Activation of lipase

Within the small intestine, bile-acid binding interferes with micelle formation. Nauss et al. [268] reported that, in vitro, chitosan binds bile acid micelles in toto, with consequent reduced assimilation of all micelle components, i.e., bile acids, cholesterol, monoglycerides and fatty acids. Moreover, in vitro, chitosan inhibits pancreatic lipase activity [269]. Dissolved chitosan may further depress the activity of lipases by acting as an alternative substrate [270]. 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

Yang and Russell [7] made comparison of lipase-catalyzed hydrolysis in three different systems organic, biphasic, and reversed micelles. They affirmed that water content is an important factor that distinctly affects every system. Their results demonstrated that activity of lipase in organic-aqueous biphasic media was lower than that obtained in reversed micelles. However, better productivities were obtained in biphasic media, which were the most suitable environment. 

An example of the appropriate application of organically-modified silica precursors is alkoxides with an alkyl group. When methyltrimethoxy- or methyl-triethoxysilane (Figure 3.2) was added in formulations to increase the hydro-phobicity of ORMOSILs, it resulted in a better enzymatic activity of lipases immobilized in the alkyl-modified silica than in a hydrophilic matrix fabricated by means ofTEOS alone [51,80,129-133]. Similarly, an increased stability of lipase from Candida antarctica B was observed after its immobilization in a silica matrix... 

This encapsulation procedure gives the highest activity for the lipases. The lecithin/ amines mixture structuring the pore network leads to a suitable phospholipids bilayer-like environment, which avoids the necessity to create an interface by substrate assembly. Monduzzi and coworkers compared the activity of lipase that was immobilized on SBA-15 physically, or chemically with glutardialdehyde [200]. 

Y. Kakizawa, K. Akiyoshi, K. Nakamura, and J. Sunamoto, in Enzymatic Activity of Lipase Complexed with Nanoparticle of Hydrophobized Polysaccharide, Kyoto, Japan, 1998, p. III-806. Society of Polymer Science, Japan (Spsj). 

Monoglyceride (MG) is one of the most important emulsifiers in food and pharmaceutical industries [280], MG is industrially produced by trans-esterification of fats and oils at high temperature with alkaline catalyst. The synthesis of MG by hydrolysis or glycerolysis of triglyceride (TG) with immobilized lipase attracted attention recently, because it has mild reaction conditions and avoids formation of side products. Silica and celite are often used as immobilization carriers [281], But the immobilized lipase particles are difficult to reuse due to adsorption of glycerol on this carriers [282], PVA/chitosan composite membrane reactor can be used for enzymatic processing of fats and oils. The immobilized activity of lipase was 2.64 IU/cm2 with a recovery of 24%. The membrane reactor was used in a two-phase system reaction to synthesize monoglyceride (MG) by hydrolysis of palm oil, which was reused for at least nine batches with yield of 32-50%. 

The lipid of morama beans is mainly ( 75%) unsaturated fatty acids, with the principal fatty acid being oleic acid (43%). The beans furthermore contain linoleic (22%) and palmitic acid (13%) as well as stearic, arachidic, linolenic, arachidonic, erucic, behenic, myristic, palmitoleic, and gadoleic acid in lower concentrations (Bousquet, 1982 Bower et ah, 1988 Engelter and Wehmeyer, 1970 Francis and Campbell, 2003 Ketshajwang et ah, 1998 Mitei et ah, 2008). The fatty acid composition resembles that of olive oil (Mitei et ah, 2008). A literature review of the fatty acid composition of morama beans is given in Table 5.3. Less than 5% of the fatty acids are present as free acids (Bower et ah, 1988 Dubois et ah, 1995), which means that the activity of lipases is negligible in dry morama beans. 

The influence of surfactant on the catalytic activity of lipases in water is well known. The addition of surfactants can enhance the activity and enantioselectivity of these enzymes in aqueous solutions [94] due to the interfacial activation and due to the emulsification of hydrophobic substrates. 

If the bonded water is extracted by dry CO2 the enzyme is denaturated and loses its activity. Therefore a certain amount of water is necessary in the supercritical fluid because acting with water-saturated CO2 again causes an inhibition of the enzyme and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Table 9.2-1 shows the residual activity of lipase from Candida cylindracea, esterase from Mucor mihei, and esterase from Porcine liver after a incubation time of 22 hours in supercritical CO2 at 40°C. It is obvious that higher water concentrations cause a strong reduction in the residual activity compared to the activity of the untreated enzyme, which was set as 100 %. Further, the system-pressure has an influence because at higher pressures the activity-loss is lower with a larger amount of water in the system [7,8],... 

Jacks, T. J., and Kircher, H. W., 1967, Fluorometric assay for hydrolytic activity of lipase using fatty acyl esters of 4-methylumbelliferone Anal. Biochem. 21 279-285. 

Valivety, R.H., Hailing, P.J., Peilow, A.D., Macrae, A.R. 1994. Relationship between water activity and catalytic activity of lipases in organic media. Effects of supports, loading and enzyme preparation. Eur. J. Biochem. 222, 461 466. 

Numerous methods have been used to measure the activity of lipases from various sources (Jensen 1983 Thomson et al., 1999 Deeth and Touch, 2000 Chen et al., 2003). They vary considerably in the substrate used, the form of the substrate, additives to the assay mix and the method for determining the extent of hydrolysis. 

True lipases show the interfacial activation phenomenon in their catalytic activity pattern. At low concentration of water-insoluble substrates, lipases are almost inactive, and the hydrolytic activity does not increase linearly. At a certain substrate concentration, however, the hydrolytic activity of lipases increases rapidly and the lipase kinetics resembles normal enzyme kinetics. This boost in activity is related to the formation of water-insoluble substrate aggregates such as micelles or another second phase. Only when this second phase is present, do lipases become fully active. This interfacial activation is caused by a large conformational change in the 3D structure of the lipases. In their water-soluble form, the active site is covered by a lid, which prevents the substrates from reaching it. At the lipidAvater interface, the lid is opened and the active site is accessible to the substrates. In addition, the now accessible area is mainly hydrophobic, which gives the open-form lipase the shape and behavior of conventional surfactant molecules with a hydrophilic and a hydrophobic moiety in one single molecule. 

The remaining activity of Lipase A was approximately 50% whereas the activity of Lipase B was almost inactivated. It is therefore assumed that Lipase A is responsible for the high thermostability observed in crude lipase preparations from C. antarctica. 

In seeds, lipases may cause fat hydrolysis unless the enzymes are destroyed by heat. Palm oil produced by primitive methods in Africa used to consist of more than 10 percent of free fatty acids. Such spoilage problems are also encountered in grains and flour. The activity of lipase in wheat and other grains is highly dependent on water content. In wheat, for example, the activity of lipase is five times higher at 15.1 percent than at 8.8 percent moisture. The lipolytic activity of oats is higher than that of most other grains. 

It can be noted that the way in which the enzyme is prepared in the dry form for catalysis in organic solvent is responsible for striking differences (up to two orders of magnitude) in the enzyme-specific activity. Furthermore, it is worth mentioning that the transesterification activity of lipase from B. cepacia entrapped in sol gel (sol gel-AK-lipase BC) was 83% of the activity in water measured using tributyrin as a substrate [6]. Analogously, in the case of CALB lyophilized with methoxypoly(ethylene glycol) (CALB -i- PEG) the activity was 51% of the activity in water in the hydrolysis of vinyl acetate [7]. It is important to note that, for both... 

Immobilization of lipases on hydrophobic supports has the potential to (1) preserve, and in some cases enhance, the activity of lipases over their free counterparts (2) increase their thermal stability (3) avoid contamination of the lipase-modified product with residual activity (4) increase system productivity per unit of lipase employed and (5) permit the development of continuous processes. As the affinity of lipases for hydrophobic interfaces constitutes an essential element of the mechanism by which these enzymes act, a promising reactor configuration for the use of immobilized lipases consists of a bundle of hollow fibers made from a microporous hydrophobic polymer (137). 

Conflicting hypotheses were also put forward with respect to the mechanism of interfacial activation. Desnuelle et al. (1960) were the first to suggest that a conformational change in the enzyme could be responsible for the enhancement of activity at the oil-water interface. There were also other hypotheses. For example. Wells (1974) suggested that the apparent activation of lipases is due to the orientation of the scissile ester bond on the surface of micelles Brockerhoff (1968), on the other hand, pointed to the possibility of differences in solvation of the ester bond in solution versus a lipid phase, whereas Brockman et al. (1973) postulated that a steep substrate concentration gradient at the interface may provide an explanation. 

Activation of lipases generates free fatty acids and lysolecithins. Lysolecithins are adsorbed within one minute by the cell wall [129], causing a reorganization of cell membrane constituents [130], recognizable by a change of the cell membrane shape [131]. Lysolecithines are reported to activate (at least in mammalian tissue) kinases [132]. 

The main response of plants to wounding either to mechanically injury but also by pathogen attack is activation of lipases, followed by those of lipoxygenases as outlined above. Lipoxygenases remove from PUFAs a hydrogen atom localized at a double allylically activated methylene group, forming a mesomeric radical (Scheme 2). 

AHAs, even at low concentrations, acidify the upper layers of the skin. Experimentally, at pH <5, this acidification causes an increase in the epidermal activity of lipases, phosphatases and transforming growth factor beta (TGF-P). At higher concentrations, the protein chains are separated from each other, and this causes epidermolysis - more or less visible exfoliation. Because of this corneocyte-shedding activity, AHAs have been used primarily to treat certain... 

Lipases are interphase-active enzymes with hydrophobic domains. The hydro-phobic surface (loop) on lipase is thought to enable lipophilic interfacial binding with substrate molecules that actually induces the conformational changes in lipases. The open conformation will provide substrate with access to the active site, and vice versa. In certain types of lipases, the movements of a short a-hehcal hydrophobic loop in the lipase structure cause a conformational change that exposes the active sites to the substrate. This movement also increases the nonpolarity of the surface surrounding the catalytic site [30, 32, 34, 35]. Obviously, the hydrophobic surface plays an important role in the activity of lipase as an enzyme. 

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