Enzymatic membrane preparation

As evident from Fig. 8.4, an increase in the selectivity has been observed in IL/ scCOj biphasic systems media (>99.5%) with respect to scCO assayed alone (95%). These results could be explained by the use of water-immiscible ILs which have a specific ability to reduce water activity in the enzyme microenvironment. The synthetic activity of the immobilized lipase in IL/scCO biphasic systems is lower than that in scCO assayed alone. Similar results were found by Mori et al. [40] in IL/ hexane biphasic systems. These authors reported that the enzymatic membranes prepared by simple adsorption of CaLB onto the surface were more reactive than membranes prepared with ILs. As can be observed in Fig. 8.4, the initial reaction rate in the assayed IL/scCO biphasic systems increased in the following sequence [bdimim ][PF ]<[bmim ][PFg ]<[bmim ][NTfj ]<[omim ] [PF ], which was practically in agreement with flie activity sequence reported by these authors using free Candida antarctica lipase B in homogeneous ionic liquid systems ([bmim ] [PF ]<[bdmim+][PFg ]<[bmim+][NTfj ]<[omim ][PF ]), with the exception of [bmim [PF ] and [bdimim+][PFg ]. These results were explained taking into account that biotransformation occurs within the ionic liquid phase, so substrates have to be transported from scCOj to the ionic liquid phase. The mechanism of substrate transport between the ionic liquid and the supercritical carbon dioxide could be by three consecutive steps diffusion of the substrates through the diffusion... 

Table 3.12 Enzymatic activities detected in bovine milk fat globule membrane preparations...

A very interesting field in membrane bioreactors is the production of cyclodextrins or oligosaccharides. In general, they have applications in food pharmaceutical, cosmetic agricultural, and plastics industries as emulsifiers, antioxidant, and stabilizing agents. In the food industry cyclodextrins are employed for the preparation of cholesterol-free products. The use of enzymatic membrane reactors to produce... 

The stability of the reagent to the assay conditions should also be ascertained. The instability of azides and diazo compounds towards thiols was mentioned earlier, and it would be prudent to ensure that all new reagents whether they contain familiar photoactivatable groups or not are stable to the prevailing chemical and enzymatic conditions. In an attempt to label the cAMP receptor of D. discoideum, Wallace and Frazier (1979b) found that 8-N rcAMP was converted to NrAMP by the phosphodiesterase of a crude membrane preparation. The NrAMP specifically labeled actin and not the cAMP receptor. 

In order to maintain the advantage of the microfabrication approach which is intended for a reproducible production of multiple devices, parallel development of membrane deposition technology is of importance. Using modified on-wafer membrane deposition techniques and commercially available compounds an improvement of the membrane thickness control as well as the membrane adhesion can be achieved. This has been presented here for three electrochemical sensors - an enzymatic glucose electrode, an amperometric free chlorine sensor and a potentiometric Ca + sensitive device based on a membrane modified ISFET. Unfortunately, the on-wafer membrane deposition technique could not yet be applied in the preparation of the glucose sensors for in vivo applications, since this particular application requires relatively thick enzymatic membranes, whilst the lift-off technique is usable only for the patterning of relatively thin membranes. 

The micropool injection method was developed by Miyahara et al. (20), and independendy by Kimura and his collaborators (15,16, 43). It was successfully used for a monolithic enzymatically coupled FET, sensitive to urea and glucose, and for the urea-, glucose-, and potassium-sensitive trifunctional FET biosensor (see Section 2.3). The thickness of an enzyme membrane is about 10 pm, and the thickness of an enzyme-immobilized membrane prepared by the ink jet-micropool injection method is 0.1 -1 pm. The lift-off method was developed by Kimura and Kuriyama s group and used for the deposition of a urease- and a glucose oxidase-immobilized membrane (16, 17) (see Fig. 8(3)). The enzyme membrane thickness is similar to the thickness of the film resist layer, about 1 pm thick. 

The human hepatic cytochromes as expressed in this system demonstrate substrate specificities and enzymatic rates which are essentially comparable to the enzymes studied in human liver micro-somes and extracts. Membrane preparations derived from the Dundee P450 System retain cytochrome activity which is useful in simple biochemical assessments of substrate-enzyme interactions. Whole bacterial cells can be manipulated to explore the effects of substrate concentration and competition for co-factors and co-en-zymes through the appliance of molecular genetics. The same clones can be directed to the analytical scale production of meta-bohtes with benefits for the progression of dmg development programmes (see Tab. 

Even though enzymatic conversion is not too effective, it is possible to prepare semipermeable membranes whose ultrafiltration yields are higher than those of passive membranes.74 75 Ultrafiltration experiments of cheese whey through cellulosic membranes to which papain was covalently bound, show that flux decay curves of enzymatic membranes are even less sensitive to pH changes.74... 

Attempts to resolve the enzyme systems, which are tightly bound to mitochondrial or endoplasmic membranes, usually yield enzymatically inactive preparations. Only for the steroid ll)6-hydroxylase system of adrenocortical mitochondria has resolution into the following three components been accomplished (IS, 21, 22, 31) (1) a particulate fraction containing cytochrome P-450 (2) a fiavine-adeninedinucleotide fiavoprotein (MW 60,000) and (3) a non-heme iron protein (MW 20,000). The latter two constitute the TPNH-cytochrome P-450 reducing system. Full 11 -hy-... 

SDS is easy to wash away from blots. In principle, this allows, us to renature SDS-denatured blotted proteins and to detect them via ligand binding or enzymatic activity. Protein/protein interactions should also be detectable, because (for example) a radioactively marked protein recognizes its binding partner on the blot of a cell lysate or a membrane preparation (Carr and... 

Adenylate cyclase is bound to the inner aspect of the plasma membrane. Its catalytic activity mediates hydrolysis of ATP to form cyclic AMP, and may be measured in fractured membranes in vitro by the conversion of radiolabelled ATP to cyclic AMP °. The activity of the enzyme may also be measured by following changes in intracellular cyclic AMP in whole cells in the absence or presence of a phosphodiesterase inhibitor . A full discussion of the relative merits of the two assay systems lies beyond the scope of the present review, but most information on the detailed biochemistry of receptor-mediated responses has been obtained by direct measurement of the enzymatic activity (i.e. conversion of labelled substrate to labelled product) in washed membrane preparations. 

A. Trusek-Holownia and A. Noworyta, Catalytic membrane preparation for enzymatic hydrolysis reactions carried out in the membrane phase contactor. Desalination 144 (2002) 427M32. 

Many researchers have used particulate preparations to study the biosynthesis of ASG. In some cases the centrifugal force used was 20,000-25,000 g and in others 100,000 g. It should be noted that such membrane preparations contain endogenous sterol acceptors (of glucose from UDP-Glc) and acyl donors. It is conceivable that other fiactions may have enzymatic activity but no endogenous substrate. Particulate preparations catalyze the formation of SG and ASG and are not stimulated by further addition of substrate. 

Membranes prepared from carrot suspension cells were found to have the capacity for a rapid in vitro phosphorylation of their lipids. This lipid phosphorylation depends absolutely on the presence of Mg2+ and can be saturated with increasing ATP concentration indicating Mg-ATP as the true enzymatic substrate [9,11]. The products of these reactions were identified by thin-layer chromatography and subsequent visualization of the labelled lipids by autoradiography as phosphatidic acid (PA), PIP, lysoPIP, and PIP2[11]. 

The membrane is a delicate yet sturdy structure which must be studied intact but cannot be isolated without disruption. In essence, membrane isolation involves the removal of all other parts of the cell. The purity of a membrane preparation is usually measured by the relative increase in specific activity of a membrane-boimd marker enzyme in the membrane preparation over the specific activity of the marker enzyme in the homogenate. In cases where specific membrane markers are lacking, enzymatic activities associated exclusively or primarily with other subcellular components must be demonstrated to be absent in order to show the preparation free from cellular contaminants. Such data, combined with phase-contrast and electron microscopic images of the preparation, are usually accepted as adequate criteria for the measurement of purity. One additional technique, at least for membranes with antigenic properties, is preparation of fluorescent antibodies to the isolated membranes and observation of the antibody localization in whole cells (Kabat and Meyer, 1971). The use of fluorescent probes in determining membrane structure and function has been reviewed (Radda, 1975 Azzi, 1975). 

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