Macromolecular substrates

The positive effect of convection of the substrate solution on mass transfer can be observed even better with macromolecular substrates that undergo processes such as protein digestion. For example, Fig. 9 compares reversed-phase chromatograms of cytochrome c digests obtained by cleavage with trypsin immobilized in both packed and molded column reactors, and clearly demonstrates the much higher activity of the monolithic device under otherwise similar circumstances [90]. 

Guisan JM, Bastida A, Blanco RM et al (1997) Immobilization of enzymes acting on macromolecular substrates. Reduction of steric problems. In Bickerstaff GF (ed) Methods in biotechnology immobilization of enzymes and cells. Humana, Totowa, NJ... 

In the case of glucoamylase, we have also seen a marked effect of dextran attachment on the ability of the enzyme to interact with starch, although not with maltose. In the case of this enzyme, however, we have gone one stage further and intentionally tried to eliminate all activity towards the macromolecular substrate by suitable choice of the coupling conditions. The results of this work are described in more detail below. 

Monitoring Enzymes That Degrade Macromolecular Substrates by Extraaive Bioconversion in Aqueous 1 vo-Phase Systems... 

In one parallel oxidation experiment of sample III-4 with the chiral oxidant 1, a poly(ester 3-sulfoxide) was obtained which does not show any appreciable optical activity. From this result we infer that even in the case of macromolecular substrates the difference in effective size of the groups directly linked to the sulfur atom plays a role in assisting the discrimination between the two enantiogroups and in determining the magnitude of the asymmetric bias (11). 

Examples are known (22-25) of the oxidation of macromolecular substrates containing sulfide functional groups, with chiral polysulfoxides being prepared by different synthetic approaches ( 6,27.). However, the asymmetric oxidation of polymeric precursors was only performed with very limited degrees of chemoselectivity and enantioselectivity (27). The asymmetric oxidation reaction employed in this work is accomplished with a moderate enantioselectivity, which is nevertheless on the order of magnitude as those obtained with low molecular weight substrates (11.28.29). 

The selectivities of enzymes that catalyze reactions involving high molecular weight substrates have been found to change when these enzymes are immobilized, because the diffusion of macromolecular substrates is slower, and because steric factors lower the activity of the enzyme by preventing free access of substrate to the enzyme s active site. For example, the enzyme ribonuclease (RNase) catalyzes the hydrolytic cleavage of phosphodiester bonds linking the nucleotides of polymeric RNA (Eq. 4.20) ... 

With the exception of enzymes such as proteases, nucleases, and amylases, which act on macromolecular substrates, enzyme molecules are considerably larger than the molecules of their substrates. Consideration of the structure of an enzyme s active site and its relationship to the structures of the enzyme s substrate(s) in its ground and transition states is necessary to understand the rate enhancement and specificity of the chemical reactions performed by the enzyme,... 

Preparation of the macromolecular substrate Dextran (Pharmacia, with an average molecular weight of 40000) is carboxymethylated with sodium chloro-acetate in 1,25 M NaOH to give about 0.2 groups/glucose residue. A,A -Bis-(3-... 

Designing derivatives of metal complexes that can form productive complexes with substrates and can manifest high effective molarities toward peptide bonds is not easy. Nature has adopted macromolecular polypeptides as the backbones of enzymes to tune the positions of catalytic elements in enzyme-substrate complexes, and thus, to achieve high effective molarities of the catalytic groups. The idea of macromolecular systems for the catalyst-substrate complexes may be applied not only to a macromolecular catalyst and a small substrate as in enzymatic systems, but also to a small catalyst and a macromolecular substrate. If a protein is used as the substrate, even a small catalyst may form a productive complex by utilizing the three dimensional (3D) structure of the protein substrate (22). 

In the previous examples the membranes have been considered generally as semiperineable barriers for the separation of small molecules from bigger ones. When in parallel to the separation a chemical reaction takes place in the bulk solution or in the membrane itself, the system may be identified as a true membrane reactor. A classical example is a stirred-tank enzymatic reactor connected by a continuous recirculation loop to an ultrafiltration or dialysis unit. Such a system, when well designed, permits the continuous removal of the reaction products from the bulk solution without loss of enzyme (or the insoluble or macromolecular substrate ). 

Much effort has gone, in recent years, in setting up alcoholic fermentations based on immobilized cell technology (J)). Some of the systems have proved to be highly productive, but are faced with drawbacks of leakage of cells, and sterical hindrances. Fermentation in two-phase system, on the other hand, has been successfully carried out with macromolecular substrates such as starch and cellulose (7 10 ). It is also easier to control a reaction system involving a number of enzymes, in a two-phase system as compared to the immobilized systems for example, there is a possibility to add more of the labile catalyst during the continuous operations. 

The microbial cells employed for the conversion have been seen to be enriched in the dextran rich bottom phase and also, the interface. The macromolecular substrates are also found located in the bottom phase. In fact, in one of the earlier studies on ethanol fermentation, starch alone constituted the lower phase of a two-phase system (.3). The solvent molecules are rather evenly distributed between the two phases. However, the partitioning behaviour of the solvent molecules can somewhat be changed by variations in the phase composition. Furthermore, a significant extraction of the solvent into the upper phase could be achieved by increasing the top to bottom phase volume ratios (1,11). Semicontinuous batch fermentations have been performed with the cells being recirculated in the bottom phase. After the conversion, the top phase with the product is removed and replaced by a fresh one along with more substrate. Alternatively, the phase is returned to the system after removal of the solvent e.g. by distillation (12,13). The... 

Macromolecular substrates such as proteins offer unique opportunities in processing modes with enzymes. Ultrafiltration membrane reactors (10) can be used to retain the protein substrate and the proteolytic enzyme in the reactor, while the hydrolytic products escape through the membrane to be collected. Using an ultrafiltration reactor, Cheftel (11) was able to solubilize 95% of FPC in 24 hr using pronase digestion. 

During the last ten years enzyme technology has moved mainly towards the development of new immobilization techniques and the improvement of those already existing. In turn, the attention of applied research has been focused on the engineering of systems based on immobilized biocatalysts. Enzymes involved in this development were enzymes catalyzing simple reactions that normally require no cofactors. A number of drawbacks affected the use of immobilized enzymatic preparations. An often dramatic reduction of initial enzyme activity due to the binding process, and the existence of diffusional resistances limits this approach with low activity enzymes, with macromolecular substrates and in general with enzymes whose cata-... 

When macromolecular substrates are involved in the transformation under study, concentration polarization phenomena affect the EMR performance more severely. Diffusion limitations of macromolecular substrates hamper the use of immobilized enzymes in the hydrolysis of high-molecu-lar-weight substrates. By selecting membranes with an appropriate molecular weight cut-off, both enzyme and substrate are retained in an EMR in touch with each other, and hydrolysis products and/or inhibitors are continuously removed from the system. Soluble enzymes can then act directly on substrate macromolecules without diffusion limitations and steric hindrance imposed by enzyme fixation to a solid support. The stirring features of CST EMRs moreover assures that substrates and/or inhibitors within the reactor vessel are maintained at the lowest possible concentration level. Such reactor configuration is then extremely useful when substrate inhibited reaction patterns are involved, or when inhibiting species are assumed to exist in the feed stream. 

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