Enzyme crystals batch

A group at Industrial Research Limited in New Zealand recently reported the results of a study to determine if a cross-linked enzyme crystal-catalyzed resolution can compete with alternative chiral technologies in the pharmaceutical and chemical process industries. The group used the enantioselective hydrolysis of a-phenylethyl acetate catalyzed by ChiroCLEC -PC as a model system (Fig. 13). Based on their results with 270 kg of racemate, they evaluated the economics of mnning the process at the 600-kg batch scale and concluded that this process is economically feasible [38],... 

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

The first cross-linked HNLs were reported by Costes et al. [72]. They compared MeHNL-CLECs with Celite -immobilized MeHNL. By cross-linking, the stability of the enzyme was improved, particularly in polar organic solvents. Furthermore, the cross-linked crystals could be reused without significant loss of activity. After six consecutive batches, 70% of the initial activity was retained, whereas the immobilized enzyme showed virtually no remaining activity (<1%). Nevertheless, crystallization and cross-linking cause a considerable loss of activity compared to the immobilization on Celite [72]. 

The hydrolysis of an IV-acylated amino acid by an enzyme provides a resolution method to amino acids. Because the starting materials are readily available in the racemic series by the Schotten-Baumann reaction, the method can be cost effective (Scheme 2.21).68-71 The L-amino acid product can be separated by crystallization, whereas the D-amino acid, which is still /V-acylated, can be recycled by being resubjected to the Schotten-Baumann conditions used for the next batch. Tanabe has developed a process with an immobilized enzyme,72 73 whereas Degussa uses the method in a membrane reactor.69 74 The process is used to make L-methionine. 

The enzyme has a monomer weight of 30 kDa and a Km and Vmax for L-pan-tolactone of 7 mM and 30 U mg-1, respectively. X-ray fluorescence spectroscopy of crystals, and renaturation of urea/EDTA-denatured Lph in the presence of Zn2+, Mn2+, Co2+, or Ni2+ indicated Lph to be a Zn2+-hydrolase. Kinetic resolution of rac-pantolactone proceeds similarly to the fungal process mentioned above except that L-pantolactone is hydrolyzed and D-pantolactone is left behind. Repeated batches with isolated Lph and enzyme recovery by membrane filtration give d-pantolactone with 50% yield and 90-95% ee over 6 days. 

Cross-linked crystals of lipase from Candida rugosa (CRL) were applied in the resolution of racemic ketoprofen chloroethyl ester. In batch-wise operation, the half-life of the catalyst was reached after about 18 cycles or, in terms of enzyme consumption, about 5.6 g of enzyme protein were consumed to prepare 1 kg of (S)-ketoprofen. CRL suffers from a low specific activity towards this poorly water-soluble substrate which may explain the high enzyme input [117]. 

Another Bristol-Meyers Squibb process represents an enzymatic route for the production of side-chain precursors of Paclitaxel (33, Scheme 10) [69]. Racemic czs-azetidinone acetate (rac-31) is subjected to the hydrolytic treatment of Pseudomonas cepacia lipase (PCL), which is used in its immobilized form on polypropylene beads. Thus, (3R,4S)-acetate 31 can be obtained in high ee as well as the remaining alcohol 32. The process takes place in 150 1 reactors where 1.2 kg mc-31/batch can be resolved with a hydrolysis rate of 0.12 g/lh. Lowering the reaction temperature to 5 °C after full conversion causes (3R,4S)-31 subsequently to crystallize. Due to the immobilization, the enzyme can be reused for at least ten cycles without any loss of activity, productivity, or optical purity of the product. Paclitaxel is finally accessible by further chemical steps. 

A production process for the synthesis of carboxylic nucleoside precursors, which can be used for the manufacture of anti-HIV-1 agents such as carbovir (128) and analogs, has been developed by Chiroscience (Scheme 39) [ 114]. The process uses jS-lactamhydrolase from Aureobacterium sp, immobilized on a glu-taraldehyde-activated solid support for the optical resolution of lactam rac-126. The biotransformation is conducted as a batch reaction and an aqueous solution of rac-126 is cycled through a fixed bed of immobilized enzyme. This setup guarantees that the enzyme can be used in a steady-state production for more than six months, limited only by the mechanical stability of the carrier. The reaction stops when (-)-126 is completely hydrolyzed and a simple addition of acetone causes only the amino acid (-)-127 to crystallize. The latter can be used... 

Table III shows the results obtained with two crystalline preparations of the polysaccharide. Single crystals and a "quench precipitate" (prepared by quickly chilling a polymer solution in ice to precipitate the polysaccharide with minimal crystallinity) were compared. The extent of degradation of lamellar crystals is highly temperature dependent, with the total fraction of polymer digested at any temperature being finite and quite reproducible from one batch of crystals to another. Incubation up to 20 hours causes no additional decrease in turbidity nor do the crystals inactivate the enzyme. The plateau in absorbance is evidence of a "two region" model of crystal morphology, i.e., one with both accessible and inaccessible zones. The extent of digestion at 20°C of the lamellar crystalline material is to be compared with that of the "quench precipitate" form at the same temperature.

The enzymatic process was scaled-up to a 640 L preparative batch using immobilized lipase PS-30 (lipase PS-30 immobilized on Accurell polypropylene). The substrate input was 4 g/L, with 0.05% water, isopropenyl acetate was the acyl donor, and toluene was the solvent. The reaction was carried out at 37 ° C, 150 RPM for 24 h. At the end of the reaction, the enzyme was filtered off with Niagara plate filter and reused for the next cycle. The toluene filtrate was extracted with methanol water (50 50 v/v). The methanol content of the recovered extract was reduced to 20% and the entire contents were extracted with ethyl acetate. The ethyl acetate layer was collected and concentrated under reduced pressure and the produrt was crystallized from heptane. The product (5R,3R)-alcohol 54a was... 

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