Biocatalyst pores

Chong, A.S.M. and Zhao, X.S. (2004) Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst. Catalysis Today, 93-95, 293-299. 

Because the monoliths allow total convection of the mobile phase through their pores, the overall mass transfer is dramatically accelerated compared to conventional porous structures. Based on the morphology and porous properties of the molded monoliths, which allow fast flow of substrate solutions, it can be safely anticipated that they would also provide outstanding supports for immobilization of biocatalysts, thus extending the original concept of monolithic materials to the area of catalysis. 

Membranes have the property to retain one or more components of a hquid mixture, whereas others may pass. The size of the biocatalyst often differs considerally from the size of the product molecules. The pore size of the membrane must thus be chosen in such a way that the product can pass the membrane, while the biocatalyst is retained. Usually membrane bioreactors consist of ultrafiltration... 

When a biocatalyst is immobihzed within a porons snpport, in addition to possible external mass-transfer effects there conld also exist resistance to internal diffusion of snbstrate, as this mnst dififnse throngh the pores in order to reach the biocatalyst. Conseqnently a snbstrate concentration gradient is established within the pores, resnlting in a concentration decreasing with increased distance (in depth) from the surface of immobihzed biocatalyst preparation. Similarly, a corresponding product concentration gradient is obtained in the opposite direction. 

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. 

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 ... 

Membrane reactors allow a different option for the separation of biocatalysts from substrates and products and for retention in the reactor. Size-specific pores allow the substrate and product molecules, but not the enzyme molecules, to pass the membrane. Membrane reactors can be operated as CSTRs with dead-end filtration (Figure 5.5e) or as loop or recycle reactors (Figure 5.5f) with tangential (crossflow) filtration. 

Membrane reactors became an option for the retention of biocatalysts when the processing of membrane materials had progressed sufficiently to control thickness and pore structure and to manufacture a membrane that was defect-free. Besides its function as a retainer the membrane also serves other functions such as (i) to stabilize the phase boundary in case of multi-phase reactions (ii) as a consequence of (i), to transport dissolved 02 preferentially over gaseous 02 and (iii) to support purification and sterilization of air and other nutrients in fermentations. 

A membrane can be generated by polymerization around a few biocatalyst molecules which surround a space of a few hundred micrometers (microencapsulation Figure 5.6, option 5), or it can be of macroscopic dimensions (Figure 5.6, option 6). In the latter case, membrane reactors can be classified according to (i) driving force, (ii) pore structure and (iii) pore size. 

Conventional filters, such as a coffee filter, termed depth filters , consist of a network of fibers and retain solute molecules through a stochastic adsorption mechanism. In contrast, most membranes for the retention of biocatalysts feature holes or pores with a comparatively narrow pore size distribution and separate exclusively on the basis of size or shape of the solute such membranes are termed membrane filters . Only membrane filters are approved by the FDA for sterilization in connection with processes applied to pharmaceuticals. Table 5.3 lists advantages and disadvantages of depth and membrane filters. 

To test the reusability of the biocatalyst, five sequential reaction cycles with CPO immobilized on SBA-16 of different pore sizes were completed [6]. The authors found that immobilization on material with larger pore, 143 A, improved the reusability of the catalyst. Enzyme immobilized by covalent attachment to silica-based materials retained a higher residual activity after five reaction cycles than the physical approach. 

In this model, the biocatalyst is entrapped in a thin layer of solution between a working electrode and a membrane with capillary pores [64]. The electrode is poised at a potential sufficiently positive to ensure that the surface concentration of reduced mediator is negligible. In addition, it is assumed that (i) the microbial activity is constant in the absence of external perturbations (ii) the concentration of reduced mediator in the external medium is negligibly different from zero (iii) the microbial cells are point sources of reduced mediator homogeneously distributed throughout the biological layer and (iv) the concentration gradients are linear. 

As will be shown later, some ceramic membranes have been used to immobilize some biocatalysts such as enzymes for increasing the reaction rate of bioreactions. Membrane pores when mostly used as catalyst carriers are advantageous over the conventional catalyst carriers in the pellet or bead form in having less mass transfer resistance and more efficient contact of the reactant(s) with the catalysL In a strict sense, the membrane material when used in this mode is not a membrane which is defined as a permselective medium. 

On the whole, SGco2 lowers appropriately the fluidity of the oil, thus allowing filtration under suitable enough conditions it also seems to improve the activity of lipase. The enzymatic membrane, in turn, represents a very good microcontacting system (particularly pore mouth) that is well adapted to elevated reaction yield because of the high probability level of contact between substrate and biocatalyst. 

When the mass of carrier material is large relative to that of the enzyme, the physical and chemical properties of the carrier (Table 6-5) will, in large part, determine properties of the resultant immobilized enzyme. Often, the carrier will impart mechanical strength to the enzyme, allowing repetitive recovery by simple filtration of the solid particles and reuse of the enzyme. The degree of porosity and pore volume will determine the resistance to diffusion and molecular size selectivity of the biocatalyst. When used in non-aqueous media, dispersion of the enzyme over a large surface area can greatly increase its activity. Table 6-3 summarizes many of the key properties and considerations for enzyme carrier materials. 

As was described above in a number of MBR processes the membrane, in addition to performing the separation functions previously discussed, also acts as a host for the biocatalysts (whole cells or enzymes) which are immobilized in the membrane s pore structure. Concerns with such MBR configurations include membrane biofouling, mass transport limitations and biocatalyst activity loss and denaturation. In the two sections that follow we discuss further some of the key aspects of MBR for biochemical synthesis. We classify these reactors into two types, namely whole-cell and enzymatic MBR. 

Though MBR offer advantages over the more conventional bioreactors, they, themselves, are not completely free of problems. One such key problem, as previously noted, relates to changes in biocatalyst activity. This is a serious concern for whole-cell MBR, when the cells are immobilized in the membrane s pore structure. Diffusional limitations for nutri-... 

The biocatalyst morphology was characterized in relation to the superficial area, pore... 

The morphologic characterization of the immobilized enzyme is important to correlate the biocatalyst performance with porous structure parameters. BET analysis, which is usually based on N2 isothermal adsorption at 77 K, allows determining the solid-specific surface area, total pore volume, pore size distribution, and mean pore diameter. It is not recommended for solids with a low specific surface area (<5 m g ). Table 2 shows the specific smface area, mean pore diameter, and total pore volume determined by BET for the pure sol-gel silica matrix having TEOS as the precursor and the same matrix with the encapsulated CGTase. 

Analyzing the curvatures in Tables 4, 5, and 6, one concludes that the POH profile approximates to the optima region for the tested oils utilizing entrapped enzyme. RSM for entrapped lipase (Fig. 2) shows that the typical POH profiles is different from that of free lipase (Fig. 1), while the pH effect is very significant for the entrapped lipase (Fig. 2) with all tested oils for the free enzyme, the POH is more affected by pH only for olive oil. Maximum hydrolysis was observed at lower pH for the entrapped lipase, whereas for the free enzyme, the maximum hydrolysis occurred at pH 7, for canola and soybean oils. POH was generally smaller for lower loadings of entrapped enzyme, as shown in Tables 3, 4, 5, and 6. This could be due to the limitation of substrate diffiision toward the biocatalyst surface and into the pores of the support because of its microporous stmcture. 

The decision as to which monolith type to use is therefore dependent on the type of application, but commercial availability and carbon type should also be evaluated. Cordierite and Mast monoliths are commercially available, whereas ACM is not. When cordierite is used in combination with CNFs, possible cracking of the support can occur. For ACM monoliths, the open structure allows high carrier loading and prevents cracking upon the growth of CNF. For integral carbon monoliths in combination with biocatalysts, the pore size and chemistry of the carbon must be tuned to match the properties of the biocatalyst. 

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