Carrier-bound enzyme

One interesting technology uses lipases in the form of cross-linked enzyme crystals (CLECs) (Margolin 1996). This immobilization method does not use any solid support and the lipase specific activity (units of activity/g of immobilized catalyst) of the immobilized lipase derivative can be enhanced by 10-fold because there is no inert support, that usually represent more than 90% of the catalyst weight in the case of carrier-bound enzymes. These cross-linked crystals have been used for the chiral resolution of commercially important organic compounds, such as ibuprofen. 

Despite the advantages of enzymes immobilized on noncatalytic matrices, the yield and productivity of the reaction can be reduced simply due to the presence of the noncatalytic mass of the carrier. There has therefore been much interest in the development of carrier-free systems, in which enzyme molecules are linked to each other to form large complexes. These are inherently immobilized because individual enzyme molecules are no longer free to diffuse in solution, but they are largely undiluted by inert molecules and therefore retain a greater degree of activity than carrier-bound enzymes. This article discusses strategies for enzyme immobilization in carrier-based and carrier-free systems and considers some of their major applications. 

The problems of working with carrier-bound enzymes and cells in biofilm reactors and the biofloc reactors are compared in Table 3.1. 

In the literature are many articles on porous diffusion, especially in connection with carrier-bound enzymes or cells (for example. Pitcher, 1978). These are directly connected to the principles expressed in Sect. 4.5 concerning the influence of internal and external mass transport. The results are presented in the same graphical form as Fig. 4.36 in which the effectiveness factor of the reaction rj. is presented as a function of the Thiele modulus. For formulating an appropriate moduls one needs knowledge of the difficult to measure value. The following equation has shown itself useful in that the volume-based reaction rate is obtainable directly from the experimental measurements (Pitcher, 1978) (cf. Equ. 4.74)... 

In summary, the electron transfer chain consists of a series of membrane-bound enzymes that possess different prosthetic groups which become alternately reduced and oxidised as they transfer electrons (or hydrogen atoms), from one carrier to the next in sequence, to oxygen to produce H2O. 

Cao, F. (2005) Carrier-Bound Immobilized Enzymes, Principles, Applications and Design, Wiley-VCH, Weinheim. 

There are, however, various types of active transport systems, involving protein carriers and known as uniports, symports, and antiports as indicated in Figure 3.7. Thus, symports and antiports involve the transport of two different molecules in either the same or a different direction. Uniports are carrier proteins, which actively or passively (see section "Facilitated Diffusion") transport one molecule through the membrane. Active transport requires a source of energy, usually ATP, which is hydrolyzed by the carrier protein, or the cotransport of ions such as Na+ or H+ down their electrochemical gradients. The transport proteins usually seem to traverse the lipid bilayer and appear to function like membrane-bound enzymes. Thus, the protein carrier has a specific binding site for the solute or solutes to be transferred. For example, with the Na+/K+ ATPase antiport, the solute (Na+) binds to the carrier on one side of... 

Cao L, van Langen L, Sheldon RA (2003) Immobilised enzymes carrier-bound or carrier-free Curr Opin Biotechnol 14 387-394... 

Abou-Rebeyeh, H., Korber, F., Schubert-Rehberg, K., Reusch, J., and Josic, D. (1991). Carrier membrane as a stationery phase for affinity chromatography and kinetic studies of membrane-bound enzymes.. Chromatogr. 566, 341-350. 

Cao, L., Van Langen, L., and Sheldon, R. 2003. Immobilised enzymes Carrier bound or carrier-free Current Opinion in Biotechnology, 14 387-94. 

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

The method is based on the principle of enzyme immunoassay with fluorimet-ric detection and is termed radial partition immunoassay. This test can analyse macromolecules and haptens rapidly and with high sensitivity in a fully mechanised analysis system. The special feature of this test is the use of carrier-bound reagents on a so-called dry tab. 

POTENTIAL USEFULNESS OF CELL MEMBRANE-BOUND ENZYME SUBSTRATES AND INHIBITORS, AND CELL MEMBRANE-BOUND RECEPTOR AGONISTS AND ANTAGONISTS AS DRUG CARRIERS WITH LUNG SPECIFICITY... 

Similar results were obtained for the immobilization of glutaryl-7-ACA-acy-lase (own laboratory experiments). Figure 6 demonstrates the decrease of activity in the supernatant of the coupling reaction mixture and the concomitant increase in carrier-bound activity. Maximum activity was measured after only 6 h, leaving about 20 % of the initial activity in solution. During the next 14 h the remaining soluble enzyme was immobilized. However, an increase in activity could not be measured under standard conditions due to diffusional limitation and internal pH-shifts in the biocatalyst particles. According to these data. 

Immobihzation entails the introduction of inert carrier materials. With the exception of enzyme crystals or enzymes within enzyme membrane reactors, the inert carrier material is usually present in excess of the active enzyme protein. The carrier for crystals is the enzyme protein itself, whose specific activity strictly determines the weight-related activity of the crystal. This is different in the case of dedicated carrier materials. The range of active enzyme can be quite broad because enzyme loading can be adjusted according to the binding capacity of the carrier material. It is therefore possible to establish a well-balanced relationship between reaction volume and carrier by adjusting the amount of bound enzyme on the carrier. 

Up to this point, we have considered the election carriers bound to the photosystem-I thylakoid membrane. They include the primary electron donor P700 and the series ofelectron acceptors Aq (Chi a). A, (phylloquinone), FeS-X, and FeS-A/B. We now turn to two mobile electron carriers around photosystem I called plastocyanin (PC), a copper protein and ferredoxin (Fd) a [2Fe-2S] irons-sulfur protein, plus the enzyme that catalyzes the reduction of NADP by ferredoxin and called the ferredoxin-NADP -reductase and abbreviated as FNR. 

The stability of the microbial lipoxygenase immobilised on polymer carrier was determined by performing measurements of the activity with the same carrier with covalently bound enzyme for a period of 20 days (Fig. 5). As can be seen from the figure, the enzyme preserved its activity almost 100% for the whole period of investigation. 

Enzyme Amount of bound enzyme, mg g dry carrier Residual activity (%) pH optimum t(°C) Optimum m[M]... 

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