Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation

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Purification and Characterization

Advanced functional polymer membranes

Roger Sheldon (1942) received a PhD in organic chemistry from the University of Leicester (UK) in 1967. This was followed by post-doctoral studies with Prof. Jay Kochi in the U.S. From 1969 to 1980 he was with Shell Research in Amsterdam and from 1980 to 1990 he was R&D Director of DSM Andeno. In 1991 he moved to his present position as Professor of organic chemistry and catalysis at the Delft University of Technology (The Netherlands). His primary research interests are in the application of catalytic methodologies—homogeneous, heterogeneous and enzymatic—in organic synthesis, particularly in relation to fine chemicals production. He developed the concepts of E factors and atom utilization for assessing the environmental impact of chemical processes.

Biocatalysis in ionic liquids

Hollow fiber membrane contactors

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1976.tb10108.x

Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Redox reactions are important steps in the metabolism and energy conversion of living cells. Besides the enzymes necessary, a number of coenzymes are involved in such reactions: ferrodoxins, lipoic acid, NAD(H), NADP(H), flavins and cytochromes. The coenzymes differ in their redox potential, in the binding constants and the mode of regeneration [1]. NADH/NADPH are the most frequently encountered coenzymes. In general they dissociate easily and need a second reaction with another metabolite for regeneration. These properties are one of the means by which nature directs the flow of intermediates in response to biosynthetic needs. Because of the spectral properties of the coenzyme moiety, dehydrogenases have been extensively studied in the past [2] and have found widespread applications in clinical and food analysis [3].

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14983.x

Dehydrogenases for the synthesis of chiral compounds

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 104 [PEG‐10,000‐NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L‐leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α‐ketoisocaproate (2‐oxo‐4‐methylpentanoic acid) to L‐leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L‐leucine. The kinetic constants Vmax and Km of enzymes are all in the same range regardless of whether native NAD(H) or PEG‐10,000‐NAD(H) is used as coenzyme. L‐leucine was produced continuously out of α‐ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space‐time yield was 324 mmol/L day (or 42.5 g/L day).

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration: CONTINUOUS ENZYMATIC TRANSFORMATION

Publisher Summary This chapter describes an enzymatic regeneration of NADH by an enzyme-catalyzed reaction. It follows the strategy developed in cellular metabolism in which NADH is produced and consumed by a series of balanced reactions. Considering a technical application, it should be noted that in addition to the anticipated conversion, a second substrate is now needed in a stoichiometric amount and two products are formed in equimolar concentration. Therefore, the cosubstrate should be cheap enough and neither cosubstrate nor coproduct should be obnoxious to the enzymes involved. It is apparent that for fast NADH recycling, ready access to both enzymes becomes crucial and reactor systems with inherent mass transfer resistances should be avoided. Therefore, an enzyme-membrane reactor was chosen for the reaction described in the chapter. In the chapter, the reaction proceeds in homogenous solution. The enzymes are retained in the reactor by virtue of their high molecular weight, utilizing an ultrafiltration membrane as a selective barrier. However, the difference in molecular weight between product and coenzyme is not sufficient to achieve satisfactory results by a membrane separation. Therefore, the molecular weight of the coenzyme is increased by attaching it covalently to a water-soluble polymer, such as polyethylene glycol (PEG).

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

The article reviews the current status of the application of aqueous two-phase systems for the extractive purification of enzymes, especially with regard to large-scale processing. The method can be used for the separation of proteins from cell debris as well as for further purification. The latter can be performed by a series of single step partitions, and apparently also by continuous multistage processes. The specificity and selectivity of extraction can be enhanced by introducing specific or general ligands. Scale-up of extractive enzyme purification is relatively simple utilizing commercially available equipment and machinery common in the chemical industry. Besides the technical performance, economic considerations also indicate the feasibility of the method at production scale.

Maria-Regina Kula

Papers

Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Dehydrogenases for the synthesis of chiral compounds

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration: CONTINUOUS ENZYMATIC TRANSFORMATION

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Purification of enzymes by liquid-liquid extraction