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Lipase substrate

Hydrolysis of substrates is performed in water, buffered aqueous solutions or biphasic mixtures of water and an organic solvent. Hydrolases tolerate low levels of polar organic solvents such as DMSO, DMF, and acetone in aqueous media. These cosolvents help to dissolve hydrophobic substrates. Although most hydrolases require soluble substrates, lipases display weak activity on soluble compounds in aqueous solutions. Their activity markedly increases when the substrate reaches the critical micellar concentration where it forms a second phase. This interfacial activation at the lipid-water interface has been explained by the presence of a... [Pg.133]

The asymmetric hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (37) to the corresponding chiral monoacetate ester (38) (Fig. 12B) has been demonstrated with lipases [61]. Lipase PS-30 from P. cepacia was most effective in asymmetric hydrolysis to obtain the desired enantiomer of monoacetate ester. The reaction yield of 75 M% and e.e. of >99% were obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/liter of the substrate. Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused (5 cycles) without loss of enzyme activity, productivity, or e.e. of product (38). The reaction process was scaled up to 80 liters (400 g of substrate) and monoacetate ester (38) was isolated in 80 M% yield with 99.3% e.e. The product was isolated in 99.5% chemical purity. The chiral monoacetate ester (38) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give chiral lactol (33) (Fig. 12B). The chiral lactol (33) obtained by this enzymatic process was used in chemoenzymatic synthesis of thromboxane A2 antagonist (35). [Pg.156]

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. [Pg.1385]

Fig. 4. Model showing the interactions between the endothelial cell surface, triacylglycerol-rich lipoproteins, apo C2, and lipoprotein lipase (LPL). Two LPL molecules are shown reacting with the same VLDL particle. These are representative of the multiple LPLs that probably react with each triacylglycerol-rich lipoprotein. The location of the recently identified glycosylphosphatidylinositol-anchored HDL binding protein-1 within the substrate-lipase complex has not yet been identified. Fig. 4. Model showing the interactions between the endothelial cell surface, triacylglycerol-rich lipoproteins, apo C2, and lipoprotein lipase (LPL). Two LPL molecules are shown reacting with the same VLDL particle. These are representative of the multiple LPLs that probably react with each triacylglycerol-rich lipoprotein. The location of the recently identified glycosylphosphatidylinositol-anchored HDL binding protein-1 within the substrate-lipase complex has not yet been identified.
The enzyme can also operate on diacetates, though with much poorer enantioselectivity. For such substrates lipases usually give better results.t l... [Pg.188]

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). [Pg.343]

Candida cylindracea, phosphate buffer pH 7, Bu20. The 6-0-acetyl of Q -methyl peracetylglucose was selectively removed. Porcine pancreatic lipase will also hydrolyze acetyl groups from carbohydrates. These lipases are not specific for acetate since they hydrolyze other esters as well. In general selectivity is dependent on the ester and the substrate. ... [Pg.90]

Janda, K.D., Benkovic, 5.J., Lerner, R.A. Catalytic antibodies with lipase activity and R or 5 substrate selectivity. Science 244 437-440, 1989. [Pg.322]

Many substrates currently produced in the chemical industry are immiscible with water, but are readily miscible with organic solvents. Most enzymes, however, will not operate efficiently, or not operate at all, in non-aqueous media. Some exceptions do exist, such as lipases and esterases, which can operate in non-aqueous environments. Currently, there is considerable interest in extending the range of enzymes that do work in organic solvents. [Pg.23]

Mixtures of triglycerides, triglycerides plus free fatty adds or triglycerides plus fatty acid alkyl esters are used as reactants in fat modification processes. These mixtures are exposed to lipases supported on macroporous particles in the presence of a small amount of water. Liquid substrates (oils) can be reacted without use of a solvent, but with solid reactants (fats) it is necessary to add a solvent to ensure that the reactants and products are completely dissolved in the organic phase. Various water immisdble solvents can be used, but hexane is preferred for commercial operation because this solvent is already used industrially for the processing of oils and fats. [Pg.332]

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. [Pg.130]

Table 5.1 presents the intrinsic kinetic parameters (Km and Vln lx) for the free lipase system and apparent kinetic parameters (K and V ) for the immobilised lipase in the EMR using fixed 2g-l 1 lipase concentration. The immobilised lipase showed higher maximum apparent reaction rate and greater enzyme-substrate (ES) affinity compared with free lipase. [Pg.131]

The inhibition analyses were examined differently for free lipase in a batch and immobilised lipase in membrane reactor system. Figure 5.14 shows the kinetics plot for substrate inhibition of the free lipase in the batch system, where [5] is the concentration of (S)-ibuprofen ester in isooctane, and v0 is the initial reaction rate for (S)-ester conversion. The data for immobilised lipase are shown in Figure 5.15 that is, the kinetics plot for substrate inhibition for immobilised lipase in the EMR system. The Hanes-Woolf plots in both systems show similar trends for substrate inhibition. The graphical presentation of rate curves for immobilised lipase shows higher values compared with free enzymes. The value for the... [Pg.131]

A single hydrolase is usually inadequate for the degradation of a carrier, but most hydrolases have unspecific activities, i.e., they split the chains of polymers that are not their typical substrates. For example, chitosan is susceptible to lipases, pectinases, amylases among others [257-260]. [Pg.187]

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]. [Pg.188]

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]. [Pg.9]

An example that refers to the third method additives can be employed is described below. Markedly enhanced enantioselectivity was reported for P. cepacia lipase and subtilisin Carlsberg with chiral substrates converted to salts by treatment with numerous Bronsted-Lowry adds or bases [63]. This effect was observed in various organic solvents but not in water, where the salts apparently dissociate to regenerate... [Pg.16]

Figure 2.11 CASTing of the lipase from Pseudomonas aeruginosa (PAL) leading to the construction of five libraries of mutants (A-E) produced by simultaneous randomization at sites composed of two amino acids. (For illustrative purposes, the binding of substrate (1) is shown) [25],... Figure 2.11 CASTing of the lipase from Pseudomonas aeruginosa (PAL) leading to the construction of five libraries of mutants (A-E) produced by simultaneous randomization at sites composed of two amino acids. (For illustrative purposes, the binding of substrate (1) is shown) [25],...
Figure 2.12 Substrate profiles of lipase variants produced by CASTing [25]. Figure 2.12 Substrate profiles of lipase variants produced by CASTing [25].
The Bacillus subtilis lipase A (BSLA) was the subject of two short directed evolution studies [19,47]. In one case systematic saturation mutagenesis at all of the ISlpositions of BSLA was performed [19]. Using meso-l,4-diacetoxy-2-cyclopentene as the substrate, reversed enantioselectivity of up to 83% ee was observed. In another study synthetic shuffling (Assembly of Designed Oligonucleotides) was tested using BSLA [47]. [Pg.38]

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... [Pg.38]

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). [Pg.69]

A novel approach was developed very recently by Kita et al. [15]. DKR of allylic alcohols was performed by combining a lipase-catalyzed acylation with a racemization through the formation of allyl vanadate intermediates. Excellent yields and enantioselectivities were obtained. An example is shown in Figure 4.4. A limitation with this approach for the substrates shown in Figure 4.4 is that the allylic alcohol must be equally disubstituted in the allylic position (R = R ) since C—C single bond rotation is required in the tertiary alkoxy intermediate. Alternatively, R or R can be H if the two allylic alcohols formed by migration of the hydroxyl group are enantiomers (e.g. cyclic allylic acetates). [Pg.93]

Very recently the Meerwein-Ponndorf-Verley-Oppenauer (MPVO) reaction has been exploited for the racemization of alcohols using inexpensive aluminum-based catalysts. Combination of these complexes with a lipase (CALB) results in an efficient DKR of sec-alcohols at ambient temperature. To increase the reactivity of the aluminum complexes, a bidentate ligand, such as binol, is required. Also, specific acyl donors need to be used for each substrate [31] (Eigure 4.9). [Pg.96]

Kanerva et al. have also reported DKR of cyanohydrins [47]. In particular, they obtained very good results with C. antartica lipase A (CAL-A) as the catalyst for the KR of a variety of substrates for which other enzymes such as CALB or PS-C do not give good results (Figure 4.22) [47a[. [Pg.103]


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See also in sourсe #XX -- [ Pg.201 ]

See also in sourсe #XX -- [ Pg.201 ]




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