Solubilization particulate enzymes

The particulate enzyme from which this solubilized form is derived contains cyt-b, and can utilize formate as reducing substrate. 

The utilization of three different detergents for the solubilization of particulate enzymes is presented to provide the reader with the scope of... 

Heller and Villemez299,300 solubilized, but did not separate, the D-mannosyltransferase and D-glucosyltransferase activities from this particulate-enzyme system, and found that, when only GDP-D-mannose is provided as the substrate, a/ -(1— 4)-linked D-mannan is synthesized and, when only GDP-D-glucose is present, ap-(l— 4)-linked D-glucan is produced. They further noted, with this transferase system, that incorporation of D-glucose from GDP-D-glucose into polysaccharide is a shortlived reaction that can be greatly extended if GDP-D-mannose is also included in the reaction mixture. 

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... 

The reason for these differences in properties between soluble and particulate enzyme was not elucidated. It may be postulated that the particles possibly contain two enzymes—one of these, stimulated by 2-acetamido-2-deoxy-D-glucose, is responsible for the synthesis of highly polymerized chitin, and the other catalyzes the synthesis of oligosaccharides only and does not require 2-acetamido-2-deoxy-D-glucose. If this hypothesis is correct, it would seem that the treatment with 1-butanol resulted in the solubilization of the second enzyme only. 

In several tissues, like gastric mucosa [6,8,46.47], pancreas [19,20], submandibular gland [22] and intestinal mucosa [36], the anion-sensitive ATPase can be solubilized by Triton X-100, a detergent/protein ratio of 3 1 being most effective [6,8,47]. The solubihzed enzyme from gastric mucosa [46] and pancreas [20] could be partially purified by gel filtration and sucrose density gradient centrifugation. The properties of the solubilized enzyme do not differ very much from those of the particulate enzyme. 

Polyphenol oxidase (PPO) (EC 1.14.18.1 monophenol monooxygenase [tyrosinase] or EC 1.10.3.2 0-diphenol 02-oxidoreductase) is one of the more important enzymes involved in the formation of black tea polyphenols. The enzyme is a metallo-protein thought to contain a binudear copper active site. The substance PPO is an oligomeric particulate protein thought to be bound to the plant membranes. The bound form of the enzyme is latent and activation is likely to be dependent upon solubilization of the protein (35). PPO is distributed throughout the plant (35) and is localized within in the mitochondria (36), the cholorplasts (37), and the peroxisomes (38). Using antibody techniques, polyphenol oxidase activity has also been localized in the epidermis palisade cells (39). Reviews on the subject of PPO are available (40—42). 

Early reports of such an enzyme system, tightly bound and occurring in a particulate fraction of mung bean, were made by Hassid and associates.292,293 The glycosyl donor was reported to be GDP-D-glucose and the product was characterized as a / -(l— 4)-linked D-glucan,292,293 Liu and Hassid294 solubilized, and partially purified, this enzyme. 

The reaction is catalyzed by a single enzyme, namely, UDP-2-acetamido-2-deoxy-D-glucose chitin 2-acetamido-2-deoxy-D-glucosyltransferase (chitin synthase, EC 2.4.1.16). Particulate preparations of chitin synthase obtained from different fungi211 did not show significant differences. The enzyme exists in the cells as inactive zymogen, and can be converted into the active form by limited proteolysis.212 The zymogenic character of chitin synthase was also confirmed in solubilized preparations,213,214 as well as in preparations from other sources.211... 

After solubilization from a particulate fraction of hog kidney cortex, the transpeptidase has been obtained in highly purified, but not yet homogeneous, form. The enzyme is active in the alkaline pH region with a maximum at about pH 8.8. Although appreciable magnesium is present in the enzyme preparation and added Mg2+ partially stimulates the catalytic action, it is not certain whether this metal is essential for activity since EDTA exerts an enhancement rather than an inhibition. 

Vetter, Y. A., and J. W. Deming. 1999. Growth rates of marine bacterial isolates on particulate organic substrates solubilized by freely released extracellular enzymes. Microbial Ecology 37 86-94. 

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. 

Some bioproducts are derived from fermentation by living organisms but immobilized enzymes can also catalyze a biotransformation. If the product is extracellular, then the initial steps of recovery include removal of the cells and other particulate matters from the broth. If the product is intracellular, it would be necessary to lyse the cells to release the product into the broth. The cell debris is then separated before the product is recovered from the broth. In certain cases, proteins produced as IBs need to be solubilized and the proteins renatured before further recovery steps. In the case of biotransformed products, the immobilized cells or enzymes and their support need to be removed initially. 

Investigations to determine whether the transfers to the various acceptors are catalyzed by one nonspecific enzyme, or by several specific enzymes, have been hampered by the particulate (microsomal) nature of active enzyme-preparations and by lability of the preparations obtained when the microsomes are solubilized by conventional methods. However, a solubilization and purification of D-glucopyranosyluronic transferase has been achieved by treating liver microsomes with the venom of Trime-resurus flavoviridis The soluble enzyme showed activity toward phenols and carboxylic acids, but not toward aniline, indicating the existence of at least two different transferases. 

Although the supernatant fraction S-1 [similar to the solubilized preparation described by Sharma et al. (29)] of the mitochondrial sonicate displayed 11 -hydroxylase activity, reaction rates were not proportional to the amount of mitochondrial protein added, but they decreased precipitously with increasing dilution of the preparation (15). This observation, indicating a dissociating enzyme system, prompted the separation of the sample into the particulate fraction and the two soluble components of the reducing system. 

On the other hand, when GDP-D-glucose was used as the substrate, both the particulate and the digitonin-solubilized enzyme systems from mung beans and from Lupinus albus yielded only (1 — 4)-/3-D-gIucan (cellulose), practically all insoluble. 

The use of enzymes to lyse cells, hydrolyze fat emulsions, solubilize proteinaceous colloids, liquify or saccharify starch gels and granules, and degrade various components of celluloslc substrates indicates that many substrates are present in a particulate form. Kinetic forms for such enzyme catalyzed reaction rates are here noted, and will be revisited in the subsequent discussion of immobilized enzyme kinetics. 

Accurate modeling of microbial solubilization of lignocellulose will be dependent on knowledge of the dynamics of microbial cell concentration over the course of bioconversion. While measurement of cell concentrations distinct from the concentration of substrate is trivial for soluble substrates, it is a substantial and not-yet-resolved challenge for fermentation of particulate substrates based on plant cell walls. Cell measurement has been approached on the basis of elemental composition (pellet nitrogen, [25]), concentration of cellular macromolecules (total protein [26] or DNA via quantitative PCR [27]), and estimated by indirect methods, such as off-gas analysis [25] and detection of enzymes (ELISA assays [28]). Future efforts using quantitative proteomics approaches also hold promise. 

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