Lipases enzymatic hydrolysis using

The type of enzyme to be used, and quantification of degradation, will depend on the polymer being screened. For example, Mochizukiet studied the effects of draw ratio of polycaprolactone (PCL) fibres on enzymatic hydrolysis by lipase. Degradability of PCL fibres was monitored... 

Rizzarelli et al. [23] synthesized a series of copolymers with units of butylene succinate (BSu) and butylene adipate (BA) with different composition. The copolymers were subjected to enzymatic hydrolysis by lipase (actually two different lipase enzymes, obtained from Mucor miehei or from Rhizopus arrhizus). The degradation products were water soluble. Thus, they were injected in an LC apparatus (the column was a Cl 8) coupled with an ESI-MS. The LC trace displayed more than 20 peaks that were easily identified using MS. These are due to the monomers (BSu and BA), the dimers, the trimers, and the tetramers. LC peaks due to the oligomers rich in BA (e.g., BA3) are weak. On the other hand, LC peaks due to the oligomers rich in BSu (e.g.,BSus) are strong. The results indicate a preferential hydrolytic cleavage. In particular, succinic ester bonds are hydrolyzed faster than adipic ester bonds in BSu-BA copolyesters. 

Figure 8. Variation of the weight of specimens of a) PPSu, PESu and PBSu during enzymatic hydrolysis using Rhizopus delemar lipase and b) PPAd, PEAd and PBSu during enzymatic hydrolysis using 0.09 mg/mL Rhizopus delemar lipase and 0.01 mg/mL of Pseudomonas Cepacia Hpase.

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). 

The above-mentioned facts have important consequences on the stereochemical outcome of the kinetic resolution of asymmetrically substituted epoxides. In the majority of kinetic resolutions of esters (e.g. by ester hydrolysis and synthesis using lipases, esterases and proteases) the absolute configuration at the stereogenic centre(s) always remains the same throughout the reaction. In contrast, the enzymatic hydrolysis of epoxides may take place via attack on either carbon of the oxirane ring (Scheme 7) and it is the structure of the substrate and of the enzyme involved which determine the regioselec-tivity of the attack [53, 58-611. As a consequence, the absolute configuration of both the product and substrate from a kinetic resolution of a racemic... 

Fig. 13.11 Schematic representation of the hollow fiber membrane biorector for the enzymatic hydrolysis of triglycerides. A hydrophilic membrane has been used, coated with lipase on the lipid side [85].

Effect of Molecular Weight of Polyester on the Hydrolysis by Rhizopus lipase. Using three kinds of polyesters, PCL-diol (I), polyhexameth-ylene adipate (II), and a copolyester (ill) made from 1,6-hexamethyl-enediol and a 70 30 molar ratio mixture of e- caprolactone and adipic acid, the effects of the of polyester on the hydrolysis by lipase were examined (Figure k) Mn did not affect the rates of hydrolysis by R. arrhizus and delemar lipases when Vln was more than about UOOO. This would indicate these lipases randomly splits ester bonds in pol-mer chains. In contrast, when TEi was less than about i4000 2 the rates of the enzymatic hydrolysis were faster with the smaller Mn of polyesters. This corresponded to the fact that Tm was lower with the smaller Mn of polyesters. 

Holmes et al. (1998) performed two enzymatic reactions, the lipase-catalyzed hydrolysis of y>-nitrophenol butyrate and lipoxygenase-catalyzed peroxidation of linoleic acid, in w/c microemulsions stabilized by a fluorinated two-chained sulfosuccinate surfactant (di-HCF4). The activity of both enzymes in the w/c microemulsion environment was found to be essentially equivalent to that in a water/heptane microemulsion stabilized by Aerosol OT, a surfactant with the same headgroup as di-HCF4. The buffer 2-(A-morpholino)ethanesulfonic acid (MES) was used to fix the pH in the range 5-6. 

Neri et al89 reported the desymmetrization of A-Boc-serinol 98 by the selective monoacetylation using PPL (porcine pancreas lipase) and vinyl acetate as the acylating agent in organic solvent. The mono acetylated product (R)-99 was obtained after 2 hours with 99% ee and isolated in 69% chemical yield. Traces of the diacetylated product 100 were observed. The cyclization of (R)-99 in basic medium afforded the racemic oxazolidinone 101. The latter was subjected to enzymatic hydrolysis in phosphate buffer affording (R)-... 

Dissolution and Separation by Special Applications Enzymes and Microextraction Several enzymes such as trypsin, protease type XIV, lipase and/or cellulase, are used for enzymatic hydrolysis. For the determination of Hg2+ and Me-Hg in fish... 

Enzymatic syntheses of CPS have also been reported, using various lipase-catalyzed transacylations [69]. Interestingly, a biotechnological process to obtain natural vanillin from CPS has been developed, capitalizing on the enzymatic hydrolysis of CPS and the oxidation of vanillamine with a flavoprotein vanillyl alcohol oxidase [70]. 

Chlorotetaine is an irreversible inhibitor of glucosamine-6-phosphate synthetase and thereby interferes with ceil wall biosynthesis. The terminal steps of a synthesis of Chlorotetaine are shown in Scheme 6.15 in which deprotection of an iV-terminal amino group is a prelude to the final enzymatic hydrolysis of a methyl ester function.43 Critical to the success of the synthesis was the suppression of easy racemisation at the ring juncture in the ester hydrolysis step by using porcine pancreatic lipase. 

There are many reports of enzymatic catalysis in scC02 performing hydrolysis, oxidations, esterifications, and franr-esterification reactions. For example, the enzymatic kinetic resolution of 1-phenylethanol with vinyl acetate in scC02 using lipase from Candida antarctica B produces (R)-l-phenyethylacetate in >99% ee (i.e., enantiomeric excess, a measure of how much of one enantiomer is present as compared to the other), as shown in Figure 12.20. 

Oxidation of chroman-4-one and its thio analogue with Mn(OAc)3 gives the 3-acetates and subsequent basic hydrolysis yields the 3-hydroxychroman-4-one. Enzymatic hydrolysis of the 0-heterocycle using Amano PS lipase in a phosphate buffer selectively cleaved the (+)-isomer <03TA1489>. Enol ethers derived from chroman-4-one are converted into the 3-hydroxy-chromanone with high enantioselectivity, optimal with the pentyl ether, using a modified Sharpless asymmetric dihydroxylation reaction <03JOC8088>. 

Recent studies have attempted to improve the efficiency of epoxidation under milder conditions that minimize the formation of byproducts. Chemo-enzymatic epoxidation uses the immobilized lipase from Candida antartica (Novozym 435) (56) to catalyze conversion of fatty acids to peracids with 60% hydrogen peroxide. The fatty acid is then self-epoxidized in an intermolecular reaction. The lipase is remarkably stable under the reaction conditions and can be recovered and reused 15 times without loss of activity. Competitive lipolysis of triacylglycerols is inhibited by small amounts of fatty acid, allowing the reaction to be carried out on intact oils (57). Rapeseed oil with 5% of rapeseed fatty acids was converted to epoxidized rapeseed oil in 91% yield with no hydroxy byproducts. Linseed oil was epoxidized in 80% yield. Methyl esters are also epoxidized without hydrolysis under these conditions. 

Haas et al. (162) have studied enzymatic phosphatidylcholine hydrolysis in organic solvents by examining selected commercially available lipases. Enzymatic hydrolysis of oat and soy lecithins, and its effect on the functional properties of lecithin, was investigated by Aura et al. (163). The phospholipase used was most effective at low enzyme and substrate concentrations. 

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