Lipase amount adsorbed

Lipases are extraordinary enzymes in the sense that the lipase is functioning on an interface between two phases (oil/water). This interface and the low amount of water are obtained by using immobilized lipase. The lipase is adsorbed on carriers such as anion exchange resins or adsorbers and covered with a thin film of water. 

About lO.Omg of thermally crosslinked PAA/PVA electrospun fibrous membrane was immersed in 2.0ml of the selected pH buffer solution with the lipase concentration at 5.0mg/ml. After the mixture was incubated in the water bath at 30°C for 24 hrs, the fibrous membrane was taken out and rinsed with the same pH buffer. The washing pH buffer was added to the residue solution left fi-om the adsorption to make up the total solution volume to be exactly 10.0ml in a volumetric flask. The concentration of lipase in the above solution was then determined by Bio-Rad (Hercules, CA) protein assay (7, //). The amount of lipase physically adsorbed by the electrospun membrane was calculated by deducting the lipase amount in the buffer solution fi om the original total amount. The average fi om measurements of three samples was reported. 

Figure 4. Amount of lipase physically adsorbed in PAA/PVA (COOH/OH molar ratio = 3.5) hydrogel fibers in buffers with different pH values.

Highly active catalysts have been produced by adsorption of lipases onto macroporous acrylate beads, polypropylene particles and phenol-formaldehyde weak anion exchange resins. Protein is bound, presumably essentially as a monolayer, within the pores of the particles. The large surface area of the particles (10m2 g 1) means that substantial amounts of protein can be adsorbed, and the pores are of sufficient size to allow easy access of reactants to this adsorbed protein. 

For lipase, initial activity corresponds to the amount of protein that was adsorbed. Specific activity is constant at 1 mmoFs gE for this carrier-enzyme system, which compares to 27% of the free enzyme activity. The trypsin system shows a lower specific activity that is only 10% of the free enzyme. The reason for the lower recovered activity of this system is not known. To rule out possible internal diffusion limitations, the Wheeler-Weisz modulus was estimated, assuming a carrier layer thickness of 0.1 mm for all carriers. Using the data of the experiments performed at 150 rpm, one finds ... 

The Accurel EPIOO carrier, macro porous polypropylene granulates, was obtained from Akzo Nobel. Particle size was in the range of 200-1000 m. 1 gram of carrier was washed with 96% ethanol and water prior to the immobilization. Lipase solution (purified and concentrated solution of Humicola lanuginosa lipase (103 KLU/ml), pH 7.5) was added to the wet carrier. The lipase loadings were in the range of 125-500 KLU/g carrier. The amount of adsorbed lipase activity was calculated from the difference in lipase activity in the supernatant before subtracted the lipase activity after adsorption. 

Experiments have been conducted to simulate leaching of phthalates from blood bags by allowing human plasma to extract added phthalates from coated Celite (Albro and Corbett 1978). It was found that more than 80% of the DEHP was associated with lipoproteins, in the order LDL > VLDL > HDL > chilomicrons. The remaining DEHP was adsorbed weakly and nonspecifically to other proteins including albumin. MEHP was in equilibrium between free in solution and adsorbed to albumin, no MEHP was bound to lipoproteins. Rock et al. (1986) reported that in human plasma, the lipase that hydrolyzes DEHP copurified with the albumin, and once in the plasma, the MEHP bound to albumin. An earlier study by Jaeger and Rubin (1972) reported that in human blood stored in PVC bags, the bulk of DEHP was associated with lipoproteins, but a substantial amount was in a fraction likely to represent DEHP soluble in plasma water as well as bound to plasma proteins and cell membranes. 

In a lipase-catalyzed reaction of a water-insoluble oily substrate, the enzyme reversibly adsorbs at the substrate-water interface, and the enzymatic reaction takes place at that interface. The interfacial area strongly depends upon some physical parameters of emulsion, such as the size of the emulsion droplets as well as the amount of the substrate present. However, the importance of the... 

Although the amount of C. antarctica lipase type B adsorbed to coconut fiber was nearly independent on the pH of adsorption (between pH 3 and 6), immobilization yield and recovered activity were dependent on the pH of adsorption because interactions between the molecule and its environment influence the structure of a protein molecule, and these interactions are pH-dependent [8],... 

Fig. 3 Effect of the pH of immobilization on the amount of adsorbed protein (closed circle) and on the hydrolytic activity (open square). Lipase was immobilized on coconut fiber by adsorption after 2 h of contact time at room temperature...

Fig. 9 Initial rate of hydrolysis of methyl butyrate by soluble lipase closed square), lipase immobilized on green coconut fiber closed circle) and Novozyme 435 closed triangle) in fully aqueous medium. The solid line represents the fit of Michaelis-Menten model to experimental data. The dashed lines represent a linear regression of the experimental data. The amount of adsorbed protein (50 mg/g of catalyser) in Novozyme 435 was determined by Secundo et al. [42]...

Table 2. Effect of fiber composition on the amount of adsorbed lipase.

As expected, the amount of lipase adsorbed in hydrogel fibers was much higher than that in the cast films with the same polymer composition because of the much larger specific surface of the fibers. For example, when the adsorption was performed in a pH 7 buffer, the amount of lipase immobilized in the cast film (3.5 COOH/OH molar ratio) was only about 58% of that in the hydrogel fibers of the same polymer composition. 

With this state of uncertainty, it is not possible to define the true structural and functional role of the chylomicron protein. Chylomicrons are generally considered as a large central sphere of glycerides with small amounts of cholesterol, phospholipid, and protein loosely adsorbed on the surface, forming, according to Lindgren and Nichols (1960), small lipoprotein subunits. Aside from physical stabilization, the adsorbed components at the surface appear to impart biochemical specificity to the chylomicron particles, as indicated by Korn s studies (1955) showing that chylomicrons, and not simple fat emulsions, form an optimal substrate for the enzyme lipoprotein lipase. 

Wang et al. (2006) immobilized the lipase enzyme from Candida rugosa by physical adsorption on the surface of polysulfone (PSF) composite nanofibrous membranes. PVP and PEG were used as additives, aiming to tailor the surface properties of the PS nanofibers, increasing their hydrophilicity. The results showed that the activity of the immobilized lipase increased with the content of PVP or PEG, whereas the adsorbed amount of lipase was unchanged. These results were attributed to the enrichment of PVP or PEG at the nanofiber surface. In another study, lipase from Pseudomonas cepacia was immobilized by physical adsorption onto electrospun PAN fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane (Sakai et al. 2010). 

Amount of adsorbed lipase, % from total lipase in the system Activity, referred to activity in homogeneous medium... 

Among spectroscopic methods, X-ray photoelectron spectroscopy (XPS), also known as electron spectroscopy for chemical analysis (ESCA), has been used for the analysis of enzyme-treated PET substrates [11, 27, 31, 102]. With XPS, the elemental composition of the top 10 nm of the PET surface is measured, and the presence of specific functional groups can be identified. XPS analysis of PET treated with a lipase from Thermomyces lanuginosus and a cutinase from Ther-mobifida fusca indicated increased amounts of hydroxyl and free acid groups, while the amount of carbon-carbon bonds decreased [11, 27], XPS has been shown to be very sensitive to contamination by residual enzyme protein strongly adsorbed to the polymer surface [31, 102]. 

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