Enzyme solubilization

The aqueous cores of reverse micelles are of particular interest because of their analogy with the water pockets in bioaggregates and the active sites of enzymes. Moreover, enzymes solubilized in reverse micelles can exhibit an enhanced catalytic efficiency. Figure B4.3.1 shows a reverse micelle of bis(2-ethylhexyl)sulfosuccinate (AOT) in heptane with three naphthalenic fluorescent probes whose excited-state pK values are much lower than the ground-state pK (see Table 4.4) 2-naphthol (NOH), sodium 2-naphthol sulfonate (NSOH), potassium 2-naphthol-6,8-disulfonate (NSOH). The spectra and the rate constants for deprotonation and back-recombination (determined by time-resolved experiments) provide information on the location of the probes and the corresponding ability of their microenvironment to accept a proton , (i) NDSOH is located around the center of the water pool, and at water contents w = [H20]/[A0T] >... 

The use of enzymes for solubilization of seed and leaf proteins has been studied as a means of overcoming difficulties presented by the varying condition of seed and leaf material available for processing (37). Ultrasonic energy was reported to increase the efficiency of enzyme solubilization procedures. The effects of various conditions of hydrolysis of cottonseed and alfalfa meal protein with trypsin were defined. 

It has been discovered that the enzymic synthesis of cellulose is specifically activated by guanosine 5 -triphosphate in the presence of a protein factor and poly(ethyleneglycol)372 or calcium ions.373 This activation results in a dramatic increase in the rate of synthesis of the polymer. The enzyme, solubilized by treatment of membrane preparation with digitonin,373 retains its regulatory properties, and does not show any requirements in lipids for its activity. 

Radioiodination of galactose oxidase. The chloramine T procedure (12) was used for the radioiodination of galactose oxidase. The enzyme, solubilized in 0.01 M sodium phosphate buffer, pH 7.4, was labeled using 1 mCi Na 125I (13-17 mCi/ngl), 0.42 mM chloramine T (Eastman), and 1.14 mM sodium metabisulfite (Baker). Unreacted iodide was separated from the iodination enzyme by dialysis, and the enzyme was diluted in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.001 M cupric sulfate. 

A variety of methods for preparing collagen films from enzyme-solubilized, monomeric collagen with diflFerent surface structures were evaluated. Our initial interest was to determine the effects of various preparative techniques on the in vivo behavior of the films. 

Figure 1 is a control (not crosslinked) film prepared by method II with enzyme-solubilized collagen. The film was removed after 7 days... 

Figure 2 is the same type of film removed after 14 days of intramuscular implantation. Here the collagen is fragmented and infiltrated with infiammatory cells. After 21 days, all control films disappeared except for the ones prepared from a mixture of insoluble collagen fiber and enzyme-solubilized collagen. Some of these remained for 30 days. 

All of the crosdinked implants lasted for at least 60 days. At this time, a few of the enzyme-solubilized collagen films crosslinked with either glutaraldehyde or dialdehyde starch appeared to be thinner. Some of the films crosslinked with glutaraldehyde appeared to be somewhat thinner after 90 days, although most were intact. 

Figure 3 is a method II enzyme-solubilized collagen film crosslinked with glutaraldehyde and implanted subcutaneously. It was unchanged after 90 days. 

A recent and exciting area of research is the solubilization of enzymes in nonaqueous solvents. One way solubilization is achieved is through noncovalent complexes of lipid (surfactant) and protein, to be referred to here as enzyme-lipid aggregates, or ELAs. Such complexes are reported to be highly active and stable. Moreover, the activity of ELAs can be significantly higher than free, suspended enzyme (in the absence or presence of surfactant), enzymes solubilized in aqueous-organic biphasic systems, or reverse micellar solutions, and can approach catalytic rates in... 

The primary structures of ctyochrome fe from several mammalian livers have been studied mainly by two groups using the enzyme-solubilized preparations which are supposed to correspond to about 70% of the... 

Modification of fish proteins by proteolytic enzymes to increase their solubilities illustrates a variety of techniques and approaches. Basically, three general enzymic methods have been used to prepare fish proteins or hydrolysates with altered solubilities and other functionalities. These methods include (a) the enzymic solubilization of fish protein concentrate prepared by hot solvent extraction of fish, (b) the enzymic modification of myofibrillar proteins extracted from fish with 0.6M NaCl, and (c) the proteolysis of whole fish to prepare biological fish protein concentrate (FPC). 

In addition to studies on enzymic solubilization of solvent-extracted FPC, the relative effectiveness of more than 20 commercially available proteolytic enzymes in the production of hydrolysates from a specially prepared haddock protein substrate has been determined (13). The concentration of enzyme, at its optimum temperature and pH, required to effect a 60% digestion in a 24-hr period was the inverse measure of enzyme activity. Pronase exhibited the greatest activity per unit weight. In general the microbial proteases ranked low in relative activity. Porcine pepsin, papain, and pancreatin combined good activity with moderate cost. 

The term maceration is frequently used for describing the degradation of plant tissue owing to enzymic solubilization of middle lamella pectate and cell wall. The author exhibits a preference for the term cell separating enzyme to macerating enzyme, because the former means an enzyme having only protopectinolytic activity. 

Optimal activity of the purified enzyme solubilized in Triton X-100 is obtained in the presence of excess phospholipids. The pH optimum of the steady-state reaction with horse heart ferrocytochrome c occurs at pH 6, yielding a turnover of about 80 electrons/sec, similar to the value obtained for the enzyme from P. denitrificans. Remarkably, a purified membrane-bound c cytochrome, identified by its N-terminal sequence as cytochrome Ci from the 6c 1 complex, stimulates the rate of electron transfer between horse heart c5d ochrome c and the cytochrome c oxidase by about a factor of two. The in vitro enzyme assay with purified cytochrome oxidase and reduced amicyanin showed no activity only after the addition of endogenous cytochrome C550 (or horse heart cjfto-chrome c) did oxidation of amicyanin occur, in agreement with the sequence of electron transfer ami — cjft C550 CCO. 

Chein, J. C. W., and Wise, W. B. (1975). Biochemistry 14, 2786. A 1 3C Nuclear Magnetic Resonance and Circular Dichroism Study of the Collagen-Gelatin Transformation in Enzyme Solubilized Collagen. 

The catalytic constant k at is quite another matter it is a first-order rate constant the dimension ([time] ) of which does not contain concentration. Therefore, this parameter in reverse micelles is not complicated by the distribution effects of the substances and may be regarded as an objective parameter, reflecting a true reactivity of the enzyme solubilized in the system of reverse micelles. 

NL Klyachko. Kinetic Peculiarities of Enzymes Solubilized by Reverse Micelles. PhD dissertation, Moscow State University, Russia, 1983. 

Luisi, P. L., Meier, P., Wolf, R. (1980) Properties of enzymes solubilized in hydrocarbons via reverse micelles. In Enzyme Engineering, Vol. 5 (Weetall, H. H., Royer, G. P., editors). New York Plenum Publishing Co... 

Enzyme applications cofactor recycling techniques, enzyme immobilization, enzyme solubilization, enzyme recycling, multi-enzyme clusters, mini- and micro-scale application tests, new enzyme analytical and separation techniques. 

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