Total turnover number TTN

In respect of designing an economic production process, the stoichiometric cofactor required in carbonyl reductions or the respective oxidation reactions needs to be minimized that is, enabled by recycling of the cofactor. The measure for the efficiency of the recycling process is the total turnover number (TTN), which describes the moles of product synthesized in relation to the moles of cofactor needed. The different approaches in cofactor recycling were recently reviewed by Goldberg et at. [12]. 

All of these oxidoreductases reduce aromatic and aliphatic a-chloropropargyhc ketones (27a-27c) with high activity. a-Bromopropargylic ketone 27d is also accepted as a substrate however, the enzymatic activity of TBADH and recLBADH decreases, probably for steric reasons. Due to the low solubility of substrate 27a about 25% of a short-chained alcohol was added. The large excess of short-chained alcohol shifted the substrate/product equilibrium toward the desired propargylic alcohol 28a, resulting in almost quantitative conversions with high total turnover numbers (TTN) of the cofactor. 

Fig. 3.1.3 Total turnover numbers (ttn) for different types of chemzymes 1, a,a-diphenyl-L-prolinol chemzyme ...

Every (bio)catalyst can be characterized by the three basic dimensions of merit -activity, selectivity and stability - as characterized by turnover frequency (tof) (= l/kcat), enantiomeric ratio (E value) or purity (e.e.), and melting point (Tm) or deactivation rate constant (kd). The dimensions of merit important for determining, evaluating, or optimizing a process are (i) product yield, (ii) (bio)catalyst productivity, (iii) (bio)catalyst stability, and (iv) reactor productivity. The pertinent quantities are turnover number (TON) (= [S]/[E]) for (ii), total turnover number (TTN) (= mole product/mole catalyst) for (iii) and space-time yield [kg (L d) 11 for iv). Threshold values for good biocatalyst performance are kcat > 1 s 1, E > 100 or e.e. > 99%, TTN > 104-105, and s.t.y. > 0.1 kg (L d). ... 

As in all catalytic processes, catalyst stability is a key process criterion. In contrast to mere temperature or storage stability, which refer to the catalyst independently of a process, the operating stability or process stability is the relevant and decisive dimension of merit. It is determined by comparing the amount of product generated with the amount of catalyst spent. The relevant quantity, also sometimes found in homogeneous catalysis, is the total turnover number (TTN) [Eq. (2.25)]. 

Relationship between Deactivation Rate Constant lrd and Total Turnover Number TTN... 

To convert the specific enzyme consumption into the total turnover number TTN, the specific enzyme consumption, sp.e.c. [U (kg product)-1] [Eq. (2.32)] first has to be converted into the enzyme consumption number e.c.n. [g enzyme (kg product)-1] [Eq. (2.26)]. 

Some of those criteria have been discussed above in the context of the catalyst or the process, such as enantioselectivity or diastereoselectivity instead of simple selectivity to product, and catalyst performance data such as turnover frequency (tof), turnover number (TON), total turnover number (TTN), and space-time-yield (s.ty.). 

While a number of dendritic catalysts have been described, catalyst recyclization in most cases is an unsolved problem. Diaminopropyl-type dendrimers bearing Pd-phosphine complexes have been retained by ultra- or nanofiltration membranes, and the constructs have been used as catalysts for allylic substitution in a continuously operating chemzyme membrane reactor (CMR) (Brinkmann, 1999). Retention rates were found to be higher than 99.9%, resulting in a sixfold increase in the total turnover number (TTN) for the Pd catalyst. 

Isolated enzymes are advantageous over whole cells owing to the absence of side activities compromising selectivity and the greater ease of repeated re-use to achieve high total turnover numbers (TTNs). Principal disadvantages are the requirement for a purification protocol, the yield loss upon purification, and the requirement for external addition of cofactors. 

The highest space-time yield (120 g 1. 1 d 1) was achieved in a continuously operated enzyme membrane reactor for the chloroperoxidase-catalyzed oxidation of indole to oxindole with H202 in aqueous t-BuOH, whereas a fed-batch reactor obtained the highest total turnover number (TTN 860 000) (Seelbach, 1997). 

In a direct comparison of the reduction of acetophenone to highly enantio-en-riched (R)-phenylethanol (94% e.e.) by heterogenized (S)-diphenyloxazaborolidine (Corey-Itsuno catalyst) or to enantiomerically pure (S)-phenylethanol (> 99% e.e.) by Candida parapsilosis carbonyl reductase (CPCR), the superior solubility of acetophenone in THF (0.25 m) versus water (0.04 m) leads to a vastly superior space-time yield of 290 g (L d) 1 in THF with the Corey-Itsuno catalyst in comparison with 27 g (L d) 1 in water with CPCR (Rissom, 1999). Conversely, the turnover frequencies (tofs) of 0.3 min-1 (Corey-Itsuno catalyst) versus 2.3 x 104 min-1 (CPCR) portend the difference in total turnover number (TTNs) of 2.4 x 108 versus 560. 

While an [S]/[C] ratio serves as a good dimension of merit for the productivity of one batch, it does not yield any information on the stability of the catalyst over its lifetime. As already discussed in Chapter 19, Section 19.3.4, the homogeneous catalysis community typically does not feel the need to recycle catalysts (Blaser, 2001), so the turnover number (TON) equals the total turnover number (TTN). The true utility and productivity advantage of biocatalysts is captured upon reuse of the catalyst, achieving catalyst lifetime productivities far in excess of catalysts used only once. 

This is the second major commercial route to i-(-)-menthol and the largest application of homogeneous asymmetric catalysis. Yearly, about 15001 (-)-menthol and other terpenes are produced by this route. The space-time yield could be improved to about 8000 mol enamine (mol Rh)-1 in 18 h. The total turnover number (TTN) was improved to an impressive 400 000. 

Low enzyme stability may be another limitation of in vitro biosystems. The total turnover number (TTN) of the enzyme represents the lifetime of the biocatalyst under its working conditions. Enzyme stability can be addressed... 

In 2013, Arnold and co-workers revisited the possibility of engineering an enzyme eatalyst for this useful transformation. Combining ortho-substituted benzenesulfonyl azides 142 with engineered P450 enzymes which include a reduced Fe center resulted in an efficient intramolecular benzylic C—H bond amination reaction. The desired aminated products 143 were produced in 73% ee with a total turnover number (TTN) of 383. In addition, expression of the eatalyst in E. coli makes the reaction proceed smoothly on a 50 mg scale with high ee value (Seheme 1.54). 

In every catalytic process, the operational stability of the catalyst under process conditions is a key parameter. It is described by the dimensionless total turnover number (TTN), which is determined by the moles of product formed by the amount of catalyst spent and - in other words - it stands for the amount of product which is produced by a given amount of catalyst during its whole lifetime. If the TONs of repetitive batches of a reaction are measured until the catalyst is dead, the sum of all TONs would equal to the TTN. TTNs are also commonly used to describe the efficiency of cofactor recycling systems. 

For example, a ferredoxin hydrogenase (EC 1.12.7.2) has been isolated recently from the hyperthermophile Pyrococcus fUriosus [38]. The performance of this biocatalyst, which showed a remarkable stability under operative conditions, has been investigated for the NADPH regeneration in the reduction of prochiral ketones catalyzed by the thermophilic NADPH-dependent ADH from Thermoanaerohium sp. Total turnover numbers (TTNs mole product/mole consumed cofactor NADP" ") of 100 and 160 could be estimated in the reduction of acetophenone and (2S)-hydroxy-l-phenyl-propanone, respectively. As a side note, it should be mentioned that, although the activity of the P. furiosus hydrogenase increased exponentially with temperature up to its maximum above 80 °C, the reactions had to be performed at much lower temperature (40 °C) because of the thermal instability of NADPH. 

Owing to the lack of stability of the monooxygenase, the hydroxylation step was achieved by applying resting cells of P. monteilii, providing access to the corresponding benzylic alcohol as an intermediate. Subsequent alcohol oxidation was performed by a cell-free extract of an ADH from I. kefir using the auxiliary co-substrate acetone to push the equilibrium of the oxidative ketone formation. The optimized process displayed an overall performance of up to 87% yield and a total turnover number (TTN) of 4200. 

Figure 7 Influence of substrate solubility on the total turnover number (ttn) of the coenzyme.

Figure 11 Increase of the total turnover number (ttn) of the coenzyme by retention and influence of the thermal deactivation. Concentrations substrate 100 nunol/L, coenzyme 0.1 mmol/L.

Figure 14 Total turnover number (ttn) as a function of the ratio of substrate/coenzyme concentration and coenzyme retention for the enzymatic synthesis of L-terf-leucine. The data points are experimental values (compare Table 5). Feed concentrations 2-oxo-3,3-dimethylbutanoic acid (A) 500 mmol/L, (B) 900 mmol/L, (C) 500 mmol/L NAD (A) 0.06 mmol/L, (B) 0.2 mmol/L, (C) 0.2 mmol/L. UF, ultrafiltration NF, nanofiltration R, retention.

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