Enzymes enzymatic reduction

For bio-transformation processes, immobilised enzymes are often used because their activity persists over a longer period of time than that of free enzymes. The reduction of enzyme activity in enzymatic reactors is a consequence of energy dissipation by sparging and stirring, which is required for instance for oxygen transport or realisation of constant reaction conditions as regards temperature and pH. In the other hand low and high pH-values leads also to a decrease of enzyme activity and increase the stress sensitivity. 

As mentioned above, Met(0) must be converted to Met before it can be incorporated into proteins. There are a wide variety of organisms that have been shown to be capable of enzymatically reducing Met(O) residues. The enzymatic reduction of free Met(O) to Met has been observed in yeast , E. cofi - , Pseudomonas , plants and animal tissues . The enzyme from E. coli has been purified about 1100-fold using a newly developed very sensitive assay . The assay involves first the conversion of [ S]Met(0) to [ S]Met by the Met(O) reductase followed by the measurement of [ S]Met-tRNA after enzymatic acylation of tRNA. Since Met(O) is not a substrate for the acylation reaction , the amount of [ S]Met-tRNA formed is proportional to the amount of [ S]Met(0) converted to [ S]Met. The assay is sensitive to Met levels of less than 1 pmol. 

Rhin(bpy)3]3+ and its derivatives are able to reduce selectively NAD+ to 1,4-NADH in aqueous buffer.48-50 It is likely that a rhodium-hydride intermediate, e.g., [Rhni(bpy)2(H20)(H)]2+, acts as a hydride transfer agent in this catalytic process. This system has been coupled internally to the enzymatic reduction of carbonyl compounds using an alcohol dehydrogenase (HLADH) as an NADH-dependent enzyme (Scheme 4). The [Rhin(bpy)3]3+ derivative containing 2,2 -bipyridine-5-sulfonic acid as ligand gave the best results in terms of turnover number (46 turnovers for the metal catalyst, 101 for the cofactor), but was handicapped by slow reaction kinetics, with a maximum of five turnovers per day.50... 

Anaerobic bio-reduction of azo dye is a nonspecific and presumably extracellular process and comprises of three different mechanisms by researchers (Fig. 1), including the direct enzymatic reduction, indirect/mediated reduction, and chemical reduction. A direct enzymatic reaction or a mediated/indirect reaction is catalyzed by biologically regenerated enzyme cofactors or other electron carriers. Moreover, azo dye chemical reduction can result from purely chemical reactions with biogenic bulk reductants like sulfide. These azo dye reduction mechanisms have been shown to be greatly accelerated by the addition of many redox-mediating compounds, such as anthraquinone-sulfonate (AQS) and anthraquinone-disulfonate (AQDS) [13-15],... 

As discussed earlier, Azo biological decolorization are mainly reduced in a direct reduction or mediated/indirect reduction with nonspecial azo reductase or reduced enzyme cofactors (Figs. 1 and 3). According to the direct enzymatic reduction mechanism, nonspecial azo reductase can catalyze the transfer of reducing equivalents originating from the oxidation of original electron donor in the azo dyes. In... 

The acceleration mechanism of redox mediators are presumed by van der Zee [15]. Redox mediators as reductase or coenzymes catalyze reactions by lowering the activation energy of the total reaction. Redox mediators, for example, artificial redox mediators such as AQDS, can accelerate both direct enzymatic reduction and mediated/indirect biological azo dye reduction (Fig. 3). In the case of direct enzymatic azo dye reduction, the accelerating effect of redox mediator will be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the azo dye. Possibly, both reactions will be catalyzed by the same nonspecific periplasmic enzymes. In the case of azo dye reduction by reduced enzyme cofactors, the accelerating effect of redox mediator will either be due to an electron shuttle between the reduced enzyme cofactor and redox mediator or be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the coenzymes. In the latter case, the addition of redox mediator simply increases the pool of electron carriers. 

Fig. 2, Principal of an enzyme-coupled cofactor regeneration system for an enzymatic reduction...

For enzymatic reductions with NAD(P)H-dependent enzymes, the electrochemical regeneration of NAD(P)H always has to be performed by indirect electrochemical methods. Direct electrochemical reduction, which requires high overpotentials, in all cases leads to varying amounts of enzymatically inactive NAD-dimers generated due to the one-electron transfer reaction. One rather complex attempt to circumvent this problem is the combination of the NAD+ reduction by electrogenerated and regenerated potassium amalgam with the electrochemical reoxidation of the enzymatically inactive species, mainly NAD dimers, back to NAD+ [51]. If one-electron... 

As we have seen a stereoselective reaction is one in which there is a preponderance of one isomer irrespective of the stereochemistry of the reactant. The enzymatic reduction of pyruvic acid is stereoselective when the chiral molecules of the enzyme complexes with achiral pyruvic acid, they given a preponderance of one form of pyruvic acid-enzyme complex which then gives a single form of lactic acid. 

Unlike the whole-cell system, enzymatic reductions require the addition of a hydride donating cofactor to regenerate the reduced form of the enzyme. Depending on the chosen ADH, the cofactor is usually NADH or NADPH, both of which are prohibitively expensive for use in stoichiometric quantities at scale. Given the criticality of cofactor cost, numerous methods of in situ cofactor regeneration, both chemical and biocatalytic, have been investigated. However, only biocatalytic regeneration has so far proven to be sufficiently selective to provide the cofactor total turnover numbers of at least 10 required in production. 

The pyridinium salt NAD 19a and its reduced form NADH 20a are important co-factors for many enzymes, fhe reduced form is involved in enzyme mediated reductions where it is converted to NAD. In natural systems, NAD is converted back to NADH by another enzyme-controlled process. Attempts to effect the direct electrochemical conversion of NAD to NADH are not very successful. Reduction on a mercury cathode at -1.1 V see on the first one-electron reduction wave leads to the radical-zwitterion, which reacts further to give dimers. Three stereoisomers of the 4,4 -dimer account for 90 % of the mixture and three 4,6 -dimers form the remainder [78]. Reduction at -1.8 V on the second reduction wave produces only 50 % of enzymatically active 1,4-NADH. The NAD analogue 19b shows related behaviour and one-electron reduction affords two diastereoisomers... 

By analogy with the enantioselective reduction of prochiral ketones to chiral alcohols an attractive method for producing enantiomerically pure amines would be enantioselective reductive amination of a ketone via enzymatic reduction of an imine intermediate (Scheme 6.11). Unfortunately the required enzymes-amine... 

Since these two biocatalysts possess complementary stereoselectivity, they enable the synthesis of both enantiomers of the desired products. The applicability of enzymatic reduction of aryl alkynones on a preparative scale was optimized with regard to the amount of cofactor and enzyme, resulting in high total turnover numbers and almost quatitative conversion [41]. 

Enzymatic reduction of 23a with recLBADH and CPCR resulted in unsatisfactory results (60% and 49% ee) as well. The results mentioned above indicate that a bulky substituent at the alkyne moiety results in a higher selectivity of the reduction. Furthermore, Bradshaw et al. reported that Lactobacillus kefir ADH, an enzyme highly homologous to LB ADH, affords (R)-4-trimethylsilyl-3-butyn-2-ol [(R)-25j with an ee of 94% in 25% yield [39bj. In our investigations ketone 23b was reduced by recLBADH with almost quantitative conversion. The enantiomeric excess and absolute configuration of the product were determined by desi-lylation with borax converting alcohol (R)-25 into enantiopure (R)-3-butyn-2-ol [(R)-24j (Scheme 2.2.7.14). 

Reduction of l-(chloro or bromo) -3-butyn-2-one (27e,f) with recLBADH affords enantiopure R-alcohols 28e,f, resulting in an interesting switch of the enantioselectivity of the enzymatic reduction. As the enantiomers (S) -28e,f can be obtained by recLBADH-catalyzed reduction of 27b-27d and subsequent removal of the si-lyl-protecting group, this enzyme offers unique access to a pair of enantiomers via the same oxidoreductase. Due to the high volatility of the substrates (27e,f) these transformations were only performed on an analytical scale. 

The enzymatic reduction of T-configured alkenones represents a valuable alternative to the enzyme-catalyzed reduction of the corresponding alkynone with subsequent partial reduction of the C-C triple bond. Nevertheless, since in our results the Z-configured vinylic alcohols are not accessible in enantiopure form by enzyme-catalyzed reduction of the olefinic ketones, for this class of compounds the two-step process via the propargylic alcohols is a highly valuable new strategy. 

Several factors may limit the overall rate of enzymatic reductive reactions. First, the electron transfer to the reactive metal (e.g., Co, Fe, or Ni) may be limiting. It is also possible that access of the organic substrates to the reduced metals contained within enzyme microenvironments may be limited. Mass transfer limitation is even more important in intact bacterial cells. For example, Castro et al. (1985) found that rates of heme-catalyzed reductive dehalogenations were independent of the heme content of the cells. 

Formation of the identical sugars of the D-series, 6-deoxymannose (rhamnose) and 6-deoxytalose, seems to proceed by a different pathway. According to Winkler and Markowitz (13), GDP-6-deoxy-D-mannose is first converted to GDP-6-deoxy-D-lyxo-4-hexulose. This 4-keto intermediate is the direct precursor for the unspecific enzymatic reduction leading to GDP-6-deoxy-D-mannose and GDP-6-deoxy-D-talose. For a pyridine-nucleotide requiring enzyme, the transformation seems to be unusual because of its lack of stereospecificity. However, closer examination and evaluation of properties of the different 4-keto-intermediate reductases must await availability of more highly purified enzyme preparations. 

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