Enzymes consumption

Table 8. Estimated Worldwide Enzyme Consumption by Product Type, 1990 ...

A great savings in enzyme consumption can be achieved by immobilizing the enzyme in the reactor (Fig. 12). In addition to the smaller amount of enzyme required, immobilization often increases the stability of the enzyme. Several designs of immobiliz-ed-enzyme reactors (lERs) have been reported, with open-tubular and packed-bed being the most popular. Open-tubular reactors offer low dispersion but have a relatively small surface area for enzyme attachment. Packed-bed reactors provide extremely high surface areas and improved mass transport at the cost of more dispersion. 

In order to improve this reaction, a proper understanding of all parameters affecting product yield is desired. Clearly, the high enzyme consumption is a major obstacle for an efficient and economically feasible process. A likely cause of the inefficient use of DERA in this conversion is enzyme deactivation resulting from a reaction of the substrates and (by-) products with the enzyme. In general, aldehydes and (z-halo carbonyls tend to denature enzymes because of irreversible reactions with amino acid residues, especially lysine residues. From the three-dimensional structure it is known that DERA contains several solvent-accessible lysine residues [25]. Moreover, the complicated reaction profile as shown in Scheme 6.5 indicates the potential pitfalls of this reaction. 

Quite common in applied biocatalysis, where the purity of biocatalyst often is not known, is the expression of biocatalyst stability as an the enzyme consumption number (e.c.n.) [Eq. (2.26)]. 

Specific enzyme consumption [U per kg product], a hands-on parameter, can be obtained from Eq. (2.32) (Kragl, 1996), where avol0 [U I. 1 is the initial volumetric... 

Product alcohol Ketone concentration [mM] Space-time yield [g L d 1] Enzyme consumption [U kg (Product)-1] ... 

Enzyme must be injected during the reaction to ensure constant activity in the case of enzyme activity losses [8]. A dropwise addition of peroxidase [86] usually results in higher enzyme consumption and, thus, lower turnover numbers than in... 

A first experiment with a single addition of MnP at the beginning of the operation was carried out in order to determine the enzyme consumption and thus the requirement of enzymatic activity. This feeding rate 37 2 U/(L h) was applied in the following experiments by means of two different strategies stepwise addition (one pulse per hour) or continuous addition [8], The continuous addition of enzyme allowed a high decolorization level (95.7%) with less enzymatic requirements than those of the stepwise addition experiment. An improvement of 69% in terms of efficiency was reached (Table 10.3). 

As far as manufacturing costs are concerned the yield of immobilized enzyme activity is mostly determined by the immobilization method and the amoimt of soluble enzyme used. Under process conditions, the resulting activity may be further reduced by mass transfer effects. More precisely, the yield of enzyme activity after immobilization depends not only on losses caused by the binding procedure but may be further reduced as a result of diminished availability of enzyme molecules within pores or from slowly diffusing substrate molecules. Such limitations, summarized as mass transfer effects, lead to lowered efficiency. On the other hand, improved stability under working conditions may compensate for such drawbacks, resulting in an overall benefit. Altogether, these interactions are a measure of productivity or of enzyme consumption, for example, expressed as enzyme units per kg of product. If we replace enzyme units by enzyme costs we obtain the essential product related costs, for example, in US per kg of product. 

Carrier-fixed penicillin G amidase in the multi-ton hydrolysis of penicillin G is a useful example to illustrate enzyme consumption. The enzyme is applied in stirred tanks with sieve plates at the bottom to retain the enzyme particles when the product solution is drained off. The pH-value is kept constant by controlled feed of ammonia solution. Fresh substrate solution is refilled about a thousand times or more. The consumption of enzyme in such a process is below 10 mg kg (0.2 ku kg ) of isolated 6-APA when the enzyme activity is determined with penicillin G solutions at 28° C and pH 8.0. Under identical conditions the consumption of soluble enzyme for each tank filling would be beyond all reasonable cost. 

Cross-linked crystals of lipase from Candida rugosa (CRL) were applied in the resolution of racemic ketoprofen chloroethyl ester. In batch-wise operation, the half-life of the catalyst was reached after about 18 cycles or, in terms of enzyme consumption, about 5.6 g of enzyme protein were consumed to prepare 1 kg of (S)-ketoprofen. CRL suffers from a low specific activity towards this poorly water-soluble substrate which may explain the high enzyme input [117]. 

If a short cycle time is chosen (e.g. 10 min.) Table VI shows that the enzyme consumption will be much higher than in the batch process, as soon as purging starts (1.8 hours from start). The enzyme consumption can be decreased by enlarging the reactor size, as enzyme concentration and reactor size are inversely proportional (eq. I, Box 1), but it will still be of the same order of magnitude as in the batch process. The increase in reactor size has, however, the disadvantage that more protein substrate is confined and lost in the end (Table VI). The concomitant loss of confined enzyme is found to be the same in both cases, which is obvious from eq. I. 

Between 15 and 20 analyzers based on enzyme electrodes are on the market worldwide. They are one-parameter instruments for the measurement of glucose, galactose, uric add, choline, ethanol, lysine, lactate, pesti-ddes, sucrose, lactose, and the activity of a-amylase (Table 23). They provide for a negligible enzyme consumption of less than 1 pg per sample. 

When cross-linked crystals of thermolysin were applied in peptide synthesis in ethyl acetate, they were stable for several hundred hours at amazingly low enzyme consumption, whereas a soluble enzyme preparation became inactive within a short period of time. Again it is worthwhile to consider the quality of the soluble enzyme preparation. When soluble thermolysin was stored in mixed aqueous-organic solutions, it lost about 50% of its activity within the first day of incubation only to be then quite stable for the next 15 days. It is possible that the initial inactivation was caused by an unstable fraction of thermolysin and that crystals of thermolysin no longer contained this unstable fraction [118]. Productivity comparable to that of crystals was achieved with thermolysin adsorbed on Amberlite XAD-7 resin which was employed in continuous plug flow reactors with ferf-amyl alcohol as solvent [119]. 

PE activity was determined with 15 ml of 1% pectin solution adjusted to pH 4.0 prior to addition of enzyme. Consumption of 0.1 N NaOH at 50 C was graphed, the slope determined by least squares, and PE units calculated by the procedure of Rouse (7). 

Enzyme consumption is defined as the number of units of enzyme consumed per unit weight of product (U kg-1) ... 

As in most cases reaction rate decreases with conversion, enzyme consumption increases to the same extent (an exceptional case is when strong substrate inhibition overcompensates product inhibition). Therefore enzyme consumption is minimal under initial reaction rate conditions (zero conversion) (Fig. 7-25). Approaching total or equilibrium conversion the reaction rate approaches zero and enzyme consumption increases rapidly. 

In contrast to enzyme consumption, substrate utilization, defined as kg substrate consumed per kg product produced, increases with increasing steady state conversion. 

Figure 7-25. Substrate utilization and enzyme consumption as a function of conversion.

Here the racemate is circulated on qtfe side of the tnpmfefane while the water necessary for hydrolysis is picked up from the other side. The resulting acid is extracted into the aqueous phase, where the pH is kept constant with, JifoOH- The enzyme is adsorbed In foe pores of foe membrane, for the pilot. scale production foe following data Were obtained reactor produefority 125 g d -nt h enzyme consumption 27 g per kg product, eaantkmierkt excess of product 95%, product yield 4 % ... 

AADH Regeneration enzyme(s) Precursor Product Product Degree cone, of con-(mM) version s.Ly. g/M) Enzyme consumptions (U kg 1) Ref. 

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