Lipases types

An interesting example of biocatalysis and chemical catalysis is the synthesis of a derivative of y-aminobutyric acid (GABA) that is an inhibitor for the treatment of neuropathic pain and epilepsy (Scheme 10.4). The key intermediate is a racemic mixture of cis- and trons-diastereoisomer esters obtained by a hydrogenation following a Horner-Emmons reaction. The enzymatic hydrolysis of both diaste-reoisomers, catalyzed by Candida antarctica lipase type B (CALB), yields the corresponding acid intermediate of the GABA derivative. It is of note that both cis- and trans-diastereoisomers of the desired enantiomer of the acid intermediate can be converted into the final product in the downstream chemistry [10]. 

In 2004, Ley et al. [45] showed a stereoselective enzymatic synthesis of cis-pellitorine [N-isobutyldeca-(2 ,4Z)-dienamide], a taste-active alkamide naturally occurring in tarragon. The reactants were ethyl ( ,Z)-2,4-decadienoate— the pear ester described before—and isobutyl amine. The reaction is catalysed by lipase type B from Candida antarctica (commercially available), which shows a remarkable selectivity towards the 2 ,4Z ester. The yield was about 80%. 

The synthesis of chiral poly(depsipeptides), polymers with alternating amide and ester bonds, by lipase-catalyzed ring opening of 3-isopropyl morpholino-2,5-dione (19) was shown by Hocker and coworkers (Scheme 11.5) [26], Various lipases were tested for the bulk polymerization of these heterocyclic monomers at temperatures of 100 °C or above. PPL and lipase type III from a pseudomonas species were shown to be effective catalysts. The isolated polymers showed Mn values of 3.5-17.5 kgmol-1. The influence of reaction temperature, the amount of enzyme and the presence of water in the reaction medium were shown to be important factors on the high molecular weight fraction and were investigated in detail [26b]. Comparison of optical rotation values for polymers prepared by... 

Name of the product Lipase Type XIII CHIRAZYME L-2, lyo... 

Lipase, type A Origin Candida antarctica Altus Industrial... 

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. 7 Operational stability of (closed square) lipase type B of Candida antarctica adsorbed on green coconut fiber and (closed circle) Novozyme 435 in subsequent batches of methyl butyrate hydrolysis...

Jensen RG, Hamosh M (1996) Selectivity of lipases, types and determination. In Malcata FK (ed). 

Attention was first drawn to this type of enzyme by Hahn in 1943 (367), who showed that injections of heparin into the blood of an animal with a high level of chylomicrons led to rapid clearing of the plasma, and that this was a result of a lipase-type action. Adition of... 

Therefore it can be concluded that PTMC homopolymer is not degraded by pure hydrolysis, but can be degraded in vivo or in vitro in the presence of lipase-type enzymes. 

Lipase activity is detected, for example, in milk, oilseeds (soybean, peanut), cereals (oats, wheat), fruits and vegetables and in the digestive tract of mammals. Many microorganisms release lipase-type enzymes into their culture media. 

Coumaric acid octanol lipase type Katsoura et al. (2006) 33... 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtihsin Novo (Genencor, California). Lipase and ceUulase types of detergent enzymes have also begun to appear. 

In addition to having the required spedfidty, lipases employed as catalysts for modification of triglycerides must be stable and active under the reaction conditions used. Lipases are usually attached to supports (ie they are immobilised). Catalyst activity and stability depend, therefore, not only on the lipase, but also the support used for its immobilisation. Interesterification reactions are generally run at temperatures up to 70°C with low water availability. Fortunately many immobilised lipases are active and resistant to heat inactivation under conditions of low water availability, but they can be susceptible to inactivation by minor components in oils and fats. If possible, lipases resistant to this type of poisoning should be selected for commercial operations. 

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). 

Esterases, proteases, and some lipases are used in stereoselective hydrolysis of esters bearing a chiral or a prochiral acyl moiety. The substrates are racemic esters and prochiral or meso-diesters. Pig liver esterase (PLE) is the most useful enzyme for this type of reaction, especially for the desymmetrization of prochiral or meso substrates. 

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]. 

FIGURE 3.2.2 Metabolic pathways of carotenoids such as p-carotene. CM = chylomicrons. VLDL = very low-density lipoproteins. LDL = low-density lipoproteins. HDL = high-density lipoproteins. BCO = p-carotene 15,15 -oxygenase. BCO2 = p-carotene 9, 10 -oxygenase. LPL = lipoprotein lipase. RBP = retinol binding protein. SR-BI = scavenger receptor class B, type I. 

We initially tested Candida antarctica lipase using imidazolium salt as solvent because CAL was found to be the best enzyme to resolve our model substrate 5-phenyl-l-penten-3-ol (la) the acylation rate was strongly dependent on the anionic part of the solvents. The best results were recorded when [bmim][BF4] was employed as the solvent, and the reaction rate was nearly equal to that of the reference reaction in diisopropyl ether. The second choice of solvent was [bmim][PFg]. On the contrary, a significant drop in the reaction rate was obtained when the reaction was carried out in TFA salt or OTf salt. From these results, we concluded that BF4 salt and PFg salt were suitable solvents for the present lipase-catalyzed reaction. Acylation of la was accomplished by these four enzymes Candida antarctica lipase, lipase QL from Alcaligenes, Lipase PS from Burkholderia cepacia and Candida rugosa lipase. In contrast, no reaction took place when PPL or PLE was used as catalyst in this solvent system. These results were established in March 2000 but we encountered a serious problem in that the results were significantly dependent on the lot of the ILs that we prepared ourselves. The problem was very serious because sometimes the reaction did not proceed at all. So we attempted to purify the ILs and established a very successful procedure (Fig. 3) the salt was first washed with a mixed solvent of hexane and ethyl acetate (2 1 or 4 1), treated with activated charcoal and passed into activated alumina neutral type I as an acetone solution. It was evaporated and dried under reduced... 

Imidazolium PFg or BF4 salts were frequently used as solvent for the present lipase-catalyzed reaction. However, these salts are very expensive, and we should develop cheaper ILs. Imidazolium alkyl sulfates might be good candidates because various types of alkyl sulfates can be easily prepared. The imidazolium alkyl sulfate was prepared starting from the corresponding ammonium alkyl sulfate as follows ammonium alkyl sulfates ([NH4][RS04]) are easily prepared by the reaction of... 

We recently prepared various types of differently fiuorinated alkyl sulfate ILs and discovered that the hydrophobicity was dependent on the content ratio of the fluorine on the alkyl sulfate anion and 2,2,3,3,4,4,5,5-octafiuoropentyl sulfate salts showed hydrophobic properties. Melting point and viscosity were also dependent on the fluorine contents of the anionic part, while conductivity was determined by the cationic part and not influenced by the fluorine contents. Efficient lipase-catalyzed transesterificafion was demonstrated using hydrophobic 1-butyl-3-methylimidazolium 2,2,3,3,4,4,5,5-octafiuoropentyl sulfate ([bmim][C5E8]) as solvent (Eig. 6). ... 

In the reaction, it was essential to use an IL as a co-solvent. Lozano, Iborra and co-workers recently reported an interesting stabilizing effect of two types of water-immiscible ILs ([emim][TFSI] and [BuMe3N][TFSI]) for CAL-B-catalyzed transesterification of vinyl butyrate. The synthetic activity and the stability of the enzyme in these IL solvent systems were markedly enhanced as compared to those in hexane. CAL-B maintained its activity higher than 75% after 4 days of incubation in [emim][TFSI] solvent, while it showed an activity of only 25% when incubated in both water and hexane media at 50°C. Comparison of the ratio of a-helix and (3-sheet by CD spectra showed the activity was closely related with a-helix content which reduced to 31% immediately after lipase was added to hexane and had reached only 2% after 4 days in hexane. On the contrary, no significant reduction of a-helix content was... 

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