Enzyme novozym Candida antarctica

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). 

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. 

For desymmetrization of diesters 3 via their hydrolysis in water, pig Hver esterase [12], o -chymotrypsin [12, 13a], and Candida antarctica Hpase (CAL-B) [14] were successfully used. However, further studies showed that respective anhydrides 5 can be used as substrates for enzyme-catalyzed desymmetrization in organic solvents [15]. The desired monoesters 4 were obtained in high yield in this way, using immobilized enzymes Novozym 435 or Chirazyme L-2 (Scheme 5.3). After the reaction, enzymes were filtered off, organic solvents were evaporated, and the crude products were crystalHzed. This was a much simpler experimental procedure in which control of the reaction progress was not necessary, and aU problems associated with extraction of products from aqueous phase and their further purification were omitted [15]. 

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). 

Initially, kinetic resolutions of 2-(trimethylsilyl)-5-[l -(2, 2, 2 -trifluoro-l -hydroxy-ethyl)]furan with a wide variety of lipases and vinyl alkanoates were examined in 1,2-dichloroethane. On the basis of these results, the system consisting of an enzyme (Novozym 435, Candida antarctica, Novo Nordisk Co. Ltd.) and vinyl propionate was sufficient to obtain optical pure alcohol and ester with a high -value. Moderate effect on the optical purity was observed on changing the organic solvents. Obviously, Novozym 435-CH2ClCH2Cl system is the most convenient practical system for obtaining the optically pure alcohol and the ester on the basis of comparison of the reaction time and the -value. 

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. 

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. 

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... 

The reaction is catalyzed by a variety of both acids and bases but simple bases such as NaOH and KOH are generally used for the industrial production of biodiesel [200, 201]. The vegetable oil feedstock, usually soybean or rapeseed oil, needs to be free of water (<0.05%) and fatty acids (<0.5%) in order to avoid catalyst consumption. This presents a possible opportunity for the application of enzymatic transesterification. For example, lipases such as Candida antarctica B lipase have been shown to be effective catalysts for the methanolysis of triglycerides. When the immobilized form, Novozyme 435, was used it could be recycled 50 times without loss of activity [201, 202]. The presence of free fatty acids in the triglyceride did not affect the enzymes performance. The methanolysis of triglycerides catalyzed by Novozyme 435 has also been successfully performed in scC02 as solvent [203]. 

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]. 

Lipase B from Candida antarctica (CALB) has been shown to be an excellent enantioselective biocatalyst for the stereo-selective acylation of racemic alcohols [14, 15]. The most often used commercial preparation of CALB is Novozym 435, where the enzyme is immobilized on a macroporous acrylic resin and the matrix presents about 90% of the total mass. 

Enzyme activity is markedly influenced by water activity (a ). Lipases from different sources have been shown to have completely different behavior some exhibit high activity at low a.., whereas others show a high activity at intermediate or very high aw (31). The a also affects the apparent and V ax values measured for lipase-catalyzed hydrolysis and esterification reactions (32). Thermodynamic a can be used to define the relationship between water and other components of the reaction system (33). Thermodynamic a of a solution is defined as the water vapor pressure over the solution divided by the water vapor pressure over pure water at a given temperature. The water content of the biocatalyst is more important in dictating enzyme activity than the total water content in the system. Novozym-435 (Novo Nordisk, Franklinton, North Carolina) from Candida antarctica has a water content of 1-2% (w/w) (34). This enzyme preparation has been shown to give high yields of TAG products under several nonaqueous reaction conditions (5, 35, 36). 

Waste oil was collected from the restaurant in South China University of Technology. The saponification value was 200.3 mg KOH/g, from which, the average molecular weight of the waste oil was known to be 840.2. Novozym 435 (lipase B from Candida antarctica, 164 U/g, limit corresponds to the amount of enzyme that produces 1 pmol methyl oleate from triolein per minute at 35 °C) was kindly donated by Novozymes Co. (Denmark). Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate and methyl heptadecanoate (as an internal standard) were purchased from Sigma (USA). All other chemicals were also obtained commercially and of analytical grade. Ultrasonic irradiation experiments were carried out using an ultrasonic bath (Type NP-B-400-15 Newpower Co. Ltd., China). 

The single enantiomer of 45 is produced by enantioselective acylation with vinyl acetate catalysed by Novozym 435, an immobilised lipase from Candida antarctica containing about 1% w/w enzyme on a macroporous polypropylic resin. You will appreciate that as both substrate and catalyst are on polymers, one at least must be soluble in the reaction mixture. The polymer is stripped from the substrate 45 with HF. 

Abstract An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t /2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction. 

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. 

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. 

Novozym 435 is already a famous biocomposite containing Candida antarctica lipase B (CAL-B) as the bioactive part. Novozym 435 is commercially available immobilized enzyme with CAL-B physically adsorbed on a macroporous resin of poly(methylmethacrylate) [82]. The enzyme versatility and the substrate affinity recommended Novozym 435 for many applications, including the biomass valorization. An example is the production of the fatty acid esters used as emollient in the cosmetic industry (ex. myristyl myristate) [83,84],... 

Enzyme-type catalyst (Novozym 435, i.e., immobilized Candida antarctica lipase B (CALB)) was used by Gubicza, Belafi-Bako, Feher, and Frater (2008) within an ESU configuration PVMR. The 1-butyl-3 methylimidazolium hexafluorophosphate. 

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