Solubilized enzymes

All the enzyme forms discussed so far remain as visible suspended solids in organic reaction mixtures. However, it is possible to treat enzymes so that they become solubilized in organic media (or at least no longer form an obvious suspension). The known methods can be classified into three categories, although these are not clearly distinct at the boundaries  

These systems have been extensively studied, particularly the last. They have considerable attractions for fundamental studies, because the more or less transparent and mobile systems permit spectroscopic studies. Thus catalytic activity may be easily followed by direct spectrophotometric measurement of substrate or product 

The specific activity of the enzymes is often good, comparable with the best alternative enzyme forms. The enzyme molecules should all be well accessible to the medium, and mass transfer limitations avoided. However, solubilized enzymes have not achieved widespread use by those mainly concerned with applications in synthesis. An extra step is required to separate the enzyme from the final reaction mixture containing the products. It may be even harder to separate solubilising additives, notably surfactants, from the products. Thus I in general would not recommend solubilized enzymes for synthetic applications. One exception is where it is wished to attack polymeric or solid-state substrates, where the enzyme molecules may need to be able to move to contact the substrate, rather than vice versa. 

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The low content of water in these formulations promotes improved stabilization of enzyme and bleach additives. The combination of LAS and AE in a low-water-content formulation is effective at solubilizing enzymes and preserving enzyme stability when the sum of the LAS and water levels ranges between 25% and 45% [53],... 

For many solubilized enzymes the greatest catalytic activity and/or changes in conformation are found at R < 12, namely, when the competition for the water in the system between surfactant head groups and biopolymers is strong. This emphasizes the importance of the hydration water surrounding the biopolymer on its reactivity and conformation [13], It has been reported that enzymes incorporated in the aqueous polar core of the reversed micelles are protected against denaturation and that the distribution of some proteins, such as chymotrypsine, ribonuclease, and cytochrome c, is well described by a Poisson distribution. The protein state and reactivity were found markedly different from those observed in bulk aqueous solution [178,179],... 

A full understanding of the role of pectin in plant development requires elucidation of the mechanisms that regulate p>ectin biosynthesis (6). Our strategy for studying the biosynthesis of HGA was to 1) establish a PGA-GalAT assay that would allow detection of synthesized HGA, 2) characterize the enzyme in microsomal membranes, 3) characterize the product synthesized by the enzyme in microsomal membranes, and 4) solubilize the enzyme and characterize the solubilized enzyme and its product. 

Further advance toward a high-resolution structure of Ca -ATPase requires three-dimensional crystals of sufficient size and quality for X-ray diffraction analysis [179]. A prerequisite for the formation of three-dimensional crystals is the solubilization of the enzyme from its membrane environment by detergents [180,181]. Since the detergent-solubilized Ca -ATPase is notoriously unstable, the first task was to find conditions that preserve the ATPase activity of solubilized enzyme for several months. 

PAN, Z., DURST, F, WERCK-REICHHART, D., GARDNER, H.W., CAMARA, B., CORNISH, K., BACKHAUS, R. A., The major protein of guayule rubber particles is a cytochrome P450. Characterization based on cDNA cloning and spectroscopic analysis of the solubilized enzyme and its reaction products, J. Bio. Chem., 1995, 270, 8487-8494. 

Figure 1. Electrophoretic profiles ofglucan synthase fractions purified from red beet. Proteins were transferred to nitrocellulose and stained by colloidal gold followed by silver overlay. Lane 1, solubilized enzyme (CSGS) Lane 2, reconstituted glucan synthase (RCGS).

Reverse micelles are microheterogenous media where solubilized enzyme molecules are subject to the partitioning between different phases. The enzyme distribution between the phases is given by [183]... 

From bisubstrate, kinetic analysis with a transferase from hen oviduct that, under the conditions of the assay, formed only GlcNAc-PP-Dol, it followed that both dolichol phosphate and UDP-GlcNAe have to he bound to the enzyme before release of the product occurs.52 However, the fact that only partially purified preparations have thus far been obtained (the preparations may also still be contaminated with substrates and product), together with experimental difficulties in handling both the substrate dolichol phosphate (which, furthermore, is not one compound, see the earlier discussion) and the unstable enzyme (enveloped in micelles of detergent), make difficult a sensible interpretation and comparison of the kinetic parameters detenuined for the different enzvme-preparations. The solubilized enzymes catalyzing reactions 1,2, and 3 have in common their alkaline pH optima and dependence on Mg2+ or Mn2+ ions. The latter fact makes (ethylenedinitrilo)tetraacetic acid (EDTA) a reversible inhibitor of enzyme activity and an important experimental tool. 

A number of cases are known in which the properties of an enzyme are markedly altered by interaction with a membrane. Of course, in some cases the normal function of an enzyme is destroyed when it is removed from the membrane. For example, the mitochondrial coupling factor cannot synthesize ATP when removed from the membrane, since coupling to a proton gradient is required. The portion of the coupling factor that is easily solubilized (F,) is an ATPase. The steady-state kinetic properties of this solubilized ATPase are appreciably changed when it is reconstituted with mitochondrial membranes The turnover numbers and pH dependencies are different the solubilized enzyme is strongly inhibited by ADP, whereas the reconstituted enzyme is not and the reconstituted enzyme is inhibited by oligomycin, whereas the solubilized enzyme is not. 

Addition of polar, and, in some cases, neutral, lipids is necessary for activity of the solubilized enzyme.416"418 Their function seems to consist in provision of a nonpolar micro-environment for the enzyme, and this was demonstrated by experiments with a fluorescent-labeled substrate 419 Studies on the influence of temperature420 and of addition of 1-butanol421 on the rate of the enzymic reaction confirmed this conclusion. 

The final enzyme in the pathway, CMP-KDO transferase, the enzyme that catalyzes the transfer of KDO from CMP-KDO to the Lipid A acceptor has been studied in Mary Jane Osborn s laboratory. We have not further purified this enzyme nor studied its properties in detail. The in vitro reaction described by Munson et al. (15) utilized a detergent solubilized enzyme preparation to catalyze the transfer of two KDO residues to the Lipid A precursor. 

Rat brain microsome preparations were conveniently stored at -10C with retention of enzyme activity. Solubilization with Triton X-100 appears to be effective and the solubilized enzyme preparation, after filtration once with Amicon XM-300 diaflo membrane, was introduced into an isoelectric focusing column (LKB) with an ampholine pH range of 3.5-10. 

ECB deacylase is produced naturally by Actinoplanes utahensis. However, very little (—0.2%) deacylase activity was detected in the culture filtrate of Actinoplanes utahensis. Less than 5% of the cell-associated deacylase activity was released by incubation of the cells at 0.01 M KH2P04, pH 6, for 1 day. A simple salt treatment with 0.8 M KC1 resulted in 60-80% recovery of soluble deacylase at a high specific activity [25], This salt-induced solubilization suggests that the deacylase is loosely bound to the membrane of A. utahensis and can be released by disruption of ionic interactions [26]. The solubilized enzyme was stable and purified to apparent homogeneity by a four-step conventional procedure [25]. 

Bognar, A.L., Paliyath, G., Rogers, L. and Kolattukudy, P.E. (1984). Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum). Arch. Biochem. Biophys., 235, 8-17. 

HMG-CoA reductase, the enzyme that catalyses the formation of mevalonate [MVA, (2)] from HMG by an irreversible reaction that is considered rate-limiting with respect to the formation of cholesterol, has received much attention. Details of the purification of the enzyme from chicken liver and baker s yeast are available,15 and the solubilized enzyme from rat liver microsomes is readily and reversibly inactivated at temperatures below 19°C.16 Cold-inactivation is an uncommon phenomenon, and all the enzymes that have been found to exhibit this behaviour have been soluble proteins. Native HMG-CoA reductase is a particulate enzyme that is probably bound to protein or lipid of the microsomal membrane, although it is not known whether the solubilized enzyme contains a lipid component. Microsomal reductase is not cold-sensitive, and the cold-inactivation of the solubilized enzyme can be completely prevented by addition to the preparation of NADP+ or (more effectively) of NAD PH.17... 

Reduction of the lipase-solubilized enzyme by NADPH is more rapid than either turnover with cytochrome c or the rates of reconstituted systems (346). In rapid reaction spectrophotometric studies, changes at 550 nm are taken is indicative of flavin radical (FIH) the oxidized (FI) and reduced (FlHj) forms of the enzyme have negligible absorbance at this wavelength. Changes at 500 nm indicate formation of FIH2 (negative) or reoxidation of FIH2 (positive) FI and FIH are isosbestic at 500 nm. Both FIH and FlHj are formed at rates consistent with their... 

During cell fractionation, nearly all the cyclopenase activity was found in a fraction containing the cell wall together with the cytoplasmic membrane (83,84). From this fraction, the enzyme could be partially solubilized by detergents (e.g., Triton X-100) to yield a protein-phospholipid complex. By treatment with M-butanol, the solubilized enzyme preparation was split into the lipid fraction and the enzyme protein which retained a considerable part of the total enzyme activity. Compared with that of the membrane-bound enzyme, the substrate affinity of the solubilized protein-lipid complex was decreased, whereas... 

It follows from the above that, if highly specific oligosaccharides need to be synthesized, glycosyltransferases [21] of the Leloir type [22] should be the enzymes of choice. These enzymes connect sugars via the activated nucleotide components with high stereo- and regioselectivity. They are substrate-specific, but, in vitro, with solubilized enzymes, it becomes possible to transfer modified donors to modified acceptors and thus broaden the scope of the synthetic applicability. 

After concentration, the solubilized enzyme was applied to a gel filtration column. The enzymes from pear and tomato were purified further (1.5-3 fold) by isoelectric focusing (20,25). Chloro-plastic hydroperoxide lyase was solubilized from tea leaves with Tween 20 and partially purified 8.5-fold with 34% recovery by hydroxyapatite column chromatography (33). Although attempts at... 

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