Enzyme Candida rugosa lipase

The outstanding influence of the anionic component on the activity and selectivity of enzymes was demonstrated in the Candida rugosa lipase-catalyzed kinetic resolution of ibuprofen, a nonsteroidal antiinflammatory drug with sales of USD 183 million in 2006 (Scheme 5.15) [63]. 

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... 

On the basis of the lipases numerous applications, also on an industrial scale, have been shown using an enzyme from this family. Table 3 summarises some applications of a popular lipase, often used in reverse micellar media, the Candida rugosa lipase. 

Another major benefit of the purity of CLCs relates to the fact that many commercially available enzyme products actually contain more than one enzyme, and these contaminating enzymes can often lead to undesired side reactions. In the case of a resolution this can mean the difference between success and failure. For example, in the resolution of racemic ketoprofen by ester hydrolysis (Fig. 1), the enantioselectivity using crude Candida rugosa lipase is poor (E = 5). 

Candida rugosa lipase (CRL) hydrolysed the other enantiomer selectively (Scheme 6.11). This proves once again that in most cases enzymes with the desired stereoselectivity are available. 

In addition, Itoh and coworkers have reported that acylation of the alcohol was accomplished by three types of enzymes Candida Antarctica lipase (CAL, Novozym 435), lipase QL Alcalgenes sp.), and lipase PS Pseudomonas cepacia). Scheme 10.5. The desired acetate showed extremely high enantioselectivity, but no reaction took place when lipase (CRL, Candida rugosa) or Procine liver lipase (PPL) was used as the catalyst in the ionic liquid (Table 10.3). 

It is generally stated that biocatalysis in organic solvents refers to those systems in which the enzymes are suspended (or, sometimes, dissolved) in neat organic solvents in the presence of enough aqueous buffer (less than 5%) to ensure enzymatic activity. However, in the case of hydrolases water is also a substrate and it might be critical to find the water activity (a ) value to which the synthetic reaction (e.g. ester formation) can be optimized. Vahvety et al. [5] found that, in some cases, the activity of Candida rugosa lipase immobihzed on different supports showed the same activity profile versus o but a different absolute rate. With hpase from Burkholderia cepacia (lipase BC), previously known as lipase from Pseudomonas cepacia, and Candida antarctica lipase B (CALB) it was found that the enzyme activity profile versus o and even more the specific activity were dependent on the way the enzyme was freeze dried or immobihzed [6, 7]. A comparison of the transesterification activity of different forms of hpase BC or CALB can be observed in Tables 5.1 and 5.2, respectively. 

An enzyme-catalyzed enantioselective esterification of racemic LA 3 with -alcohols containing one to eight carbon atoms in hexane as a solvent, using Candida rugosa lipase <1997TA337>, has been described (Scheme 24). The best selectivity at 30% conversion was obtained with -hexanol, yielding the (3)-ester 176 with 72% ee, along with (K)-lipoic acid (20% ee) this decreased with -octanol (58% ee for an ester and 24% for acid) and there was a drastic drop... 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 1 reaction 2). These enzymes also catalyze transesterifications and the asymmetrization of meso -substrates (Section 13.2.3.1.1). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities. As more and more research has been conducted with these enzymes, a less empirical approach is being taken due to the different substrate profiles amassed for various enzymes. These profiles have been used to construct active site models for such enzymes as pig liver esterase (PLE) (EC 3.1.1.1) and the microbial lipases (EC 3.1.1.3) Pseudomonas cepacia lipase (PCL), formerly P.fluorescens lipase, Candida rugosa lipase (CRL), formerly C. cylindracea lipase, lipase SAM-2 from Pseudomonas sp., and Rhizopus oryzae lipase (ROL) [108-116]. In addition, x-ray crystal structure information on PCL and CRL has been most helpful in predicting substrate activities and isomer preferences [117-119]. 

Ulbert O, Frdter T, Belafi-Bako K, Gubicza L (2004) Enhanced enantioselectivity of Candida rugosa lipase in ionic liquids as compared to organic solvents. J Mol Catal B Enzym 31 39 5... 

Amano G and Lipozyme RM IM) [20], The highest yield of terpinyl acetate was obtained by using Candida rugosa type VII. Near cero yield of terpinyl acetate was obtained with Amano PS and Amano G. An esterification up to of 53.0% was obtained under optimized conditions in continuous operation using acetic anhydride as acyl donor and Candida rugosa lipase as enzyme at 10 MPa and 50°C for 1.5 h. However, the enzyme activity decreased up to 50% after 10.5 h repeated esterification in a batch. 

Mobile SS, Potdar MK, Harjani JR et al (2004) Ionic liquids efficient additives for Candida rugosa lipase-catalysed enantioselective hydrolysis of butyl 2-(4-chlorophenoxy)propionate. J Mol Catal B Enzym 30 185-188... 

Candida rugosa lipase Fe304-chitosan NPs Enzyme immobilization [177]... 

Candida rugosa lipase Acetylated y-Fe203 MNPs Enzyme immobilization [178]... 

Candida rugosa lipase Ionic liquid functionalized magnetic silica NPs Enzyme immobilization [179]... 

Esterases and polyurethanases can hydrolyze polyurethanes resulting in changes on the surface of PU film. In vivo, cholesterol esterase can initiate polyurethane degradation while a number of PU-degrading enzymes have been described from micro-organisms, such as Candida rugosa lipase or esterases from Pseudomonas chlororaphis, for example, polyurethanase [105]. 

Novo Nordisk showed in the synthesis of (-)-Ormeloxifene (48), a drug candidate for the treatment and prevention of osteoporosis, that the enzymatic asymmetric hydrolysis of potential intermediates can be carried out using Candida rugosa lipase (CRT) immobilized on Accurel (Scheme 15) [74]. Racemic czs-hexanoate mc-46 was subjected to enzymatic hydrolysis in aqueous acetonitrile and gave the phenol ds-(3R,4S)-47 in 95% ee. This transformation is a nice example to demonstrate that the enzyme can recognize and use remote stereocenters. The reaction was run on a 10 g scale, and it was even possible to recycle hexanoate (3S,4R)-46. Simple recovery of the immobilized enzyme by... 

The attachment and use of enzymes on hydrophilic supports covered with a dense layer of highly hydrophobic groups has been shown by Vita-Invest in recent years [78]. Various lipases were immobilized on Octyl-Sepharose CL-4B, and in certain cases an increase of activity of more than 100 times could be observed. The usefulness of these supported enzymes was demonstrated in first experiments by the regioselective hydrolysis of glucose pentaacetate (57) to glucose tetraacetate with Candida rugosa lipase (Scheme 18). The regioselectivity was found to be pH dependent, and the 2-0-deacylated derivative 58 could be obtained at pH 5, whereas the 6-0-deacylated product 59 was formed at pH 7. 

Hsu AF, Jones KC, Foglia TA et al. (2004) Transesterification activity of lipases immobilized in a phyllosilicate sol-gel matrix. Biotechnol Lett 26 917-921 Hung TC, Giridhar R, Chiou SH et al. (2003) Binary immobilization of Candida rugosa lipase on chitosan. J Mol Catal B Enzym 26 69-78... 

Various materials have been used as supports for the immobilization of a Candida rugosa lipase (Lipase The enzyme adsorbed on silica gel... 

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