Candida Antarctica Lipase B CALB

The basidomycetous yeast Candida antarctica produces two different lipases, A and B. This yeast was originally isolated in Antarctica with the aim of finding enzymes with extreme properties. Both the lipase A and lipase B have been purified and characterized. The two lipases are very different. The lipase B is a little less thermostable and is less active toward large triglycerides but very active 

CALB is industrially produced by submerged fermentation of a genetically modified Aspergillus micro-organism. The active site in CALB is deep and narrow, which is believed to be the reason for the substrate specificity and pronounced stereo-specificity of this lipase [10]. The role and even presence of a lid in CALB has been much debated, but it is now believed that CALB in fact has a lid like other Upases [11], 

Immobilization is a technique to physically confine or localize an enzyme with retention of its activity (see also Chapter 2). In immobilization, the enzyme is localized on a matrix or carrier through different modes of attachment. The main purposes of enzyme immobilization include (i) enzyme stabilization (ii) improvement of enzyme performance by increasing the contact area of enzyme and substrate and (iii) re-use or continuous use of enzymes in several reactions or over an extended time [12, 13]. 

In Novozym 435, the enzyme is not covalently linked to the carrier, but merely adsorbed. Therefore, as mentioned, enzyme leaching is a known issue with this product. The problem has been addressed by several authors [20, 21], Even though the enzyme performance has been satisfactory, there has been a need specifically for a non-leaching immobilized formulation. It has been shown that several physical and chemical treatments such as coating the immobilized particles can enhance the stability of Novozym 435 [20-22], 

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The use of an enzyme in a cascade using nanoencapsulation has also been demonstrated [23]. In this case, the dynamic kinetic resolution (DKR) of secondary alcohols was achieved with an acidic zeolite and an incompatible enzyme, Candida antarctica lipase B (CALB) (Scheme 5.8). 

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

Biphasic systems consisting of ionic liquids and supercritical CO2 showed dramatic enhancement in the operational stability of both free and immobilized Candida antarctica lipase B (CALB) in the catalyzed kinetic resolution of rac- -phenylethanol with vinyl propionate at 10 MPa and temperatures between 120 and 150°C (Scheme 30) 275). Hydrophobic ionic liquids, [EMIM]Tf2N or [BMIM]Tf2N, were shown to be essential for the stability of the enzyme in the biotransformation. Notwithstanding the extreme conditions, both the free and isolated enzymes were able specifically to catalyze the synthesis of (J )-l-phenylethyl propionate. The maximum enantiomeric excess needed for satisfactory product purity (ee >99.9%) was maintained. The (S)-l-phenylethanol reactant was not esterified. The authors suggested that the ionic liquids provide protection against enzyme denaturation by CO2 and heat. When the free enzyme was used, [EMIM]Tf2N appeared to be the best ionic liquid to protect the enzyme, which... 

Kinetic resolution technology has also been applied to the duloxetine problem (Scheme 14.13). In this case, chloroketone 41 was converted to racemic alcohol 43 using sodium borohydride. The racemate was then treated with vinyl butanoate in hexanes, in the presence of catalytic immobilized Candida antarctica Lipase B (CALB). The reaction was stopped after reaching 50% conversion, leading to the isolation of the desired (5)-chloroalcohoI 43a, as well as the (Zf)-ester 45 in good yields and excellent enantiomeric excesses. Chloroalcohol 43a was converted to duloxetine (3) via the... 

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

The activity of three ester spHtting enzymes, Candida antarctica lipase B (CALB), Mucor miehei lipase (MML) and esterase, towards the carbonate surfactant was studied. While CALB and esterase were found to catalyze the hydrolysis of the carbonate bond, MML showed no activity. 

The first example of chemoenzymatic DKR of allylic alcohol derivatives was reported by Williams et al. [37]. Cyclic allylic acetates were deracemized by combining a lipase-catalyzed hydrolysis with a racemization via transposition of the acetate group, catalyzed by a Pd(II) complex. Despite a limitation of the process, i.e. long reaction times (19 days), this work was a significant step forward in the combination of enzymes and metals in one pot Some years later, Kim et al. considerably improved the DKR of allylic acetates using a Pd(0) complex for the racemization, which occurs through Tt-allyl(palladium) intermediates. The transesterification is catalyzed by a lipase (Candida antarctica lipase B, CALB) using isopropanol as acyl acceptor (Scheme 5.19) [38]. 

The same concept is applicable to allylic alcohols, ketones, or ketoximes. Enol acetates or ketones were successfully converted in multi-step reactions to chiral acetates in high yields and optical yields through catalysis by Candida antarctica lipase B (CALB, Novozyme 435) and a ruthenium complex. 2,6-Dimethylheptan-4-ol served as a hydrogen donor and 4-chlorophenyl acetate as an acyl donor for the conversion of the ketones (Jung, 2000a). 

Recent studies in the pharmaceutical field using MBR technology are related to optical resolution of racemic mixtures or esters synthesis. The kinetic resolution of (R,S)-naproxen methyl esters to produce (S)-naproxen in emulsion enzyme membrane reactors (E-EMRs) where emulsion is produced by crossflow membrane emulsification [38, 39], and of racemic ibuprofen ester [40] were developed. The esters synthesis, like for example butyl laurate, by a covalent attachment of Candida antarctica lipase B (CALB) onto a ceramic support previously coated by polymers was recently described [41]. An enzymatic membrane reactor based on the immobilization of lipase on a ceramic support was used to perform interesterification between castor oil triglycerides and methyl oleate, reducing the viscosity of the substrate by injecting supercritical CO2 [42],... 

Csajagi et al. (2008) recently demonstrated the enantioselective acylation of racemic alcohols in a continuous flow bioreactor, using Candida antarctica lipase B (CaLB) 167. Employing a packed-bed reactor, containing 0.40 g of enzyme 167, and pumping a solution of rac-phenyl-1-ethanol 119 (10 mg ml-1) in hexane THF vinyl acetate 168 (2 1 1) at a flow rate of 100 gl min-1 (at 25 °C), the authors found the reactor reached steady state after 30 min of operation. Under the aforementioned conditions, the... 

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. 

Inhibition of lipases, both by the substrate or the product, has been observed. In alcoholysis of methyl propanoate with n-propanol catalyzed by Candida antarctica lipase B (CALB), the alcohol was found to inhibit the enzyme resulting in a deadend complex [21]. Phosphate- and phosphonate-conlaining inhibitors are known to inhibit proteases. Studies of the inhibition of CALB have shown inhibition by diethyl p-nitrophenyl phosphate. The inactivation of the enzyme was caused by covalent binding of diethyl p-nitrophenyl phosphate in the active site [22]. 

C and 80 bars. For that, four ionic liquids, namely [bmim INTf "], [bmim ] [PFg"]) [bdimim ][PFg ] and [omim+][PF ], were used. Figure 8.4 shows the synthetic activity and selectivity of immobilized Candida antarctica lipase B, CaLB, on ceramic membranes in scCO medium as well as in four different IL/scCO biphasic systems. 

Although there are notable exceptions as given below, the most common lipase-catalyst used for polyester synthesis is Candida antarctica lipase B (CALB) (please refer to Chapter 14 for more information on the structure and reaction mechanisms of CALB). The immobilized CALB catalyst that has been primarily used is Novozym 435, manufactured by Novozymes (Bagsvaerd, Denmark). Novozym 435 consists of CALB physically adsorbed within the macroporous resin Lewatit VPOC 1600 (poly[methyl methacrylate-co-butyl methacrylate], supplied by Bayer) (please refer to Chapter 3 for more information on Novozym 435). 

Polyacrylic resins were employed to study how immobilization resin particle size influences Candida antarctica Lipase B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed more rapidly on smaller beads. Saturation occurred in less than 30 seconds and 48 h for beads with diameters 35 and 560-710 pm, respectively. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560-710 and 120 pm while CALB appears evenly distributed throughout 35 pm resins. The fraction of active CALB molecules adsorbed onto resins not influenced by particle size was less than 50 %. At about 5% w/w CALB loading, decrease in the immobilization support diameter from 560-710 to 120,75 and 35 pm increased conversion of s-CL to polyester (20 to 36, 42 and 61%, respectively, at 80 min). Similar trends were observed for condensation polymerizations of 1,8-octanediol and adipic acid. 

Candida antarctica Lipase B (CALB) is atfracting increasing attention as a biocatalyst for the synthesis of low molar mass and polymeric molecules. Almost all publications on immobilized CALB use the commercially available catalyst Novozym 435, which consists of CALB physically adsorbed onto a macroporous acrylic polymer resin (Lewatit VP OC 1600, Bayer). Primarily, commercial uses of CALB are limited to production of high-priced specialty chemicals because of the high cost of commercially available CALB preparations Novozym 435 (Novozymes A/S) and Chirazyme (Roche Molecular Biochemicals). Studies to better correlate enzyme activity to support parameters will lead to improved catalysts that have acceptable price-performance characteristics for an expanded range of industrial processes. 

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