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Item type:Publication, Access status: without files , Mechanistic and kinetics elucidation of Mg2+/ATP molar ratio effect on glycerol kinaseDetailed kinetics and mechanistic analyses of glycerol kinase from Cellulomonas sp. with respect to Mg2+ to ATP molar ratio were performed. The enzyme essentially requires Mg2+ for its activity and shows maximum activity at the optimum Mg2+ to ATP molar ratio of [0.12–0.3]. Subsequent increase of Mg2+ to ATP molar ratio higher than the values in the optimum region suppresses the enzyme activity to a non-zero asymptotic value. The enzyme exhibits two-step kinetics as a function of ATP at a fixed Mg2+ concentration due to the formation of multiple Mg-ATP complexes at different Mg2+ to ATP molar ratio. The addition of inorganic polyphosphate (PPin) inhibits or activates glycerol kinase due to the complexation of PPin with Mg2+ that shifts Mg2+ to ATP molar ratio below or to the optimum level. The change in all 31P NMR signals of ATP (i.e. α-, β- and γ-phosphate) by the addition of Mg2+ reveals that all of them are involved in Mg-ATP complex formation. The 1H NMR signals of CH2-protons on the ribose moiety of ATP become nearly equivalent after the addition of Mg2+ establishing the upper limit of optimum Mg2+ to ATP molar ratio for the enzyme activity. Therefore, it is concluded that the active site of glycerol kinase shows different catalytic property with respect to different Mg-ATP complexes. Glycerol kinase exhibits high affinity (low Km) and less activity (low kcat) for complexes with a stoichiometric or over-stoichiometric constitution like Mg2ATP. On the other hand, the enzyme shows less affinity (high Km) and high activity (high kcat) for unsaturated complexes like [Mg(ATP)2]−6.Publicationtype: Journal Article146 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Biocatalytic phosphorylations of metabolites: past, present, and futurePhosphorus is a key element for life that occurs in living cells as a structural part of many key biochemicals. Phosphorus plays important functional roles in biocatalysis, such as making metabolic reaction steps irreversible by transferring energy-rich groups, using phosphate groups as leaving groups, or selectively hydrolyzing phosphorus–oxygen or phosphorus–nitrogen bonds. The large number of biocatalysts known to catalyze the transfer of phosphorus-containing groups from donor molecules to acceptor molecules covers a wide variety of reaction classes. Bioprocessing technology can and should replicate these naturally occurring reactions. We illuminate some strategic advantages of this promising technology for developing biocatalytic processes and separation steps. Constraints, opportunities, and outlooks for implementing green engineering procedures to establish sustainable production processes are discussed.Publicationtype: Review Article173 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Bioreaction engineering leading to efficient synthesis of L-glyceraldehyd-3-phosphate(Wiley-VCH, 2016-12-19); ; ; ; Enantiopure L-glyceraldehyde-3-phosphate (L-GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L-glyceraldehyde (L-GA) by ATP is used for the synthesis of L-GAP. L-GAP has a half-life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg2+ to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca2+, but not by L-GAP and inorganic orthophosphate. Moreover, equal amount of Ca2+ exerts a different degree of inhibition relative to the activity without the addition of Ca2+ depending on the Mg2+ to ATP molar ratio. If the Mg2+ to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred-tank reactor is the most efficient process for the synthesis of L-GAP.Publicationtype: Journal Article111 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Biocatalytic phosphorylation of metabolitesThis chapter focusses on selective biocatalytic phosphorylation reactions of metabolites. Phosphoryl group transfer reactions are of fundamental importance for living organisms and a variety of enzyme classes are involved in catalyzing these phosphorylation reactions. A large number of phosphorylases, phosphatases, and phosphotransferases/ kinases have been described as enzymes for the biocatalytic formation of new O-P- bonds, while the number of enzymes catalyzing the formation of N-P-, S-P-, and C-P-bonds is much smaller. Analytical method development, in particular separation and detection methodologies, for the direct analysis of biocatalytic phosphorylation reactions, are described as decisive steps for the assay and screening of novel phosphorylating biocatalysts, for investigating their functional properties and for process design. It is equally important for obtaining product stability data, as the areas of stability for the phosphorylated metabolites are best determined by experiments. As high-energy phosphate compounds are key players in life processes, the most important phosphate compounds are discussed in this chapter. Even though ATP is preferable in most enzymatic phosphorylation reactions, additional phosphoryl donors with varying energy level, availability, and specificity are of great interest. The stereo- and regioselectivity of phosphorylating enzymes offers benefits for the production of phosphate esters. A selection of new emerging phosphorylation reactions is shown. The subchapter on reaction engineering for biocatalytic phosphorylation processes provides basic reaction engineering principles, reaction kinetics, reaction system and process optimization strategies for biocatalytic phosphorylation reactions. The transition from stoichiometric phosphorylation reactions to biocatalytic phosphorylation reactions improves selectivity, safety, health, and environment, reduces the number of reaction steps in existing phosphoryla-tion routes, and decreases the waste-to-product ratio, if the phosphate donor is either recycled or used up in the reaction.Publicationtype: Book Part73 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , One-pot enzymatic reaction sequence for the syntheses of d-glyceraldehyde 3-phosphate and l-glycerol 3-phosphate(2016-02-01); ; A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate (d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose 1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows 98.5% correlation between experimental data and numerically simulated data matrices. The evaluation of different types of reactor was performed by combining the reaction kinetics model, mass balances and kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.Publicationtype: Journal Article91