Candida antarctica immobilized lipase from

Many acetate esters (such as those of isoamyl, benzyl, citroneUyl, and geranyl alcohols) are components of natural flavors. They can be obtained by Upase-cata-lyzed esteriflcation in organic solvents, but the major problem with enzymatic acetylations is deactivation of lipases by acetic acid [8, 9]. Most of the Upase-catalyzed syntheses of esters have been carried out by transesterification to avoid free acid toxicity and water formation. Claon and Akoh [10] found that immobilized lipases from Candida antarctica promote highly effective direct esterification of geraniol and citronellol with acetic acid. 

The same methodology was also applied to the DKR of (5-hydroxyesters. In the latter case, the reaction was carried out in tandem with an aldol reaction and the P-hydroxyester formed, after neutralization, underwent DKR using the immobilized lipase from Candida antarctica and a ruthenium catalyst [8]. 

Enzymatic resolution of racemic functionalized isoxazolines is a valuable technique for the preparation of enantio-merically enriched and pure 4,5-dihydroisoxazoles. These enzymatic resolutions exploit a variety of transformations of functional groups resident on the side-chains of the isoxazoline ring. The multipolymer enzymatic resolution of soluble polymer-supported alcohols 307 and 308 was achieved using an immobilized lipase from Candida antarctica (Novozym 435). The (R)-alcohol 309 was obtained in enantiomerically pure form (>99% ee) after its cleavage from the poly(ethylene glycol) (PEG) scaffold (Scheme 70) <2000JOG8527>. 

Fig. 5 Generation of the -enantiomer of the monoacetate precursor of an antifungal agent from a diol as mediated by an immobilized lipase from Candida S-monoacetate antarctica, fraction B.

Novozym 435, immobilized lipase from Candida antarctica, type B could efficiently catalyze the transesterification of waste edible oil with methyl acetate. However, FFA affected the reaction clearly, and the ME yield drops sharply with increasing FFA concentration. Acetic acid, the by-product formed in the transesterification of FFA with methyl acetate, was proved responsible for the decrease of reaction rate and ME yield. Addition of organic base to the reaction system could not only speed up the reaction, but also improve the ME yield. This protocol seems to be an efficient alternative for the conversion of waste edible oil to corresponding methyl esters. 

In addition to the use of enzyme and transition metal combinations for the dynamic resolution of alcohols, there has been a brief report of the use of amines as substrates. In 1996, Reetz and Schimossek reported the combination of palladium on carbon with an immobilized lipase (from Candida antarctica) in the dynamic... 

SP 382 Immobilized lipase from Candida antarctica, containing type A B discontinued... 

This work was partially supported by a Grant-in-Aid for General Scientific Research and by a Grant-in-Aid for the 21st Century COE Program KEIO LCC from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Immobilized lipase from Candida antarctica (CA, Novozym 435) was kindly supplied by Novozymes Japan Ltd. (Chiba, Japan). 

A new way to prepare peroxy acids was discovered by Novo Nordisk, DK (9-11). They showed that some lipases catalyze the conversion of fatty acids with hydrogen peroxide (preferably 60%) to peroxy fatty acids Novozym 435, an immobilized lipase from Candida antarctica on polyacrylic Lewatit, is the most active and stable biocatalyst for this purpose (Scheme 1). Recently we found that Novozym 435 is also capable of catalyzing perhydrolysis (12), i.e., the reaction of carboxylic acid esters with hydrogen peroxide to percarboxylic acids (Scheme 2). 

Substituted acrylic monomers. The reactants (methyl acrylate plus alcohol or amine) were added neat or in a non-aqueous solvent together with Novozym 435 immobilized lipase from Candida antarctica as a catalyst. Molecular sieves (4A) were used to remove water in order to shift the reaction equilibrium to product formation, and also to eliminate side reactions due to Michael addition that was usually enhanced by the presence of water or methanol. Unreacted starting materials were removed by evaporation, and the monomer products obtained without further purification. TLC analysis indicated that the desired products had formed. The purity of the products was confirmed by NMR and IR analysis. The two monomers were successfully polymerized in a separate step. 

A combination of different enzymes has been shown to increase yield in the enzymatic production of EAAE. Eor example, Tiirkan and Kalay (2006) used three different immobilized lipases from RML, TLL and Candida. They found lipases from RML and TLL catalyzed the first step (TAG to DAG) of transesterification faster while hpase from Candida antarctica catalyzed the second (DAG to MAG) and third (MAG to FAAE and glycerol) steps faster. 

Synthesis of amino acid surfactants has also been achieved using certain lipases. Studies with immobilized lipases from Candida antarctica and Rhizopus miehei have shown that the enzymes could accept At-Cbz amino acids as acyl donors and catalyse the esterification with long-chain fatty alcohols with high yields [55, 56]. Removal of water produced during the reaction was essential to shift the equilibrium towards ester synthesis. Synthesis of At-acyl amino acids was also done by lipase-catalysed direct transacylation of amino acids with triglycerides or vegetable oils (e.g. soya bean, palm oil) [57-59]. 

Until now, thoroughly investigated commercial immobilized lipases are Novozym 435 (Hemandez-Martin and Otero, 2008), Lipozyme TLIM (Wang et al., 2008), and Lip-ozyme RM IM (Aguieiras et al., 2013). AU of them are extracellular enzymes. The most widely used are Novozym 435, from Candida antarctica, immobilized on a mac-roporous acryhc resin Lipozyme RM IM, from Rhizomucor miehei, immobilized on an anionic resin and Lipozyme TL IM, from Thermomyces lanuginosus, immobilized on a gel of granulated sUica. 

Lipase from Rhizomucor miehei has recently been used for the selective N-acylation of 1-deoxy-l-methylamino-D-glucitol by fatty acids in hexane [17]. The methodology has been extended further to the enzymatic synthesis of biosurfactants (glycamides) by transacylation reaction (Fig. 9) catalyzed by immobilized lipase from Candida antarctica in organic media [18]. 

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... 

The concept of zeolite action was tested in a particular reaction where the enzyme is exposed from the beginning to an acidic environment the esterification of geraniol with acetic acid catalyzed by Candida antarctica lipase B immobilized on zeolite NaA [219]. Lipases have been used for the hydrolysis of triglycerides and due to their ambivalent hydrophobic/hydrophilic properties they are effective biocatalysts for the hydrolysis of hydrophobic substrates [220]. When water-soluble lipases are used in organic media they have to be immobilized on solid supports in order to exhibit significant catalytic activity. 

The one-pot dynamic kinetic resolution (DKR) of ( )-l-phenylethanol lipase esterification in the presence of zeolite beta followed by saponification leads to (R)-l phenylethanol in 70 % isolated yield at a multi-gram scale. The DKR consists of two parallel reactions kinetic resolution by transesterification with an immobilized biocatalyst (lipase B from Candida antarctica) and in situ racemization over a zeolite beta (Si/Al = 150). With vinyl octanoate as the acyl donor, the desired ester of (R)-l-phenylethanol was obtained with a yield of 80 % and an ee of 98 %. The chiral secondary alcohol can be regenerated from the ester without loss of optical purity. The advantages of this method are that it uses a single liquid phase and both catalysts are solids which can be easily removed by filtration. This makes the method suitable for scale-up. The examples given here describe the multi-gram synthesis of (R)-l-phenylethyl octanoate and the hydrolysis of the ester to obtain pure (R)-l-phenylethanol. 

A novel continuous-flow SCCO2 process for the kinetic resolution of 1-phenyethanol enantiomers (Figure 30) using Novozym 435 immobilized enzyme from Candida antarctica was described by Matsuda et al. [51], The lipase enzyme, selectively acetylated the R)-alcohol component. A mixture of starting material and vinyl acetate was passed through the enzyme with supercritical carbon-dioxide (Figure 31). The reaction zone was pressurized and heated, so the reaction could be performed imder supercritical conditions, synthesizing the desired (i )-acetate with 99.7% ee. and 47% yield. 

Scheme 2.2 Enantioselective kinetic resolution of cis/ttoris (IR,5R)-bicyclo[3.2.0]hept-6-ylidene-acetate ethyl ester, 1, catalyzed by immobilized lipase from Candida antarctica (Novozyme 435). The reaction is run in a pH-stat at pH 8.0 and...

An example where a transition metal catalyst is used in combination with an enzyme has been described (Scheme 19.26).207 The racemic alcohol 50 was converted to the (A1)-acetate 51, using a ruthenium catalyst along with Novozym 435 (immobilized Lipase B from Candida antarctica), 3 equivalents of p-chlorophenylacetate in t-BuOH, and 1 equivalent of 1-indanone. The reaction yield was 81% with an optical purity of >99.5% ee. 

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