-F16BP. A reaction kinetics model defining the entire reaction cascade was developed and validation of the model shows 98.5% 2-D correlation of experimental and numerically simulated data matrices. The evaluation of different reactor types was performed. Batch-wise operation in STR is the most convenient procedure for the
one-pot enzymatic synthesis of D-GAP.
D-glyceraldehyde-3-phosphate (D-GAP).
Asymmetric phosphorylation of glycerol catalyzed by glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) utilizing ATP as a phosphoryl donor and Mg2+ as an essential activator was used for the synthesis of sn-G3P. This enzyme exhibits maximum activity at the optimum Mg2+ to ATP molar ratio of [0.12 to 0.3]. The enzyme shows Michaelis-Menten kinetics with respect to glycerol as well as ATP maintaining constant Mg2+ to ATP molar ratio and two-step kinetics with respect to ATP at a fixed concentration of Mg2+. Detailed kinetics and mechanistic analyses were performed applying 31P and 1H NMR. The two-step kinetics with respect to ATP at the fixed Mg2+ concentration is due to the formation of multiple Mg-ATP complexes. The active site of glycerol kinase exhibits different catalytic property with respect to different Mg-ATP complex species. Validation of reaction kinetics models shows 96.8% and 98.8% 2-D correlation of experimental and numerically simulated data matrices.
Glycerol kinase from Cellulomonas sp. catalyzed phosphorylation of L-glyceraldehyde by ATP was developed for the synthesis of L-GAP. L-GAP decomposes at the reaction of pH 8 and shows a half-life of 6.86 h. The enzyme exhibits maximum activity at the optimum Mg2+ to ATP molar ratio of 0.7. Validation of reaction kinetics model shows 99.9% 2-D correlation of experimental and numerically simulated data matrices. Experimental and numerical evaluations of different reactor types reveal that batch-wise operation is the most convenient process for the synthesis of L-GAP.
A one-pot enzymatic reaction sequence was designed for the synthesis of D-GAP using
fructose-1,6-bisphosphate aldolase from rabbit muscle (RAMA) (EC 4.1.2.13),
sn-glycerol-3-phosphate dehydrogenase (sn-G3PDH) from rabbit muscle (EC 1.1.1.8) and formate dehydrogenase from Candida boidinii (FDH) (EC 1.2.1.2). The reaction sequence circumvents the thermodynamic limitation of aldol cleavage of D-fructose-1,6-bisphosphate
(D-F16BP) that leads to 100% conversion of D-F16BP. A reaction kinetics model defining the entire reaction cascade was developed and validation of the model shows 98.5% 2-D correlation of experimental and numerically simulated data matrices. The evaluation of different reactor types was performed. Batch-wise operation in STR is the most convenient procedure for the
one-pot enzymatic synthesis of D-GAP.
Schlagworte
Synthesis of sn-glycerol-3-phosphate
Synthesis of L-glyceraldehyde-3-phosphate
Synthesis of D-glyceraldehyde-3-phosphate
Mechanistic elucidation of the Mg2+ to ATP ratio effect by NMR
In situ ATP regeneration
DDC Class
540: Chemie
More Funding Information
Federal Ministry of Education and Research of Germany (BMBF), P28, project number: 0316055)
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