Biocatalysts substrate concentrations

No enzymatic side effects are observed and substrate concentrations up to 20% by weight can be used without affecting the enzyme activity. The biocatalyst is used in soluble form in a batch wise process, thus poorly soluble amino adds can be resolved without technical difficulties. Re-use of the biocatalyst is in prindple possible. 

However, in most cases enzymes show lower activity in organic media than in water. This behavior has been ascribed to different causes such as diffusional limitations, high saturating substrate concentrations, restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation by lyophilization that becomes irreversible in anhydrous organic media, and, last but not least, nonoptimal hydration of the biocatalyst [12d]. Numerous methods have been developed to activate enzymes for optimal use in organic media [13]. 

Aqueous-organic two-phase reaction has been widely performed [18]. One of the purposes of using two-phase reaction system is to control the substrate concentration in aqueous phase where the biocatalysts exist. Hydrophobic substrate and products dissolve easily in the organic phase, so that the concentration in the aqueous phase decreases. The merits of controlling and decreasing the substrate concentration in the aqueous phase are as follows ... 

This example involves the same diffusion-reaction situation as in the previous example, ENZSPLIT, except that here a dynamic solution is obtained, using the method of finite differencing. The substrate concentration profile in the porous biocatalyst is shown in Fig. 5.252. 

Km The Michaelis-Menten constant, which is defined as the substrate concentration at which the biocatalyst is working at half its maximum rate (V max). In practice, Km gives a measure of the binding affinity between the substrate and biocatalyst the smaller the value, the tighter the binding in the complex. 

Figure 11.20 Schematic plot of the overall rate of reaction catalyzed by a surface-bound biocatalyst against the bulk substrate concentration.

Unlike external diffnsion, internal mass transfer occnrs simultaneously with the biocatalyst reaction and takes into acconnt the depletion of snbstrate within the pores with increasing distance from the snrface of the snpport. The rate of reaction will also decrease, for the same reason. The reaction is dependent on the substrate concentration and thns the distance from the ontside snpport snrface. 

The internal mass-transfer effects can be reduced, however, by decreasing the particle dimensions of the porous support containing the biocatalyst. Particle-diameter decrease results in a reduction of the distance from the outer support surface that the substrate must cross and, consequently, also results in a decrease of the substrate concentration gradient. 

Enzymatic kinetic resolution is a key step in the synthesis of the platelet aggregation inhibitor Lotrafiban (Figure 10.11). A disclosed process involves CaLB in tert-butyl alcohol/water (88 12) at 50 °C the substrate concentration was only r> g I 1 owing to its low solubility in this medium [122]. By exploiting the higher solubility in 88% [BMIm][PF6] and the better thermal stability of the biocatalyst in this medium, a higher rate was observed, the reaction was performed at 40 54 1. 1 at 75 °C, and the biocatalyst (Novozym 435) could be recycled 10 times. 

Fig. 5.49. Dimensionless plot of overall reaction rate against bulk substrate concentration for a surface immobilised biocatalyst...

The rate of reaction of a surface-immobilised biocatalyst may be determined graphically, as shown in Fig. 5.52. The curve marked is that relating the substrate concentration to the rate of reaction when there is no diffusional... 

In the case of gel entrapped biocatalysts, or where the biocatalyst has been immobilised in the pores of the carrier, then the reaction is unlikely to occur solely at the surface. Similarly, the consumption of substrate by a microbial film or floe would be expected to occur at some depth into the microbial mass. The situation is more complex than in the case of surface immobilisation since, in this case, transport and reaction occur in parallel. By analogy with the case of heterogeneous catalysis, which is discussed in Chapter 3, the flux of substrate is related to the rate of reaction by the use of an effectiveness factor rj. The rate of reaction is itself expressed in terms of the surface substrate concentration which in many instances will be very close to the bulk substrate concentration. In general, the flux of substrate will be given by ... 

The influence of eutectic media on the kinetics and productivity of biocatalysts has yet to be fully elucidated. Syntheses in eutectic suspensions have been scaled up to the pilot scale in a rotating drum reactor. The bioactive peptide Na-Cbz-L-Lys(Ne-Cbz)-Gly-L-Asp(OAll)-L-Glu(OAll)OEt was synthesized via a sequential N-to-C strategy in a heterogeneous solid-liquid mixture of the substrates in the presence of chymopapain and subtilisin as well as 16-20% (w/w) water and ethanol (Gill, 2002). At substrate concentrations of around 1 m, yields of 67-74% per step at product concentrations of 0.36, 0.49, and 0.48 kg kg-1 were achieved. The corresponding space-time yields were between 0.30 and 0.64 kg (kg d)-1 and biocatalyst reuse provided productivities of 166-312 kg product (kg enzyme)-1. 

Enzymes are biocatalysts, as such they facilitate rates of biochemical reactions. Some of the important characteristics of enzymes are summarized. Enzyme kinetics is a detailed stepwise study of enzyme catalysis as affected by enzyme concentration, substrate concentrations, and environmental factors such as temperature, pH, and so on. Two general approaches to treat initial rate enzyme kinetics, quasi-equilibrium and steady-state, are discussed. Cleland s nomenclature is presented. Computer search for enzyme data via the Internet and analysis of kinetic data with Leonora are described. 

The electrochemical activation of enzyme electrodes results in the electrobiocata-lyzed oxidation or reduction of a substrate specific to the biocatalyst. The rate of the biotransformation is dependent on the substrate concentration, hence these assemblies provide a basis for the construction of analytical biosensors [160]. The... 

Need for purified substrates and water to avoid poisoning of biocatalysts. Low concentration product stream. 

In certain cases, restriction of the experimental conditions to low substrate concentrations (cs< Km) is an acceptable condition for the investigation of biocatalyst properties. In this case, the enzyme kinetics can be simplified to the form of a pseudo-first order kinetics expressed by the relation... 

In spite of usefulness of the simplification obtained by decreasing the experimental substrate concentration, many studies are aimed at the investigation of kinetic properties of immobilized biocatalysts within broader concentration ranges. In a previous paper [29], cells of Escherichia coli with penicillin acylase activity were immobilized by entrapment in calcium pectate gel and tested on the transformation of penicillin G to 6-amino penicillanic acid. Figure 9 shows experimental data from a microcalorimetric investigation of the penicillin G transformation in steady state. As appreciable particle-mass transfer was expected, the mathematical model that includes particle-mass balance was used. 

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