Lipase Chirazyme

FIGURE 8.4 Effect of solvent removal and the stepwise addition of fructose on the percent conversion of saccharide (a) and on the solubility of fructose (b) for lipase-catalyzed fructose-oleic acid esterification. ( ) Fructose was added to 50 mmol oleic acid and 13.4 g tert-butanol in 5 mmol increments for 2-h cycles until the net amount of fructose added was 25 mmol tert-butanol underwent free evaporation for up to 10 h, at which time, the reaction was stopped, solvent was removed completely by rotary evaporation, then the reaction was continued. (A) Fructose (25 mmol) was added at time 0 to 50 mmol of oleic acid and 13.4 g (46.9 wt.% fructose-free basis) tert-butanol solvent freely evaporated away throughout at a rate of 0.47 g per h. Both reactions were operated in stirred batch mode using approximately 0.5 g immobilized C. antarctica lipase, (Chirazyme L-2, s.-f, c2, Lyo., Boehringer-Mannheim, Indianapolis, IN), a stir rate of 450 rev min and a reaction temperature of 65°C. (From Walde, P. et al., Chem. Phys. Lipids, 53, 265-288, 1990. With permission.)... 

CALB Candida antarctica lipase (Chirazyme L-2, Novozym 435b)... 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Enzymes PPL, lipase from Pseudomonas fluorescens F-AP, lipase from Rhizopus orizae AP-6, lipase from Aspergillus niger, SP-254, lipase from Aspergillus oryzae P-2, Chirazyme WCPC, whole cell cultures of Penicillium citrinum WCPFL, whole cell cultures of Pseudomona fluorescens CAL-B, lipase from Candida antarctica B PS-C, lipase from Pseudomonas cepacia GCL, lipase from Geotrichum candidum. n.r. not reported. 

Candida antarctica lipase A (CAL-A, Chirazyme L-5, C2), 100 mg ethyl acetate... 

Two 2-hydroxyaldehydes protected as acetals (63) have been resolved by lipase-catalyzed acylation with vinyl acetate as reagent and solvent (Scheme 4.24) [84]. For the vinyl derivative (n = 0) Chirazyme L2 (CALB) gives the best E, whereas Pseudomonas fluorescens lipase (PFL) provides the best E for n = 1 [84]. 

Schemem 4.29 Lipase-catalyzed purification by removal of undesired stereoisomers from the product. Chirazyme L2 (CALB), vinyl acetate, n-heptane, mol. sieves , 25°C.

Candida antarcdca lipase B (CAL-B) (Chirazyme L2, Roche, Basel, Switzerland)... 

To achieve better results a series of lipases was screened using the Chirazyme screening kit and analysis by gas chromatography with a chiral stationary phase. Candida antartica Lipase B, available as Novozyme 435, was chosen for further development with the acyl transfer agent vinyl propionate. 

FIG. 2. Relationship between product yields and the percentage of hexane in ethyl methylketone. Reaction condition 0.5 mmol glucose, 0.5 mmol fatty acids, 50 mg Chirazyme L-2 lipase, 0-0.4 ml ethyl methylketone and/or hexane, 59°C, 650 rpm, 48 h. St solidification time ns no solidification. 

Chirazyme Lipases Product Information, Boehringer Mannheim GmbH... 

The most important factor for adsorption is the choice of immobilization matrix. Several commercially available matrices commonly employed for nonaqneous enzymology are listed in Table 8.3. They consist of macroporons millimeter-sized particulates that are hydrophilic with the exception of Accnrel (polypropylene). Celite (diatomaceons earth) is probably, the most commonly employed matrix of those listed in Table 8.3. In addition, a few enzymes are commercially available in immobilized forms, inclnding Lipozyme IM and Novozyme from Novo-Nordisk, the Chirazyme prodnct line from Roche Molecular Biochemicals, and Pseudomonas cepacia lipase immobilized in Sol-Gel AK (Fluka). 

The synthetic compounds are obviously esters of a cyclopropane carboxylic acid and a cyanohydrin 202. Enantioselective transesterification of butanol and the acetate 201 of the cyanohydrin using immobilised lipases gives the required (S)-alcohol 202 and the unreacted enantiomer of the acetate (R) -201 easily racemised with Et3N. Reports using different lipases appeared at the same time CHIRAZYME L-6, the lipase from Pseudomonas immobilised on sephadex DEAE was used in i-Pr20 and the racemisation carried out with Et3N under reflux in the same solvent.55... 

Name of the product Lipase Type XIII CHIRAZYME L-2, lyo... 

SP 523 Lipase powder from Thermomy-ces lanuginosus (formerly Humi-cola lanuginosa) CHIRAZYME L-8, lyo Roche Diagnostics... 

SP 524 Lipase powder from Rhizomucor miehei, rec. in Aspergillus oryzae CHIRAZYME L-9, lyo Roche Diagnostics... 

Lipases Esterases Screening Set Origin mammalian sources and microorganisms Roche Diagnostics CHIRAZYME Lipases Esterases, Screening Set Industrial Enzymes 2... 

Lipase Origin Candida antarctica, type A Roche Diagnostics CHIRAZYME L-5, lyo. Industrial... 

Lipase, immobilized Origin Mucor miehei Roche Diagnostics CHIRAZYME L-9, c.-f., C2, lyo. 

Lipase Origin porcine pancreas Roche Diagnostics CHIRAZYME L-7, lyo. 

Screening kits/sets containing samples of the normal commercially available enzymes are also provided by other enzyme suppliers, such as Boehringer Mannheim/Roche (Chirazyme sets for lipases/esterases, aldol reaction kits), Altus Biologies (ChiroScreen Kits TE and EH (based on CLECs, see section 5) for the chiral resolution of alcohols, amines, and esters), Biocatalysts (kits with alcohol dehydrogenases), Enzymatix (lipase biotransformation research kit), and others. 

Screening experiments utilized process stream ester feed, which consisted of about 22% w/v (0.91M) of the ester in toluene. Since toluene precluded the use of free ammonia due to its low solubility in toluene, solid ammonium carbamate was employed. Reactions were performed using a mixture of neat process feed, ammonium carbamate (71 g/L, 2 mol eq of ammonia), and biocatalyst (25 g/L), shaken at 400 rpm, 50°C. Under these conditions, CALB and its immobilized forms Novozym 435 and Chirazyme L2 provided racemization-free amide with yields of 69%, 43%, and 40%, together with 21%, 18%, and 22% of side products (by HPLC), respectively, while all other biocatalysts (lipases) furnished less than 5% of the desired product [42]. The ammonolysis reaction with free CALB was then optimized with regard to the temperature and the CALB and ammonium carbamate loads to increase yield from 56% to 71%, with side products varying from 7% to 19%. 

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. 

Chirazyme L2-C2 (CAL-B) proved to be a very useful enzyme for the development of an acylation process for the large-scale production of vitamin A (retinol, 91) at Roche (Scheme 27) [90,91]. In the plant process of vitamin A, intermediate 88 is partially acylated and then subjected to acid-catalyzed dehydration and isomerization to yield the vitamin A ester 90 via acetate 89. Contrary to the chemical acylation, an enzymatic approach allowed for a highly selective monoacylation of 88, and Chirazyme L2-C2 showed a very high conversion rate at 30% (w/w) substrate concentration. A first continuous process on the laboratory scale was set up with a 15 ml fixed-bed reactor containing 5.0-8.0 g of immobilized biocatalyst 4.9 kg of 89 was synthesized within 100 days in 99% yield and with 97% selectivity for the primary hydroxyl group. The laboratory process was implemented in a miniplant (120 g of biocatalyst), which could convert 1.4 kg of 88 into 1.6 kg 89 per day. After 74 days the conversion efficiency was still 99.4%. Further development of this transformation led to a modified process, which uses Thermomyces lanuginosus lipase immobilized on Accurel MPlOOl for the continuous production of 89 [92]. 

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