Novozym Candida antarctica lipase

In conventional synthetic transformations, enzymes are normally used in aqueous or organic solvent at moderate temperatures to preserve the activity of enzymes. Consequently, some of these reactions require longer reaction times. In view of the newer developments wherein enzymes can be immobilized on solid supports [183], they are amenable to relatively higher temperature reaction with adequate pH control. The application of MW irradiation has been explored with two enzyme systems namely Pseudomonas lipase dispersed in Hyflo Super Cell and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin). 

Systems such as Pseudomonas lipase dispersed inside Hyflo Supercell (a diatoma-ceous silica of pH 8.5-9) and SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin) are thermally stable and have optimum activity in the range 80-100 °C. They can therefore be used with conventional or microwave heating if the temperature is strictly controlled. 

Candida antarctica lipase B (CAL-B), Novozyme 435 (from Sigma) (2 g) vinylbutyrate (630 p, 4.96 mmol) diisopropylether (DIPE) (30 mL)... 

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. 

Since then, the process has been extended to a wide variety of lactones of different size and to several lipases, as recently reviewed [93-96]. Interestingly, large-membered lactones, which are very difficult to polymerize by usual anionic and coordination polymerizations due to the low ring strain, are successfully polymerized by enzymes. Among the different lipases available, that fi om Candida antarctica (lipase CA, CALB or Novozym 435) is the most widely used due to its high activity. An alcohol can purposely be added to the reaction medium to initiate the polymerization instead of water. The polymerization can be carried out in bulk, in organic solvents, in water, and in ionic liquids. Interestingly, Kobayashi and coworkers reported in 2001 the ROP of lactones by lipase CA in supercritical CO2... 

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 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). 

Various lipases and esterases have been used for the enantioselective esterification of alcohols and hydrolysis of esters. For example, Burkholderia cepacia lipases (PS, Amano Enzyme Inc.) and Candida antarctica lipase (CAL, Novozymes) have been widely used for its wide substrate specificities, high activities and chemo, regio and enantioselectivities. Fundamentals and some selected applications are shown in this section. The origins and abbreviations of lipases introduced here are as follows. 

Irimescu, R., Furihata, K., Hata, K., Iwasaki, Y., and Yamane, T. 2001. Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of l,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol. J. Am. Oil Chem. Soc., 78, 285-289. 

Adsorption of enzymes to various polymeric resins is a straightforward means for immobilization. Zwitterionic molecules such as proteins can bind to both anionic and cationic ion exchange resins. Hydrophobic macroporous resins are also useful for immobilizing many enzymes, particularly lipases. For example, an immobilized form of Candida antarctica lipase B (CAL-B) on acrylic resin has been sold for many years under the name, Novozym 435 (N435). The enzyme is produced in a modified Aspergillus organism by submerged fermentation and is subsequently adsorbed onto a macroporous... 

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). 

The application of enzymes as catalysts in organic chemistry is closely linked to their immobilization. Indeed, many enzymes are only available in an immobilized form. The immobilized enzymes can be used as received, greatly easing their application. Numerous of these readily available immobilized enzymes are now the working horses of biocatalysis. This has even led to the incorrect use of the abbreviation of an enzyme name for a specific enzyme preparation, that is CALB for the immobilized form of Candida antarctica lipase B on cross-linked polymethacrylate (also known as Novozym 435). Vice versa the commercial name of an enzyme preparation-Amano PS-has taken the place of the enzyme (Burkhdderia cepacia lipase on dextrin or diatomaceous earth). Surprisingly, often no attention is paid to the fact that the enzyme is immobilized [1]. 

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). 

Novozym 435 is Candida antarctica lipase B (Novo Nordisk A/S) PS-C (type II) from Amano is Pseudomonas cepacia lipase... 

In our study, we conducted the enzyme-catalyzed methanolysis of rapeseed oil using Novozym 435, a well-known nonspecific lipase. Novozym 435 facilitates reactions between a wide variety of alcohols and is also a remarkably heat-tolerant enzyme [6, 8], Watanabe et al. [9] previously reported that immobilized Candida antarctica lipase was inactivated in the presence of more than half the stoichiometric amount of methanol against total fatty acids in the oil. This disadvantage was surmounted by the utilization of three-step methanolysis, in which only one third of the total amount of methanol was added in each stage [7, 9]. 

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. 

Here, we will discuss some fundamental aspects of the selectivity of Novozym 435 —Candida antarctica Lipase B immobilised on an acrylic resin— for (o-substituted lactones. This selectivity determines the polymerisability of a (D-substituted lactone employing Novozym 435 as the catalyst. We will first address the influence of the conformation of the ester bond in the lactone cisoid or transoid) on the selectivity of the ring-opening of a selection of lactones with ring sizes vaiying from a 4- to 13-membered ring. Then, we will address the influence of the size of the substituents at the co-position on the selectivity and... 

Dimethyl adipate (39.40 g, 0.30 mol), triethylene tetraamine (43.87 g, 0.30 mol) and 2.0 g of Novozym 435 (immobilized Candida antarctica lipase) were mixed in a 250 ml flask and heated in an oil bath to 80°C. The mixture was stirred at 80°C and the color of the reaction mixture turned to light brown in one hour. After being stirred at this temperature for 16 hrs in an open vessel, the reaction mixture solidified. 150 ml of methanol was added to dissolve the polyamide product. The immobilized enzyme was insoluble in the methanol solution and was removed by filtration. Methanol was removed by evaporation using a rotary evaporator under reduced pressures to give the product as a brown solid. The yield was 50 g M, 8000 Mw/M , 2.10. 

The immobilized Candida Antarctica lipase (Novozym 435)-catalyzed esterification of methyl a-D-glucopyranoside 65, a-glucose and a,a-trehalose 68 with dodecanoic acid 66 has been studied under microwave irradiation (see... 

The separation of monoacid TAG from among the sTAG group showed that Candida antarctica lipase (Novozyme ) synthesized EPA-enriched TGA in 98% yield. 

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