Lipase enzyme solubilization

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

For some foods, incomplete extraction of color is obtained, probably due to the high binding affinity of dyes to the bulk of the food matrix, especially to proteins, lipids, and carbohydrates (156,161,162). This problem can be overcome by the use of selected solvents or enzymes to digest the food prior to extraction. Petroleum ether can be used to extract lipids (163). Acetone can be used to remove lipids and coagulate protein (164). Enzymes, such as amyloglucosidase (165,166), papain (167), lipase, pectinase, cellulase, and phospholipase, added to the sample and incubated under optimum pH and temperature conditions release synthetic colors bound to or associated with the food matrix. Furthermore, enzyme digestion can solubilize some foods, enabling analysis to be continued (156). 

There is considerable difference in the system behavior at high pressure batch and continuous performance. In the continuous system the decrease in conversion between 80 bar and 150 bar is about 30% at 50°C and not only some percent as in the batch system. The difference may be that in the continuous system at high pressure the water is removed from the enzyme and in the batch system, although it is solubilized in SC CO2, it remains in the system and the activity of the lipase only slightly decreases. 

Not all ILs are good solvents for proteins, however. There is the interesting example of lipase. Lipase is soluble in both aqueous and organic solvents, so it can be easily solubilized in ILs. Certain lipases even become dispersed or dissolved in some ILs. Since lipase is a very stable enzyme, it catalyzes the hydrolysis of lipids. Enzymatic activity is reported to be maintained in ILs [1]. There is not much published on the solubilization of biomaterials in ILs. In the present chapter we introduce a procedure to use in solubilizing biomaterials in ILs. First we consider the preparation of the IL, and then the chemical modification of biomaterials suitable for dissolution. We have found this procedure helpful when we tried to use electrochemicaUy active biomaterials in ILs. 

The lipase-solubilized reductase is inhibited by p-mercuribenzoate, is protected from this inhibition by NADPH, and the inhibition is relieved by thiols (10). Careful titration of this enzyme with p-mercuribenzoate at pH 6.5 results in an almost 3-fold stimulation upon addition of 2 moles of mercurial per flavin the control activity is again observed when 7 equivalents have been added. At pH 7.7, a stimulation of 70% is seen with 1 equivalent and loss of activity is complete (extrapolated) with 6 equivalents (245). The protection of the enzyme by NADPH against mercurial inhibition is reminiscent of the effects with NADH cytochrome 63 reductase (360). 

Studies on the mechanism of NADPH-cytochrome P-450 reductase have been carried out thus far only with the trypsin- or lipase-solubilized forms. Assuming that this enzyme is composed of several semi-autonomous domains, and assuming further that modification during solubilization is restricted to the domain involved in the interaction with cytochrome P-450, then, as was the case with NADH-cytochrome bs reductase, mechanism studies on the soluble enzyme will contribute to the ultimate understanding of the operation of the reconstituted system. The fact that the soluble reductase is composed of a single polypeptide chain gives hope that the modification is a subtle one. 

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... 

A reexanaination of the mechanism of NADPH-cytochrome P-450 reductase has followed the crucial finding that all forms of this enzyme (detergent-, lipase-, and trypsin-solubilized) contain equimolar amounts of FAD and FMN suggesting that the flavins might have distinct roles (878). Distinct roles have been found for the FAD and FMN in sulfite oxidase (40O, 4OI). The static spectral results with the lipase-solubilized... 

Lower lipase activity observed in the transesterification of methylmethacrylate with 2-ethylhexanol in SCCO2 was attributed to the formation of carbamates between CO2 and free amino groups on the enzyme surface (187, 188). The decrease in pH as a result of CO2 solubilization in the microaqueous environment surrounding the enzyme is also thought to contribute to a decrease in enzyme activity (188). 

Water affects the reaction rate through its effect on reaction kinetics and protein hydration, which is required for optimal enzyme conformation and activity. Enzymes need a small amount of water to maintain their activity however, increasing the water content can decrease the reaction rate as a result of hydrophilic hin-drance/barrier to the hydrophobic substrate, or because of denaturation of the enzyme (189). These opposite effects result in an optimum water content for each enzyme. In SCFs, both the water content of the enzyme support and water solubilized in the supercritical phase determine the enzyme activity. Water content of the enzyme support is, in turn, determined by the distribution/partition of water between the enzyme and solvent, which can be estimated from water adsorption isotherms (141, 152). The solubility of water in the supercritical phase, operating conditions, and composition of the system (i.e., ethanol content) can affect the water distribution and, hence, determine the total amount of water that needs to be introduced into the system to attain the optimum water content of the support. The optimum water content of the enzyme is not affected by the reaction media, as demonstrated by Marty et al. (152), for esterification reaction using immobilized lipase in n-hexane and SCC02- Enzyme activity in different solvents should, thus, be compared at similar water content of the enzyme support. 

In some instances, water may not be necessary at all for the solubilization of the enzyme in a hydrocarbon solvent. A striking example has been provided by Okahata and coworkers [78] who solubilized lipase in benzene or n-hexane by coating the enzyme with the nonionic surfactant (24) or 2Ci6Br. The lipid-coated lipase showed activity for the synthesis of di- and triglycerides from monoglycerides and aliphatic acids. 

IL has been used to solubilize and stabilize a diverse number of proteins and enzymes. Remarkable enhancement in activity and stability has been observed in neat ILs or composites in many cases. However, it is important to note some disparity in results reported especially in the case of resolution of phenylethanol by lipases [ 18,19]. The contradictory results were ascribed to the purity of ILs used [87, 123, 124]. In particular, halides or HF contamination are known to decrease the activity of the enzyme [87]. With regard to enzyme compatibility of ILs, though hydrophilic ILs dissolve the enzymes, enzyme-IL interaction is strong enough to strip off the essential water... 

While it is a powerful solvent, DMSO is undesirable for industrial use. Instead, the surfactant Triton X-100 can be an effective replacement for DMSO in enzymatic hydrolyses [14], and in our case, worked very well. Triton X-100 forms micelles when mixed with water and would solubilize low concentrations of ester for hydrolysis. However, increasing the surfactant charge did not accelerate hydrolysis. In an attempt to reduce the somewhat long reaction time, we screened the enzymes Amano PS Lipase XIII, Pseudomonas jluorescens and Candida cylindracea versus Amano PS-30. PS-30 was still considered the best choice of enzyme when... 

Results of in vitro and model experiments indicate that proteases and lipases attack the cuticle earlier than the chitinases and that, together, those enzymes act in a synergistic manner to solubilize the cuticle. When grown in cultures containing comminuted cuticle, fungi produce endoproteases, exoproteases, lipases, esterases, chitinases and B-N-acetylglucosaminidases, with... 

Microsomal cytochrome 65 has been solubilized by treatment with pancreatic lipase 124-129), proteolytic enzymes 127-129), or detergents 130,130a). Solubilization by the lipase was suggested to result from the action of proteolytic enzymes contained in the lipase preparation or in the microsomal suspensions. 128). All methods for the purification of the solubilized cytochrome hs adopt, as main procedures, ammonium sulfate fractionation and chromatography on DEAE-cellulose as introduced by Strittmatter and co-workers 126, 131,132). DEAE Sephadex chromatography was also used for the separation of two forms of cytochrome 65 after treatment with trypsin 129,130). 

The fatty acid binding protein (AFABP/aP2) facilitates the intracellular solubilization and diffusion of fatty acids produced by lipolysis. The N-terminal domain of HSL is a docking site for interaction with AFABP/aP2. AFABP/aP2 stimulates the activity of HSL in vitro by relieving product inhibition by fatty acids, however, this activity seems to be independent of the interaction between AFABP/aP2 and HSL. The AFABP/aP2 may also shuttle fatty acids produced by ATGL or the monoacylglycerol lipase as well, but there is no evidence for physical association with these enzymes. Consistent with the maiel of AFABP/aP2 as a fatty acid shuttle facilitating efflux, AFABP/aP2 null mice exhibit reduced lipolysis while fatty acids accumulate intracellularly (D.A. Bemlohr, 1999). 

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